culture media manual
TRANSCRIPT
A member company of International Diagnostics Group plc
CultureMedia
Manual
CultureMedia
Manual
LAB M has specialised in microbiologicaldiagnostic products since 1973. The quality of itsdehydrated culture media has an internationalreputation, particularly for anaerobic media (FAA,FAB) and innovative media for improveddetection of Salmonella.
Key services such as SMART QA proficiencytesting scheme and QC Assays media controlcultures are assisting LAB M customers in the
achievement of laboratory accreditation tonational and international standards. Customerscan also rely upon the broad base of skills andexperience both within LAB M and across theInternational Diagnostics Group to find theappropriate solution for your microbiologicalproblems
i
PROFILE
The MALTHUS System enables automatedanalysis of microbial activity, by measurement ofchanges in the electrical conductance of culturemedia. Since metabolic activity, rather thanbiomass is being measured, Malthus can provide afaster and more accurate result than available withtraditional methods.
MALTHUS Systems can reduce analysis time andlabour costs for many routine QC tests in food,toiletries and other industrial samples. The ability
to handle large sample numbers and the flexibilityto add and remove individual samples, withoutaffecting others, makes it an ideal screening tool.
MALTHUS is recognised as an officiallyapproved method by the Association of theOfficial Analytical Chemists (AOAC), the UKMinistry of Agriculture Fisheries and Foods(MAFF), the British Standards Institute (BSI) andthe United States Department of Agriculture(USDA).
PROFILE
ii
Q Laboratories is a leading UKAS accreditedlaboratory, providing a range of services tofacilitate the production of microbiologically safeproducts.
Routine testing for pathogens and spoilageorganisms is a central part of the business. Theexpertise from Q Laboratories and othercompanies in the International Diagnostics Group,enables an extensive range of tests to be offeredacross a wide range of food, pharmaceutical andhousehold products.
Testing, by itself, does not solve all quality issues.Q Laboratories specialises in evaluation ofprocesses, in development of HACCP (HazardAnalysis Critical Control Point) systems andadvice on all aspects of factory hygiene, whichultimately contributes to product quality.
Q Laboratories’ customer base includes manybrand leaders. With a testing base across the UKand worldwide demand for its consultancy,training and seminar services, Q Laboratories canprovide the specialist support increasinglyrequired to meet quality standards imposed byregulations, and more importantly, those expectedby consumers.
PROFILE
IDG Research services the group’s growingdemand for new product development and thisexpertise is extended to external companiesthrough contract development services.
IDG Research currently produce immunoassaykits for a leading European producer of foodtesting equipment. The kits are based upon
magnetic particle technology, which haveapplications in traditional microbiology culture,immunoassay and DNA capture technologies.
The facility has recently established a molecularbiology laboratory to develop technologies toenhance the specificity of pathogen detection infood and other industrial applications.
PROFILE
LABORATORIES
RESEARCH
Index by product code v
Index by product name vii
Index by organism ix
Manufacturing process pages 5-8Flow chart 5
Quality criteria 6
The application of growth rate analysers 7-8
Preparing culture media pages 9-13Dehydrated Media 9
storage 9
weighing out 9
pH of culture media 9
glassware 10
addition of powder to water 10
heat 10
dispensing prior to sterilisation 10
Autoclaving Culture Media 10
total heat input 10
measurement points 11
volume of medium 11
heat conductance of agar 11
load distribution / composition 11
agitation 11
equipment 11
Sterility Indicators 12
adhesive tape 12
Browne’s tubes 12
Spore indicators 12
Molten Media 12
re-melting agar 12
pouring plates 12
drying plates 12
Sterile Supplements 13
Storage of Prepared Media 13
Troubleshooting Guide 13
Quality control of culture media pages 14-21The ecometric technique 14
Productivity ratio 15
Liquid media 15
Templates / suggested records 16-19
Preservation of stock cultures 19
Laboratory accreditation 20
HACCP 21
Microbiology methods pages 23-27Sources of information 23
TVC 23
Coliforms / enterobacteriaceae 23
E. Coli
presence or absence 24
enumeration using membranes 24
enumeration without membranes 24
O157:H7 24
Yeasts and moulds 25
Staph. aureus 25
Pseudomonas spp. 25
Bacillus cereus 25
C. perfringens 26
Listeria
FDA method 26
modified USDA method 26
Salmonella
semi-solid method 26
conventional method 27
Campylobacter 27
Culture media information pages 29-95Format and abbreviation guide 29
Dehydrated media guide 29
Alphabetical Listing of Products 31
amies transport - FAA 31-50
FAB orange serum agar 51-70
OGYE - yersinia selective agar 71-96
Lyophilised selective agents pages 97-104Alphabetical listing of products 97
Agars, peptones and media constituents pages 105-109Alphabetical listing 105
iii
CONTENTS
LAB001 Columbia Agar Base 43
LAB002 MacConkey Agar (without salt) 60
LAB003 DCLS Agar 45
LAB004 Tryptone Soy Broth USP 15, 89
LAB005 MacConkey Broth Purple 24, 61
LAB006 CLED Medium (Bevis) 42
LAB007 Mannitol Salt Agar 62
LAB008 Nutrient Agar 69
LAB009 Sabouraud Dextrose Agar 78
LAB010 Plate Count Agar APHA 23, 73
LAB011 Tryptone Soy Agar USP 15, 88
LAB012 Sensitivity Test Agar (STA) 80
LAB013A Bismuth Sulphite Agar Base A 34
LAB013B Bismuth Sulphite Agar Base B 34
LAB014 Nutrient Broth No. 2 BP 69
LAB015 Blood Agar Base No. 2 35
LAB016 Fluorescence Agar 52
LAB018 Yeast Extract Agar 23, 95
LAB019 Milk Agar 23, 63
LAB020 Dextrose Tryptone Agar 46
LAB022 Reinforced Clostridial Medium (Broth) 76
LAB023 Reinforced Clostridial Agar 76
LAB024 Cooked Meat Granules (2 x 250g) 43
LAB024 Meat Granules 43
LAB024Z Cooked Meat Medium Tablets 44
LAB025 Fluid Thioglycollate Medium USP 15, 51
LAB027 Hoyle’s Medium 54
LAB028 Blood Agar Base 34
LAB029 Desoxycholate Citrate Agar (DCA) 44
LAB030 MacConkey Agar (with salt) 59
LAB031 Violet Red Bile Agar (VRBA) 23, 92
LAB032 XLD Agar 26, 27, 94
LAB033 Sabouraud Liquid Medium USP 78
LAB034 Brilliant Green Agar (Modified) 26, 27, 37
LAB035 TYC Medium 90
LAB036 Rose Bengal Chloramphenicol Agar 25, 77
LAB037 Malt Extract Agar 25, 61
LAB038 Wort Agar 93
LAB039 Mueller Hinton Agar II 67
LAB041 CLED Medium (single indicator) 42
LAB042 Mueller Kauffman Tetrathionate Broth 27, 68
LAB044A Selenite Broth Base 79
LAB044B Sodium Biselenite 27, 79
LAB045 MacConkey Agar No. 3 60
LAB046 Buffered Peptone Water 26, 27, 39
LAB048 Brain Heart Infusion Agar 35
LAB049 Brain Heart Infusion Broth 14, 19, 36
LAB051 Brilliant Green Bile 2% Broth 24, 37
LAB052 SS Agar (Modified) 9, 83
LAB053 Triple Sugar Iron Agar 86
LAB055A Selenite Cystine Broth Base 27, 80
LAB059 Kligler Iron Agar 56
LAB060 Endo Agar 49
LAB061 Eosin Methylene Blue Agar (Levine) 50
LAB062 Tryptose Phosphate Broth 89
LAB063 Tryptone Glucose Extract Agar 88
LAB064 Thioglycollate Medium (Brewer) 85
LAB065 Desoxycholate Citrate Agar (Hynes) 45
LAB066 Anaerobe Identification Medium Base 32
LAB067 GC Agar Base 53
LAB068 Nutrient Broth ‘E’ 69
LAB069 Simmons Citrate Agar 80
LAB071 Fastidious Anaerobe Broth (FAB) 15, 51
LAB072 Tryptone Bile Agar 24, 87
LAB073 Bacillus Cereus Medium (PREP) 25, 33
LAB074 Nusens Agar 68
LAB075 Todd Hewitt Broth 85
LAB078 CEMO Agar 40
LAB079 WL Nutrient Agar 93
LAB080A Minerals Modified Glutamate Medium 24, 64
LAB080B Sodium Glutamate 24, 64
LAB082 Membrane Lauryl Sulphate Broth 63
LAB084 Single Step Staph Selective Agar (4S) 81
LAB085 Baird Parker Medium 25, 33
LAB086 Rappaport Vassiliadis Medium (RVS) 27, 75
LAB087 Sugar Free Agar 83
LAB088 Violet Red Bile Glucose Agar (VRBGA) 23, 92
LAB089 Oxytetracycline Glucose Yeast Extract Agar 25, 71
LAB090 Fastidious Anaerobe Agar (FAA) 50
LAB091 E.E. Broth 49
LAB092 M17 Agar 59
LAB093 MRS Agar 65
LAB094 MRS Broth 66
LAB095 DN’ase Agar 47
LAB096 TCBS Cholera Medium 84
LAB097 Tetrathionate Broth (APHA) 27, 84
LAB098 Potato Dextrose Agar 74
LAB099 Wort Broth 94
LAB100 Ringers Solution 1/4 Strength 9,77
LAB100Z Ringers Solution 1/4 Strength Tablets 9, 77
LAB101 Ringers Solution (Calgon) 9, 77
LAB102 Ringers Solution (Thiosulphate) 9, 77
LAB103 Maximum Recovery Diluent 9, 23-26, 62
LAB104 Peptone Water 72
LAB105 China Blue Lactose Agar 41
LAB106 Kanamycin Aesculin Azide Agar (complete) 55
LAB107 Kanamycin Aesculin Azide Broth (complete) 55
LAB108 Pseudomonas Agar Base 25, 74
LAB109 Perfringens Agar OPSP 26, 72
LAB110 Hektoen Enteric Medium 53
LAB111 Sabouraud Maltose Agar 78
LAB112 Campylobacter Blood Free (CCDA) 14, 39
LAB113Z Salt Meat Broth Tablets 79
LAB114 Mueller Hinton Broth II 67
LAB115 Milk Plate Count Agar 23, 64
LAB116 MLCB Agar 65
iv
INDEX by product code
LAB117 DTM Dermatophyte Test Medium 46
LAB119 Yeast Extract Dextrose Chloramphenicol Agar 95
LAB120 Yersinia CIN Agar Base 96
LAB121 Bromocresol Purple Lactose Agar 38
LAB122 Listeria Isolation Medium (Oxford) 26, 58
LAB123 Kirchners T.B. Enrichment Medium 56
LAB124 Amies Transport Medium With Charcoal 31
LAB125 Amies Transport Medium Without Charcoal 31
LAB126 Lactose Broth 57
LAB127 Cooked Meat Medium 44
LAB129 Tryptone Water 24, 89
LAB130 Urea Agar Base 90
LAB131 Urea Broth Base 91
LAB133 Cetrimide Agar 40
LAB135 Campylobacter Enrichment Broth 40
LAB136 Easter - Gibson Pre-enrichment Medium 48
LAB137 Easter - Gibson Salmonella Medium 48
LAB138 Listeria Enrichment Broth (FDA) 26, 58
LAB139 Buffered Listeria Broth 26, 39
LAB140 Helicobacter Pylori Agar Base 54
LAB144 Palcam Broth 72
LAB147 Orange Serum Agar 70
LAB148 Palcam Agar Base 26, 71
LAB149 Plate Count Agar 23, 73
LAB150 MSRV Medium 26, 66
LAB155 UVM Base 26, 91
LAB157 Aseptic Commissioning Broth 32
LAB158 Liquid Baird Parker Medium 57
LAB159 Malt Extract Broth 62
LAB160 Brazier’s CCEY Agar 36
LAB161 Sorbitol MacConkey Agar 24, 82
LAB162 Tryptone Bile Glucuronide Agar 24, 87
LAB163 R2A Medium 75
LAB164 Fraser Broth 26, 52
LAB166 Slanetz & Barley Medium 82
LAB165 O157 Broth MTSB 24, 70
LAB167 Aeromonas Agar 31
LAB537 Diassalm 26, 47
MC001 Yeast Extract 108
MC002 Agar No. 1 Bacteriological - High Clarity 105
MC003 Soy Peptone 107
MC004 Balanced Peptone No. 1 106
MC005 Tryptone 108
MC006 Agar No. 2 Bacteriological - Gen. Purpose 24, 105
MC007 Acid Hydrolysed Casein 105
MC008 Tryptose 108
MC009 Mycological Peptone 107
MC011 Proteose Peptone A 107
MC019 Beef Extract 106
MC023 Malt Extract 107
MC024 Bacteriological Peptone 106
MC025 Bile Salts No. 3 106
MC026 Sodium Desoxycholate 107
MC027 Skim Milk Powder (2 x 250g) 107
MC029 Agar No. 4 - Plant Tissue Culture Grade 106
X009 Chloramphenicol 46, 77, 78, 95, 104
X011 Colistin, Nalidixic Acid 35, 43, 99
X012 Colistin, Nalidixic Acid 35, 43, 100
X013 Colistin, Oxolinic Acid 35, 43, 104
X015 Neomycin 75 35, 42, 97
X016 Neomycin 100 35, 42, 98
X018 Kanamycin 75 98
X019 P-INC Supplement 103
X040 VCA 54, 100
X068 VCNT (for Thayer Martin) 102
X069 LCT (for New York medium) 102
X070 LCAT (for New York medium) 53, 101
X073 Egg Yolk Emulsion 33, 36, 81, 98
X074 Polymixin B 33, 98
X085 Egg Yolk Tellurite 33, 103
X089 Oxytetracycline (for OGYE) 71, 104
X090 Nalidixic Acid, Vancomycin 50, 97
X091 Nalidixic Acid 50, 97
X092 Metronidazole, Nalidixic Acid 50, 97
X093 Cycloserine, Cefoxitin 36, 50, 99
X107 CN (for Ps. aeruginosa) 74, 102
X108 CFC (for Pseudomonas spp) 25, 74, 102
X109 Sulphadiazine 72, 99
X110 Oleandomycin, Polymixin 72, 99
X112 Cefoperazone, Amphotericin 39, 98
X120 CIN Selective Supplements 91, 104
X122 CCCAF (for Oxford medium) 58, 100
X131 CVTC 40, 98
X137 TMAO Selenite (as X136) 48, 100
X138 NAC (for FDA broth) 39, 58, 101
X140 Cepacia Selective Supplement 102
X144 PAC (for Palcam media) 71, 72, 101
X150 Novobiocin (for MSRV/Diassalm/O157 Broth) 47, 66, 70, 99, 103
X155 UVM I Supplement 91, 101
X156 UVM II Supplement 91, 101
X161 Cefixime Tellurite (for SMAC) 70, 82, 99
X164 Fraser Supplement (1/2 strength) 52, 101
X165 Fraser Supplement (full strength) 52, 101
X207 Methicillin (for MRSA) 103
X209 Chloramphenicol 104
X212 Cefoperazone, Amphotericin 98
X214 Skirrows 98
X215 Neomycin 75 97
X219 P-INC Supplement 103
X260 Bacitracin 100
X268 VCNT (for Thayer Martin) 102
X269 LCT (for New York medium) 102
X270 LCAT (for New York medium) 101
X271 GC Growth Supplement 102
X290 Nalidixic Acid, Vancomycin 97
X291 Nalidixic Acid 97
X546 VCC Supplement 99
X555 UVM I Supplement 101
X564 Fraser Supplement (1/2 strength) 52, 101v
Acid Hydrolysed Casein MC007 105
Aeromonas Agar LAB167 31
Agar No. 1 Bacteriological - High Clarity MC002 105
Agar No. 2 Bacteriological - Gen. Purpose MC006 105
Agar No. 4 - Plant Tissue Culture Grade MC029 106
Amies Transport Medium With Charcoal LAB124 31
Amies Transport Medium Without Charcoal LAB125 31
Anaerobe Identification Medium Base LAB066 31
Aseptic Commissioning Broth LAB157 32
Bacillus Cereus Medium (PREP) LAB073 25, 33
Bacitracin X260 100
Bacteriological Peptone MC024 106
Baird Parker Medium LAB085 25, 33
Balanced Peptone No. 1 MC004 106
Beef Extract MC019 106
Bile Salts No. 3 MC025 106
Bismuth Sulphite Agar Base A LAB013A 34
Bismuth Sulphite Agar Base B LAB013B 34
Blood Agar Base LAB028 34
Blood Agar Base No. 2 LAB015 35
Brain Heart Infusion Agar LAB048 35
Brain Heart Infusion Broth LAB049 14, 19, 36
Brazier’s CCEY Agar LAB160 36
Brilliant Green Agar (Modified) LAB034 26, 27, 37
Brilliant Green Bile 2% Broth LAB051 24, 38
Bromocresol Purple Lactose Agar LAB121 38
Buffered Listeria Broth LAB139 26, 39
Buffered Peptone Water LAB046 26, 27, 39
Campylobacter Blood Free (CCDA) LAB112 14, 39
Campylobacter Enrichment Broth LAB135 40
CCCAF (for Oxford medium) X122 58, 100
Cefixime Tellurite (for SMAC) X161 70, 82, 99
Cefoperazone, Amphotericin X112 39, 98
Cefoperazone, Amphotericin X212 98
CEMO Agar LAB078 40
Cepacia Selective Supplement X140 102
Cetrimide Agar LAB133 41
CFC (for Pseudomonas spp) X108 25, 74, 102
China Blue Lactose Agar LAB105 41
Chloramphenicol X009 46, 77, 78, 95, 104
Chloramphenicol X209 104
CIN Selective Supplements X120 95, 104
CLED Medium (Bevis) LAB006 42
CLED Medium (single indicator) LAB041 42
CN (for Ps. aeruginosa) X107 74, 102
Colistin, Nalidixic Acid X011 35, 43, 99
Colistin, Nalidixic Acid X012 35, 43, 100
Colistin, Oxolinic Acid X013 35, 43, 104
Columbia Agar Base LAB001 43
Cooked Meat Granules (2 x 250g) LAB024 43
Cooked Meat Medium LAB127 44
Cooked Meat Medium Tablets LAB024Z 44
CVTC X131 40, 98
Cycloserine, Cefoxitin X093 36, 50, 99
DCLS Agar LAB003 45
Desoxycholate Citrate Agar (DCA) LAB029 44
Desoxycholate Citrate Agar (Hynes) LAB065 45
Dextrose Tryptone Agar LAB020 46
Diassalm LAB537 26, 47
DN’ase Agar LAB095 47
DTM Dermatophyte Test Medium LAB117 46
E.E. Broth LAB091 49
Easter - Gibson Pre-enrichment Medium LAB136 48
Easter - Gibson Salmonella Medium LAB137 48
Egg Yolk Emulsion X073 33, 36, 81, 98
Egg Yolk Tellurite X085 33, 103
Endo Agar LAB060 49
Eosin Methylene Blue Agar (Levine) LAB061 50
Fastidious Anaerobe Agar (FAA) LAB090 50
Fastidious Anaerobe Broth (FAB) LAB071 15, 51
Fluid Thioglycollate Medium USP LAB025 15, 51
Fluorescence Agar LAB016 52
Fraser Broth LAB164 26, 52
Fraser Supplement (1/2 strength) X164 52, 101
Fraser Supplement (1/2 strength) X564 52, 101
Fraser Supplement (full strength) X165 52, 101
GC Agar Base LAB067 53
GC Growth Supplement X271 102
Hektoen Enteric Medium LAB110 53
Helicobacter Pylori Agar Base LAB140 54
Hoyle’s Medium LAB027 54
Kanamycin 75 X018 98
Kanamycin Aesculin Azide Agar (complete) LAB106 55
Kanamycin Aesculin Azide Broth (complete) LAB107 55
Kligler Iron Agar LAB059 56
Kirchners T.B. Enrichment Medium LAB123 56
Lactose Broth LAB126 57
LCAT (for New York medium) X070 53, 101
LCAT (for New York medium) X270 101
LCT (for New York medium) X069 102
LCT (for New York medium) X269 102
Liquid Baird Parker Medium LAB158 57
Listeria Enrichment Broth (FDA) LAB138 26, 58
Listeria Isolation Medium (Oxford) LAB122 26, 58
M17 Agar LAB092 59
MacConkey Agar (without salt) LAB002 60
MacConkey Agar (Sorbitol) LAB161 82
MacConkey Agar (with salt) LAB030 59
MacConkey Agar No. 3 LAB045 60
MacConkey Broth Purple LAB005 24, 61
Malt Extract MC023 107
Malt Extract Agar LAB037 25, 61
Malt Extract Broth LAB159 62
Mannitol Salt Agar LAB007 62
Maximum Recovery Diluent LAB103 9, 23-26, 62
Meat Granules LAB024 43
Membrane Lauryl Sulphate Broth LAB082 63vi
INDEX by product name
Methicillin (for MRSA) X207 103
Metronidazole, Nalidixic Acid X092 50, 97
Milk Agar LAB019 23, 63
Milk Plate Count Agar LAB115 23, 64
Minerals Modified Glutamate Medium LAB080A 24, 64
MLCB Agar LAB116 65
MRS Agar LAB093 65
MRS Broth LAB094 66
MSRV medium LAB150 26, 66
Mueller Hinton Agar II LAB039 67
Mueller Hinton Broth II LAB114 67
Mueller Kauffman Tetrathionate Broth LAB042 27, 68
Mycological Peptone MC009 107
NAC (for FDA broth) X138 39, 58, 101
Nalidixic Acid X091 50, 97
Nalidixic Acid X291 97
Nalidixic Acid, Vancomycin X090 50, 97
Nalidixic Acid, Vancomycin X290 97
Neomycin 100 X016 35, 42, 98
Neomycin 75 X015 35, 42, 97
Neomycin 75 X215 97
Novobiocin (for MSRV/Diassalm/ 47, 66,O157 Broth) X150 70, 99, 103
Nusens Agar LAB074 68
Nutrient Agar LAB008 69
Nutrient Broth ‘E’ LAB068 69
Nutrient Broth No. 2 BP LAB014 69
O157 Broth MTSB LAB165 24, 70
Oleandomycin, Polymixin X110 72, 99
Orange Serum Agar LAB147 70
Oxytetracycline (for OGYE) X089 71, 104
Oxytetracycline Glucose Yeast Extract Agar LAB089 25, 71
P-INC Supplement X019 103
P-INC Supplement X219 103
PAC (for Palcam media) X144 71, 72, 101
Palcam Agar Base LAB148 26, 71
Palcam Broth LAB144 72
Peptone Water LAB104 72
Perfringens Agar OPSP LAB109 26, 72
Plate Count Agar LAB149 23, 73
Plate Count Agar APHA LAB010 23, 73
Polymixin B X074 33, 98
Potato Dextrose Agar LAB098 74
PREP Agar (B.cereus) LAB073 33
Proteose Peptone A MC011 107
Pseudomonas Agar Base LAB108 25, 74
R2A Medium LAB163 75
Rappaport Vassiliadis Medium (RVS) LAB086 27, 75
Rappaport Vassiliadis Medium, semi-solid LAB150 26, 66
Reinforced Clostridial Agar LAB023 76
Reinforced Clostridial Medium (Broth) LAB022 76
Ringers Solution (Calgon) LAB101 9, 77
Ringers Solution (Thiosulphate) LAB102 9, 77
Ringers Solution 1/4 Strength LAB100 9,77
Ringers Solution 1/4 Strength Tablets LAB100Z 9, 77
Rose Bengal Chloramphenicol Agar LAB036 25, 77
Sabouraud Dextrose Agar LAB009 78
Sabouraud Liquid Medium USP LAB033 78
Sabouraud Maltose Agar LAB111 78
Salt Meat Broth Tablets LAB113Z 79
Selenite Broth Base LAB044A 79
Selenite Cystine Broth Base LAB055A 27, 80
Sensitivity Test Agar (STA) LAB012 80
Simmons Citrate Agar LAB069 81
Single Step Staph Selective Agar (4S) LAB084 81
Skim Milk Powder MC027 107
Skirrows X214 98
Slanetz & Barley Medium LAB166 82
Sodium Biselenite LAB044B 27, 79
Sodium Desoxycholate MC026 107
Sodium Glutamate LAB080B 24, 64
Sorbitol MacConkey Agar LAB161 24, 82
Soy Peptone MC003 107
SS Agar (Modified) LAB052 9, 83
Sugar Free Agar LAB087 83
Sulphadiazine X109 72, 99
TCBS Cholera Medium LAB096 84
Tetrathionate Broth (APHA) LAB097 27, 84
Tetrathionate Broth (Mueller Kauffmann) LAB042 27, 68
Thioglycollate Medium (Brewer) LAB064 85
Todd Hewitt Broth LAB075 85
TMAO Selenite X137 48, 100
Triple Sugar Iron Agar LAB053 86
Tryptone MC005 108
Tryptone Bile Agar LAB072 24, 87
Tryptone Bile Glucuronide Agar LAB162 24, 87
Tryptone Glucose Extract Agar LAB063 88
Tryptone Soy Agar USP LAB011 15, 88
Tryptone Soy Broth USP LAB004 15, 89
Tryptone Water LAB129 24, 89
Tryptose MC008 108
Tryptose Phosphate Broth LAB062 89
TYC Medium LAB035 90
Urea Agar Base LAB130 90
Urea Broth Base LAB131 91
UVM Base LAB155 26, 91
UVM I Supplement X155 91, 101
UVM I Supplement X555 101
UVM II Supplement X156 91, 101
VCA X040 54, 100
VCC Supplement X546 99
VCNT (for Thayer Martin) X068 102
VCNT (for Thayer Martin) X268 102
Violet Red Bile Agar (VRBA) LAB031 23, 92
Violet Red Bile Glucose Agar (VRBGA) LAB088 23, 92
WL Nutrient Agar LAB079 93
Wort Agar LAB038 93
Wort Broth LAB099 94
XLD Agar LAB032 26, 27, 94
Yeast Extract MC001 108
Yeast Extract Agar LAB018 23, 95
Yeast Extract Dextrose Chloramphenicol Agar LAB119 95
Yersinia CIN Agar Base LAB120 96vii
Actinomyces 35, 97
Aeromonas 19, 31
Aspergillus niger 20, 60
Bacillus cereus 19, 25, 33, 98
Bacillus stearothermophilus 12, 46
Bacteroides fragilis 19, 31, 97
Blastomyces 46
Campylobacter 14, 19, 39, 40, 98
Candida 19
Citrobacter 19
Clostridium difficile 36, 99
Clostridium perfringens 19, 26, 34, 35, 43, 72, 76, 99,
Clostridium sporogenes 19
Coccidiodes 46
Coliforms 23, 38, 41, 42, 49, 50, 57, 61, 63, 64, 89, 92
Corynebacterium 19
Dermatophytes 46
Enterobacteriacieae 23, 38, 49, 60, 81, 85, 92
Enterococcus 55, 82
Escherichia coli 19, 24, 50, 61, 87
Escherichia coli O157:H7 24, 70, 82, 99
Eubacterium 50
Fusobacterium 50
Gardnerella 35, 43, 99
Haemophilus 13, 19, 88, 100
Helicobacter 54, 100
Histoplasma 36, 46
Klebsiella 19
Lactobacillus 19, 65, 66, 70, 93
Listeria 26, 39, 52, 58, 71, 72, 91, 100, 101
Micrococcus 19, 79
Mycobacterium 56
Neisseria 19, 31, 43, 53, 67, 101, 102
Peptostreptococcus 50
Porphyromonas 50
Propionibacterium 50
Proteus 19, 42, 90
Pseudomonas 19, 25, 41, 52, 74, 102
Saccharomyces 20
Salmonella 9, 14, 19, 34, 37, 39, 48,53, 65, 68, 75, 79,
80, 83, 100, 103
Salmonella spp 19, 26, 27, 44, 45, 47, 84, 86, 94
Salmonella typhi 49, 65, 68, 84
Serratia 20
Shigella 14, 20, 44, 45, 53, 83, 94
Staphylococcus aureus 20, 25, 33, 47, 57, 62, 79, 81, 103
Staphylococcus epidermidis 20
Streptococcus 20, 35, 41, 42, 59, 70, 89, 104
Taylorella 40
TVC 23, 63, 64, 73, 75, 88, 95
Vibrio 20, 84
Yeasts & Moulds 25, 61, 62, 71, 74, 78, 93, 94, 104
Yersinia 20, 96, 104
viii
INDEX by organism
5
RAW MATERIALSAgars, Peptones, Extracts, Dyes, Chemicals etc.
Each component is individually tested for suitability. Growth promoting components are selected with the help ofan automated growth rate analyser (Malthus Instruments)
PRODUCTIONWeighing, Milling, Blending
A production batch is made from raw materials of specifiedbatch number which have been pre-tested for compatibility.The components are individually milled to ensure uniform
particle size. Weighings are double checked before thecomponents are blended.
QUALITY CONTROLPhysical, Biological parameters. Comparison with previous
batch and competition
Quality control first checks that the batch is completelyblended, then a series of physical and biological tests are
performed to ensure the product meets the exactingstandards required by our customers. Comparisons withprevious and competitor’s batches are made. Results are
recorded and a reference sample stored.
BOTTLINGInto 500g sealed containers, or bulk containers at request
Automated equipment delivers pre-weighed amounts into containers which are hermetically sealed.
Each container is immediately labelled with product details,code and batch number.
CUSTOMERSLAB M products are dispatched all over the world tomicrobiologists in all types of laboratory. Strict batch
traceability in accordance with ISO9001 ensures we can recallall products if necessary, safeguarding your products/process.
LAB M Culture Media: The Process Outline
This section intentionally starts at page 5
LAB M Culture Media – The quality criteria
Raw materialsPeptones and Extracts – Clarity, pH, moisture, growth promotingproperties with Gram positive and Gram negative organismsaerobically and anaerobically, freedom from toxicity. Compatibilitywith other components, haemolysis patterns, antibiotic antagonists.
Agar – Clarity, pH, gel strength, melting point, setting point, heavymetal content (particularly Ca++, Mg++) compatibility with othercomponents. Clarity on re-melt.
Bile Salts – Clarity, pH, thin layer chromatography, compatibilitywith other components.
Dyes & Chemicals – pH, chemical parameters, growth promotion/inhibition, properties after incorporation into culture media.
Selection of ingredientsLAB M pioneered the use of impedance technology for the selectionof Culture Media ingredients.
We were the first Company to utilise automated growth rate analysersto ensure only the most suitable ingredients available are chosen. Forexample with the use of growth rate analysis, we can select thosepeptones that trigger the exponential growth of inoculated micro-organisms in the shortest possible time; this technology is also usefulin minimising batch to batch variation.
Further information on this technique is on page 5 and 6.
ProductionAll components from specified batch numbers. All componentsweighed accurately and checked.
Components milled to uniform particle size. Components blended forspecified time, multiple samples taken to ensure thorough blending.
Quality controlPhysical – pH, clarity, gel strength, colour, heat stability, viscosity,redox.
Biological – Growth characteristics, productivity ratio, chemicalreactions and colour changes, comparison with previous batch andcompetition.
6
Choosing peptones for enrichment media - peptone A is chosen because of a faster response time.The survival time of the test organisms is also taken into consideration.
The application of growth rateanalysers in the manufacture of
bacteriological culture mediaby W.A. Hyde and K. Denton
IntroductionOne of the fundamental performance parameters of a bacteriologicalculture medium is its ability to promote the early and rapid growth ofmicro-organisms. There have been many methods devised to assessparameters, most of which rely on the viable count.
The viable count gives information on the state of the culture at asingle moment in time but gives no indication of the growth rate,unless serial viable counts are performed. Methods based on theclassical method of Lister are used for estimating growth rate. Usingthis method he was able to plot the classic growth rate curve of abacterial culture.
Unfortunately this technique is both laborious and inaccurate.Halverston and Ziegler in 19331 showed the technique to be subjectto very large experimental errors. For example, with five tubescounted from each dilution, variation can be from -70 to +260 percent!
Other variations on the Lister technique for assessing substrateperformance are:
a) Agar dilution techniques
b) Surface inoculation techniques such as the drop method of Milesand Misra2 and the ecometric technique of Mossel et al3
c) The novel spiral plater technique4
d) Photometric and nephelometric techniques
Techniques a-c still suffer from giving a point source of informationin what is a dynamic analysis.
The nephelometric/photometric techniques are relatively insensitiveand give little information about the log phase, and they cannot beused with materials which produce turbidity.
The introduction of electrical methods into the field of culture mediaperformance analysis has provided microbiologists with the means toaccurately record development of a culture over a large section of thegrowth curve. The main electrical parameters measured, areconductance and impedance. There are advantages in measuringeither of these parameters, and these are outlined in the excellentbook by Eden, R. and G5.
Impedance/conductance techniquesIn 1898, Stewart showed that bacterial growth in fluid could bedetected by changes in the electrical properties of that fluid. Sincethis time several workers have made use of conductance andimpedance measurements in the analysis of bacterial growth, finallycrystallising in the 1970’s with papers by Ur and Brown (1975)6 andCady (1975)7 describing the use of continuous impedance monitoringas a tool with wide potential in microbiology.
In 1977, workers at the Torry Research Station in Aberdeen producedequipment capable of accurately plotting the growth curve of bacteriain various fluid culture media using impedance techniques8.
The curves produced on the equipment could be directly related to thetotal viable count.
Multi-channel growth rate analysers are commercially available, egthe Malthus system9.
PeptonesThe peptones, infusions and extracts that are used by microbiologistsare manufactured from biological raw materials and these are subjectto variations and inconsistency (e.g. meat, milk, plants). These aremanufactured by acid or enzyme digestion processes which arecumbersome and difficult to control.
It is not surprising, therefore, that products described as ‘meatpeptone’, for example, are much more variable in performance thanreagent grade chemicals. (Figure 1) Because of this variation it isstandard practice for culture media manufacturers to test samples ofthese raw materials from many sources in order to maintain a supplyof products with suitable performance characteristics.
Normally pre-shipment samples will be evaluated, both alone andmade up into typical formulations, and both performance andcomparison with ‘stock’ material will be taken into account before adecision to purchase is made. Full quality control of the bulk materialis then also carried out.
LAB M introduced an eight channel Malthus Growth Rate Analyserinto its raw material selection and quality control procedures in 1982.This has enabled closer control of the performance characteristics ofthe individual peptones and culture media formulations availablefrom the company.
Method of use
The eight channel machine is used to compare the performance oftwo substrates inoculated with three to four organisms or whentesting finished product to compare the new batch of productagainst a stock batch, and against a competitor’s product.
The organisms are inoculated into 2ml or 10ml volumes of thesterile substrate in tubes which contain the electrodes. The tubesare then placed in an accurately controlled water bath andconnections are made between the electrodes and the analyser. Thebase lines for each channel are set on the chart recorder and thegrowth curves for each test are recorded.
The speed of the chart and the sensitivity of the instrument can beadjusted in order to accommodate the growth characteristics ofvarious organisms. Using this technique it is possible with someorganisms and substrates to obtain a result in less than half aworking day.
7
Figure 1
Growth curve informationThere are several parameters which can be assessed from the growthcurve produced by the Malthus system. These include:
Lag time – The time that elapses before a change in electricalproperties can be detected. Rapid metabolism causing changes inelectrical properties may be detected before cell division begins.
Detection time or take off – When the curve shifts from the baselinelag phase into the log phase, this information can be used as ameasure of the number of organisms present in a sample with the aidof suitable calibration graphs (Figure 2).
Log phase - The log phase gradient is a measure of the rate of ametabolism in the multiplying bacteria.
Maximum peak of the curve - This is related to the maximum‘number’ of bacteria achieved in the culture. The information can beused in a number of ways, for example:
a) The peptone with the shortest lag time can be chosen for thosemedia that are required to give rapid growth from a smallinoculum, e.g. sterility test media, blood culture media, andfermentation media.
b) For culture media that demand standardisation of performancefrom batch to batch, it is possible to choose peptones with aspecified lag time growth rate.
c) The maximum peak is of interest when choosing a raw materialfor production of large numbers of bacteria such as for vaccines.
As equipment based on impedance methods became established inthe microbiological quality control procedures of the food industry, anew aspect of culture media quality control is opened up, and needsto be tested for, these are the electrical properties of the product.Only by the use of impedance/conductance measuring equipment inquality control, can performance of the culture media on similarequipment in customers’ laboratories be assured.
ConclusionElectrical methods of monitoring bacterial growth have been knownsince the latter part of the 19th Century, however, only in the last fewyears with the development of micro-processors and sophisticatedelectronics are these methods beginning to reach their full potential inGrowth Rate Analysers.
In culture media manufacture the Growth Rate Analyser providesinformation that makes a significant contribution to improving boththe performance and standardisation of products manufactured fromvariable raw materials. (Figure 3)
References1. Halvorson, H.O., Ziegler, N.R. (1933a) J. Bact. 25, 101, (1933b) I.Bid., 26, 331, 559
2. Miles, A.A., Misra, S.S. (1938) J. hyg., Camb., 38, 732
3. Mossel, D.A.A., Van Rossem, F., Koopmans, M., Hendriks, M.,Verdouden M., Eelderink, I. (1980) J. Appl. Bact., 49, 439-454
4. Gilchrist, J.E., Donelly, C.B., Peeler, J.T., Delany, J.M. (1973)Appl. Microbiol. 25, 244-252
5. Eden, R. Firstenberg, Eden, G., Impedance Microbiology (1984)Research Studies Press, John Wiley and Sons, New York
6. Ur, A., Brown, D.F. J., (1975) New Approaches to theIdentification of Microorganisms (edited by C.G. Heden and T. Illeni)P.61-71 John Wiley and Sons, New York.
7. Cady, P. (1975) (edited by C.G. Heden and T.Illeni, New York).P.73-99, John Wiley and Sons, New York
8. Hobbs, G., Gibson, D. M. (1977) J. Appl. Biol. 43, 3
9. Malthus Instruments Ltd., William Clowes St., Burslem, Stoke onTrent.
First published in International Labmate 1987 Vol.12 Issue 2 pp 36-37
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Figure 2
Figure 3
Preparing Culture Media
Quality AssuredBefore each batch of LAB M Culture media is passed for sale itundergoes a rigorous quality control procedure to ensure it givesmaximum recovery and reproducibility. Reconstitution of media inthe users laboratory must be done with care to ensure the same highstandards of performance.
The following section outlines the correct procedures which willensure high quality reconstituted products, and suggests simplequality control techniques that can be used to check the performanceof prepared media.
Dehydrated Culture Media
StorageDehydrated media stored unopened under optimal conditions have ashelf life of 3-5 years but once the container is opened the contentsshould be used within six months. In-house quality control by theuser will help determine the condition of product in openedcontainers. The best conditions for storing dehydrated media are in acool, even temperature away from any sources of moisture such aswashing up areas or laboratory autoclaves and away from stronglight. Storage in a refrigerator is generally not recommended as thereis the risk of condensation on the container when it is brought out ofthe refrigerator.
TABLE 1 – Deterioration of SS Agar stored in various conditionsfor 6 months.
Storage conditions moisture gain %
Unopened bottle stored in cool,
dark, dry conditions 0
Loose cap, stored in light on bench 1.1
Loose cap, stored in light in autoclave room 4.4
The effect of the moisture gain on the performance of the agar can bequite dramatic. A 1.1% gain in moisture on storage will lead to a 53%reduction in the numbers of Salmonella isolated. Similarly a 4.4%gain in moisture will result in a 78% reduction in isolation rate. Thisdemonstrates the importance of ensuring the container lid is tightlyclosed and the pot stored in cool, dry, dark conditions. Barry, A. L.and Fay, G. D. A review of some common sources of error in thePreparation of Agar Media. 1972. Am. J. Med. Tech. Vol. 38 No. 7.
When a container is opened for the first time the date should be notedon the container. Dehydrated media should not be used if it shows anysign of moisture gain i.e. become lumpy or discoloured. The lid onthe container should be replaced quickly after media has been takenout and closed tightly.
Weighing OutUsing a top-pan balance with an accuracy of ±0.1 gram the powdershould be spooned onto a weighing boat or clean beaker. Do not tipthe media out of the container as this will cause excess dust whichmay be irritant and will certainly need cleaning up. The componentsof some formulations can be irritant so the wearing of a face mask atthis stage is advisable.
WaterPurification by distillation or deionisation is advisable. It is importantthat the equipment is properly maintained; the output of ion resinsneed to be electronically monitored and microbial colonisation of theresin and tubing must be avoided. Storage vessels for purified watermust also be monitored for microbial colonisation. It is advisable touse only fresh purified water with a conductivity of less than 10microsiemens. Stored water tends to become acidic because itabsorbs atmospheric CO2. Tap water is not recommended because ofthe potential presence of heavy metal ions which can cause inhibitionand precipitation problems.
pH of culture mediaMeter Sensitive to one decimal place.
Electrodes Calomel, gel filled unbreakable, combinationelectrodes shielded with detachable shield forcleaning. Flat electrodes may be used, butsome problems have been found by users.
Short-term storage pH 4.0 bufferof electrode
Electrode faults Indicated by slow or erratic readings andinability to obtain two points on the meterwithout adjustment.
Cleaning of 0.5% pepsin in N HCl for 1-2 hours.electrode Wash in deionised water, soak in pH 7.0
buffer for 2 hours.
Daily checks Use pH 4.0 and pH 7.0 buffer and, if possible,pH 10.0.
Testing meter pH calibrating or checking attachment.(Available from BDH.).
Water Deionised or double-distilled water is onlyweakly dissociated. Measure conductivity < 10 micro Siemens. To check pH add 0.3ml.saturated KCl to 100ml water.
Culture media Acidity: Bile salts precipitated; H2S reactionsreactions affected depressed; sugar fermentation; antibiotics lessby pH active (aminoglycocides, cephalosporins,
macrolides).
Alkalinity: Potentiates aminoglycocides; sugar fermentation; antibiotics less active (fusidin, tetracycline, penicillins).
Media difficult to Ringers, Maximal Recovery Diluent pH because oflow ionic strength
Effect of Autoclaving or irradiation will lower the pHautoclaving or about 0.2 - 0.5 units, but not predictable.irradiation
Effect of volume Mixed volume loads should be avoided. Timeof media on pH allowance must be made for various volume
sizes of media to ensure that overheatingleading to acid formation and caramelisationdoes not occur. Further details are given underAutoclaving Culture Media.
Temperature These adjust for changes within the electrodecompensator only. They DO NOT compensate for changes
in ionisation of solution at high temperatures.
Optimum 20 - 25˚C. or according to mediatemperature to manufacturer’s instructions.pH culture media
Acceptable ± 0.2 is usual. This assumes 1 litre volumepH variations produced strictly according to media
manufacturer’s instructions.
Adjustment of pH Should not be necessary if all systems correct, of media e.g. water quality, balance, volume etc., and
sterilisation is carried out precisely tomanufacturer’s instructions.
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pH out of If pH needs to be adjusted becausetolerance manufacturer’s instructions cannot be
followed, titration of small additions of N/10HCl. or N/10 NaOH into aliquots of mediashould be made, media processed and pHmeasured when cooled and appropriatecalculations carried out.
QUALITY CONTROL USING MICROBIOLOGICALPERFORMANCE CRITERIA ARE ESSENTIAL
GlasswareAll glassware must be undamaged and clean having been rinsed with purified water before storage. Borosilicate glass is preferable tosoda-glass because the latter may leach alkali into any solutioncontained in it.
Addition of Powder to WaterThere are a number of ways to do this, attempting to mix the waterand powder too quickly can cause the formation of lumps which aredifficult to disperse.
1. Pour approximately 1/3 of the volume of water required into a flask,add the powder slowly whilst constantly swirling, then addremaining water.
2. Pour the full amount of powder onto the full amount of water in aflask. Allow to stand for 10 minutes before swirling to mix.
3. Add the powder to the flask and slowly pour on the water withfrequent swirling.
NOTE: The water should always be measured before adding to theflask. Never heat water before adding to the medium.
HeatRemember that heat denatures the nutrients and the agar in culturemedia. Heat is necessary for sterilisation and for dissolving the agarif the medium is to be distributed into tubes or bottles prior tosterilisation. However these processes must be carefully controlled toensure the minimum possible heat input is used. Heat input is less inequipment that allows rapid heating and rapid cooling, e.g. mediapreparators. Media should never be left at high temperatures forprolonged periods for example holding in a 56˚C water bathovernight will noticeably reduce a culture medium’s performance andgel strength.
Dispensing prior to SterilisationMost broth media are readily soluble at room temperature or with theaid of gentle heat. Ensure a clear, well mixed, solution is obtainedbefore dispensing into final containers and sterilising. Agarcontaining media need to be brought to the boil to take the agar intosolution. This boiling should be done with frequent agitation toensure even heat distribution. As soon as the medium begins to boilit should be removed from the source of heat.
CAUTION: Agar media, particularly those with low agar content,may boil unexpectedly and overflow out of the flask. To prevent thisagitate frequently and gently as the medium approaches boiling andthe agar begins to dissolve. Allow the medium to cool to 47˚C beforedispensing with constant mixing into final containers for sterilisation.
Autoclaving culture media
To achieve optimum performance from reconstituted culture media itis important to ensure sufficient heat input to kill all spores whilstprotecting the medium from excessive heat input that would damagethe nutrient and gelling properties of the medium. In our experiencethe commonest cause of problems with culture media is the heatsterilisation, a dynamic process which has many variables that mustbe considered. These are:
Total Heat Input
The total heat input of a sterilisation cycle can be shown graphically.
The total time above 50˚C is important because both nutrients andagar will be undergoing a denaturation process. The higher thetemperature the faster the denaturation, i.e. 30 minutes at 121˚C willnoticeably affect the gel properties of most agars whilst the mediumcould be held at 50˚C for several hours before denaturation can bedetected. Total heat input should be controlled to protect themedium from denaturation. Prolonged heating and cooling canlead to excessive heat input.
The total time spent above 100˚C is important, because not only isthis the temperature at which spores are being killed, but it is thetemperature with the potential to cause most damage to the medium’sperformance. The aim of effective sterilisation is to ensure all sporesare destroyed with the minimum necessary heat input in order toensure no ill effects are caused to the medium. This can be done byusing the lethal rate equation or tables (Stumbo 1973). From a chartrecording of the sterilisation process (the recording must be from athermocouple placed inside the medium during processing), theportion of the graph between 100˚C and 121˚C for both the heatingand cooling stages is divided into 1 minute intervals, and using theequation or table, (shown below) can be converted into an equivalenttime at 121˚C. The time is added together and the total time removedfrom the holding time. This ensures that the heating process issufficient to kill the spores, but keeps damaging heat input to aminimum. Thus the instruction to ‘autoclave at 121˚C for 15 minutes’should be taken to mean ‘sterilise by an equivalent heat process to121˚C for 15 minutes’. Once calculated the new holding time can beused for future cycles, but only for the same volume of medium.Different types of media differ in heat penetration i.e. agars andbroths so the reduction in holding time must be calculated fordifferent volumes of both.
Lethal Rate Equation:L = Log-1 (T - 121.1)
Z
Where L = Equivalent time at 121˚C
T = Actual temperature for 1 minute
Z = Temperature coefficient (= 10˚C for spores)
Log-1 = antilog of the number. Using this equation, a lethal rate table can be produced: (see facing page)
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Figure 4 Schematic diagram of heat input during sterilisation
TABLE 2 – Lethal Rate Table:
Measurement PointsSome autoclaves will measure chamber temperature, some willmeasure the temperature of the medium (by use of a thermocouple),some will measure both. It is important to remember that thetemperature of the medium will lag behind that of the chamber bothin heating up and cooling down.
Volume of MediumThe heating and cooling periods of large volumes of media are longerthan for small volumes of media. Therefore, the total heat treatmentin cycles, based on holding time alone, can be significantly greaterfor large volumes than for small volumes. Adjustments have to bemade to the fluid heat penetration time in the sterilisation cycleshown below. Brecker & Bridson gave the following guide times forheat penetration at 121˚C in glass bottles:
TABLE 3 – Heat Penetration Times
Volume Time to reach 121˚C
500ml 18 minutes
1 litre 22 minutes
2 litre 27 minutes
5 litre 37 minutes
The thickness of the glass and the shape of the container were notspecified; both these factors affect the rate of heat penetration. Thesetimes seem to be based on both autoclave and medium being heatedfrom cold to 121˚C without the effective ‘holding’ at 100˚C thatoccurs with modern pulsing autoclaves or with traditional autoclavesbefore the outlet valve is closed.
In our experience, with a 5 minute hold at 100˚C, a 1 litre Pyrex flaskof medium will attain 121˚C within 5 minutes of the chamberreaching that temperature. Be aware that a sterilisation cycle that is optimum for 1 litre of medium will be excessive for l00ml of medium.
Heat Conductance of AgarAgar is a very poor conductor of heat and heat penetration into agarcontaining media is significantly slower than into non-agarcontaining media. Brecker & Bridson showed that 500mls of agar ina thin-walled bottle took 6 minutes longer to reach 121˚C than did500mls of water in an identical bottle. If a thermocouple is used in a500ml bottle of water to control the sterilisation of 500mls of agarmedium, the difference in heat penetration must be taken intoaccount. The poor heat conductivity of agar has significant effectswhen trying to sterilise large volumes of medium (e.g. 2 litres ormore). Pre-heating of the medium to get the agar into solution beforeautoclaving is recommended. Brecker & Bridson found that: “In 4litres of medium, with the agar settling undissolved to the bottom, thetemperature at the centre of the agar mass had not reached 121˚C 1 hour after the autoclave chamber had.”
Load Distribution / CompositionHeat penetration in an overloaded autoclave will be hampered as willevacuation of air. Ensure sufficient space is left between items in aload to allow free passage of steam. As covered in point 3, thevolume of medium in the bottles is directly related to the heat input.It is therefore impossible to properly control a cycle if mixed volumes(e.g. 1 litre flasks and 20ml bottles) are put together in the same load.Mixed volume loads should therefore be avoided if at all possible.
AgitationThe continuous agitation of a medium during sterilisation greatlyshortens the heating and cooling times. Modern media preparatorshave this ability which gives them a significant advantage overgeneral purpose autoclaves.
EquipmentThe equipment used for autoclaving varies from the very simple tovery complex, although they all operate by using steam underpressure to attain temperatures above 100˚C. The crudest autoclave isthe domestic pressure cooker. We consider the lack of proper controlsmake it unsuitable for routine use. Simple bench top autoclaves arelarger versions of pressure cookers but with the ability to measure thetemperature and pressure of the chamber. This equipment is suitablefor the sterilisation of small to medium sized volumes and the heatingand cooling periods are short. It is important to allow this type ofequipment to free steam before closing the outlet valve, otherwise,not all the air will be flushed out of the chamber, reducing theeffectiveness of the equipment.
Larger laboratory autoclaves now have separate steam generationfacilities allowing rapid heating and some models evacuate the air byvacuum and then inject the steam under pressure ensuring there areno air pockets causing cold spots. This type of equipment will havebuilt-in thermocouples and chart recorders capable of monitoringboth chamber and load temperatures throughout the cycle. This typeof equipment also incorporates a cooling jacket through which coldwater is run after the completion of the holding time. For safetyreasons the door will not open until the chamber has cooled to 80˚C- this can prolong cooling times.
Media preparators are dedicated to sterilising media. The mainadvantage of this equipment is that they agitate the medium speedingup heating and cooling and ensuring thorough mixing. They are wellequipped with both time and temperature controls. The cooling isbrought about by use of a water jacket. The only disadvantage ofmedia preparators is that they can only handle one medium at a timewith a cycle time of just under 1 hour. Corry et al made the followingcomment. “General purpose lab autoclaves are difficult to standardiseand more consistent results are obtained by purpose-built mediasterilisers.”
It is important that whatever equipment is used is properlymaintained to ensure accuracy of gauges.
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Figure 5 Schematic diagram showing the lag of mediumtemperature compared to chamber temperature.
Temp Time Temp Time
100 0.008 111 0.098
101 0.010 112 0.123
102 0.012 113 0.155
103 0.015 114 0.195
104 0.019 115 0.245
105 0.025 116 0.309
106 0.031 117 0.389
107 0.039 118 0.490
108 0.049 119 0.617
109 0.062 120 0.776
110 0.078 121 1.000
Temp = Temperature for 1 minute interval
Time = The equivalent time at 121˚C
References:Stumbo C.R. (1973) Thermobacteriology in Food Processing. 2ndEdition. Academic Press, New York.
Bridson E.Y. and Brecker A. (1970) Design and Formulation ofMicrobial Culture Media. Methods in Microbiology Volume 39edited by Norris S.R. and Ribbon D.W. Academic Press, New York.
Gardner J.F. and Peel M.M. (1986) Introduction to Sterilisation andDisinfection. Churchill Livingstone.
Sterility indicators
Sterility indicators can be a useful tool to highlight deficiencies insterilising equipment, but only if they are used correctly and theirlimitations are fully understood. Use of an inappropriate indicator forthe product or equipment can lead to the mistaken belief that theprocess is being properly monitored. The major drawback of sterilityindicators is that even when used carefully for the correct application,they will show that the minimum conditions have been achieved, butgive no information about the total heat input. Consequently theygive no indication as to whether the medium has been subjected toexcessive heat resulting in loss of performance of the sterilisedproduct. Wherever possible an appropriate equivalent heat processshould be calculated for the load to be sterilised.
Three types of indicator are commonly used, physical, chemical andbiological, examples of which are given below:
Adhesive TapeBowie-Dick tape is classed as a physical indicator even though itrelies upon a chemical change to produce the black diagonal stripeswhich indicate the tape has been subjected to heat. More commonlyreferred to as autoclave tape, it was designed for use in thedemonstration of adequate steam penetration. A cross of the tape wasplaced on a sheet of steam permeable paper across a towel at the mid-point of a stack of towels, and this was examined aftersterilisation to make sure the diagonal stripes changed to blackuniformly along the tape cross. It is therefore only an indicator ofsteam penetration, as the stripes will change colour before the fullcycle is complete. In most of the applications to which Bowie-Dicktape is put in the microbiology laboratory, it will be a veryunsatisfactory method for demonstrating the achievement of sterileconditions.
Browne’s TubesThe Browne’s tube is a chemical indicator comprising a heat sensitivesolution within a glass tube, which changes colour from red to greenwhen the tube is subjected to a high temperature for the requiredlength of time. In this respect it is a more reliable indicator of sterilitythan Bowie-Dick tape, as it shows both temperature and time aresufficient for sterility, whereas Bowie-Dick tape merely indicates aheating process has occurred. However the tube only indicates thatthe correct conditions have been achieved at that particular site in thesteriliser. The use of multiple tubes throughout the load is moresatisfactory, but again no information is given about the total heatinput. Furthermore, the tubes can deteriorate on storage which maylead to premature colour change and false information as to theperformance of the sterilising equipment.
Spore IndicatorsThe spores of Bacillus stearothermophilus are extremely resistant toheat and can thus be used as a biological indicator of sterilisation.Spore strips prepared in the laboratory or commercially producedstrips, are placed in various parts of the load. After the cycle iscomplete they are removed aseptically and placed in a bottle ofthioglycollate medium or cooked meat medium, and incubated at55˚C for 7 days, during which they are examined for growth. Again
this type of indicator shows if the time and temperature requirementshave been achieved but not if excessive heat has been used. LikeBrown’s tubes, spore strips only show ‘spot’ conditions, and maydeteriorate on storage giving false results. However, the length oftime needed to obtain a result means they are inconvenient for routineuse.
All the above indicators are a compromise in the absence ofaccurate steriliser control, and none can demonstrateoverheating of the medium. The only way to ensure sterilisationwithout overheating, is to use thermocouples introduced into themedium being processed, linked to chart recorders, and inaccordance with a previously determined equivalent heat processfor the equipment and volume of media being sterilised.
Molten Media
Holding molten media in water baths at 47 ˚C or above for more thana few hours should be avoided. The extra heat input will damage boththe nutrient and gelling properties of the medium. Holding at 47˚Covernight will result in noticeable denaturation of agar gel . Ideally,media should be poured as soon as possible; holding at 47˚C for 4hours should be the maximum holding time any medium is subjectedto. The addition of extra agar to compensate for the reduction ingelling caused by overheating is not recommended as the overheatingwill have also damaged the nutrient properties.
Re-melting AgarMost media will stand re-melting once in a boiling water bath. Again,the total heat input into the medium has to be considered. If a mediumis likely to be allowed to set then re-melted, it is doubly importantthat holding at 47˚C in a water bath is minimised. Never re-melt anagar more than once.
Pouring PlatesMedia should be cooled, in a water bath, to 47˚C before pouring.Immediately before pouring the medium should be swirled gently toensure thorough mixing. Plates should contain at least 5mm. ofmedium if they are to be stored before use to minimise the effects ofthe drying that will occur during storage. Single vent petri dishes losemoisture more slowly than triple vent dishes but they are also moreprone to condensation problems if not adequately dried. The amountof medium specified for poured plate techniques is generally lessbecause the medium is not stored in vented petri dishes. Whenlayered plates are used the total depth should not be less than 5 mm.
NB: -It is important to note that the tubing through which the mediumis dispensed can retain inhibitory substances on the inner surfaces.This means it is necessary to use separate tubing for non-selectivemedia and for inhibitory/antibiotic supplemented media. Failure to dothis can result in traces of inhibitory substances being incorporatedinto non-selective agars, thereby reducing their ability to support thegrowth of fastidious organisms.
Drying PlatesIn order to achieve well isolated colonies on agar plates streaked forsingle colonies, it is necessary to make sure the agar is free fromsurface moisture by drying, but caution must be taken to ensure overdrying does not occur as this can be detrimental to the performanceof the agar. It is worth noting that numerous procedures in the stagesof preparation of agar plates can result in loss of moisture, and thesum of these can impair performance if not considered and kept to aminimum. Boiling of the medium to dissolve agar before autoclaving,autoclaving, and pouring at temperatures above 50˚C can all result inmoisture loss, and so media should only be boiled before autoclavingif absolutely necessary, the autoclaving carefully controlled in anequivalent heat process, and cooled to 47˚C before pouring.
There are 2 commonly used methods of drying plates:
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1. Carefully place the plate in the incubator with the mediumcontaining side up. Lift the base of the dish up and rest it on thelid as in the diagram. This allows excess moisture to evaporatewhilst minimising the possibility of contaminating the agar.
2. Plates are allowed to stand (with lids on) overnight on the bench.The effectiveness of this technique will depend on the venting ofthe petri dishes, the humidity of the surroundings and the amountof excess moisture present. The practice of incubating platesovernight could lead to excessive moisture loss and reducedmedium performance. To check for sterility, incubate arepresentative sample at the temperature and time parameters usedfor performing the test for which the medium is employed.
Sterile Supplements
The addition of sterile supplements is performed after the mediumhas been sterilised and cooled to approximately 47˚C. Most sterilesupplements such as blood, serum and antibiotics are denatured athigher temperatures. The supplement itself should be warmed up toroom temperature before adding to the medium and it should beadded whilst mixing to prevent the formation of ‘cold spots’ andpremature gelling of the agar. Care must be taken to observe strictaseptic precautions whilst adding the supplement. The supplementedmedium should be thoroughly mixed by swirling before any platesare poured. Any antibiotic supplement reconstituted but not usedshould be thrown away. Antibiotics vary widely in their stability oncereconstituted and can deteriorate rapidly - even deep frozen. Serumproducts are best stored frozen then thawed. It is wise to aliquotserum on arrival into suitable volumes to be frozen until required. Toproduce ‘chocolate’ or heated blood plates add the sterile blood to themedium at 80˚C or add at a lower temperature and gently re-heat withfrequent swirling until the medium ‘chocolates’. This chocolating ofthe blood destroys enzymes which would otherwise inactivate thenicotinamide adenosinedinucleotide (NAD or V factor) required forgrowth by Haemophilus spp.
Storage of prepared media
The shelf life of prepared media is dependent upon the compositionof the medium, the form in which it is stored and the conditions ofstorage. All media should be stored in the dark to prevent theformation of bacteriostatic and bactericidal substances (e.g.peroxides).
Each time a batch of medium is prepared some form of performancequality control should be carried out. It is unwise to use mediabeyond their minimum shelf life without repeating the quality controland comparing it with the initial result.
PlatesMost plates stored medium side up at 4˚C in the dark will have aminimum life of 7 days. This can be extended up to 3-4 weeks forsimple nutrient media by using some form of airtight packing. Platescontaining antibiotics have a shelf life governed by the stability of theantibiotics. Generally speaking it is unwise to extend the shelf life ofan antibiotic containing medium beyond 7 days. As a medium losesmoisture the ingredients of the medium will be concentrated makingselective media progressively more selective. A plate with an originalmedium depth of 5mm will have its ingredients concentrated 20% bythe time its gel has shrunk to a depth of 4 mm. Plates showing visiblesigns of shrinkage (drying) should not be used. Plates should bebrought up to room temperature before use to avoid any ‘thermal’shock to the bacteria. Any plates left on the bench for more than 8hours should be discarded as unsuitable for use.
Bottled MediaAny medium in an airtight capped container will have a longer shelflife than in a plate. Many simple nutrient media can be stored at 15-20˚C for 3 months in the dark. Indeed, many can be stored for longerbut we advise repeat of a simple Q.C. procedure after 3 months. If amedium is stored as a gel and then is re-heated before use, repeat ofa simple Q.C. procedure is recommended. Bottled media containingantibiotics will have shelf lives governed by the activity of theantibiotic.
Troubleshooting Guide
The laboratory that routinely quality controls the media it produceswill occasionally find it has produced a batch that is not up tostandard. The following is a list of potential problems and theirpossible causes:
FAULT POSSIBLE CAUSESoft Gel Excess heat, pH too low causing acid
hydrolysis, inaccurate weighing, inadequatemixing, agar not dissolved.
pH incorrect Alkaline glassware, impure water, overheating,chemical contamination, pH taken at wrongtemperature, pH equipment faulty or poorlystandardised, deterioration of dehydratedmedium. (See specific section on pH of culturemedia)
Abnormal colour Impure water, dirty glassware, deterioration ofdehydrated medium, excess heat, pH wrong.
Darkening Excess heat, deterioration of dehydratedmedium.
Precipitation Excess heat, deterioration of dehydratedmedium, impure water or glassware.
Toxicity Excess heat (scorching or burning),deterioration of dehydrated medium.
Poor growth Contaminated water or glassware, deteriorationof dehydrated medium, incorrect weighing andmixing. Excess heat.
Poor selective or Contaminated water or glassware, incorrectdifferential weighing and mixing, deterioration ofproperties dehydrated medium. Excess heat.
13
Fig. 6 Rapid drying of plates
Lid
agar
Quality Control of Culture Media
The routine quality control of culture media is an essential ‘goodlaboratory practice’ necessary to maintain the standards andperformance of any bacteriological culture technique. More recentlyit is a requirement of many laboratory accreditation schemes such asNAMAS, and CLAS etc.
We recommend the following: A full quality control when a newbatch of dehydrated medium is introduced into the laboratory. Thisfull Q.C. to be repeated every 3-4 months on opened containers. Ashort-form quality control when a new batch of medium isreconstituted from dehydrated media previously quality controlled.A short-form quality control whenever prepared media are usedbeyond their minimum shelf life or are re-heated more than once.
Keeping of records - both full and short-form quality controls - sothat trends, e.g. fall off in performance can be detected. Each mediumused in the laboratory should have its own quality control protocoland the necessary organisms should be maintained.
TechniquesThe parameters of growth on culture media are:
● Lag time
● Organisms grown from known inoculum
● Organisms inhibited from known inoculum
● Comparative growth with standard inoculum
● Comparative inhibition with standard inoculum
● Colony size
● Colonial appearance
In practice absolute measurements of growth are time-consuming orrequire sophisticated equipment whilst colonial appearance issubjective and difficult to record. Colony size is easily measured butis an insensitive indicator of performance. Comparative methods arethe most suitable ones for routine quality control of culture mediathey can be used for comparisons of growth and inhibition. Theecometric technique of Mossel is simple and gives numericalreadings that can form the basis of records suitable for trend analysis.Both absolute growth index (AGI) and relative growth index (RGI)can be obtained by this method.
The Ecometric TechniqueThis plating technique is simple enough to use in both full and short-form quality controls. It is based on streaking an inoculum toextinction. The results obtained can be compared with previousbatches of the same medium or with batches of the same mediumfrom different manufacturers. The results can also be compared withresults obtained using the same organisms on non-selective media.
Method1. Inoculate 5mls. of Brain Heart Infusion Broth (LAB 49) with a
loopful of the chosen test organism (see individual products) andincubate for 4 hours.
2. Divide the plate to be tested into quarters designated A, B, C andD as shown below:
3. Charge a 1 microlitre loop with the incubated 4-hour culture andspread the test plate, going from Al - Bl - Cl - Dl - A2 - B2 etc.without flaming or recharging the loop and finish at D5.
4. Repeat this process with a control plate. For a batch of dehydratedmedia new to the laboratory, use a suitable non-inhibitory plate.For a new batch of plates from the laboratory stock of dehydratedmedium, use a plate of the same medium from the last batch tocheck consistency. Incubate both plates for 18 hours.
5. Note the last point at which growth occurs, on the test and controlplates, and record the segment and line, e.g. C4 or D5 etc. This isthe end point and can be used to calculate the absolute growthindex (AGI) and relative growth index (RGI) of a medium. TheAGI is obtained by noting the end point in Table 4. below and thisgives the AGI. e.g. if the end point is C4 then the AGI is 75.
TABLE 4 – Absolute Growth Index (AGI)
Al = 5 Bl = 10 Cl = 15 Dl = 20
A2 = 25 B2 = 30 C2 = 35 D2 = 40
A3 = 45 B3 = 50 C3 = 55 D3 = 60
A4 = 65 B4 = 70 C4 = 75 D4 = 80
A5 = 85 B5 = 90 C5 = 95 D5 = 100
The RGI is a comparison of the AGI of the test plate and that of thecontrol plate. For example:
Plate A—non-selective control—end point A5, AGI = 85
Plate B—selective agar test —end point C4, AGI = 75
Using the formula RGI = AGI Test x 100AGI Control
RGI = 75 x 10085
= 88.24 %
Thus, for this particular organism the test plate was 88.24% asefficient as the non-selective control plate. The performance of aselective agar can be thoroughly tested using an organism which ithas been designed to isolate and one which it is designed to inhibit.For the former the RGI should be as close to 100 as possible and thelatter as close to 0 as possible. The very nature of selective agars andthe bacteria which must be selected or repressed means thateffectiveness of different media will vary quite considerably. Forexample, Campylobacter Selective Agar (LAB 112) is a very goodselective medium as it will give high RGI’s for Campylobacter spp.and achieve suppression of bacteria without inhibiting the organismwhich is to be isolated. Examples of less effective selection occurswith Salmonella media, where there is a close relationship betweenpathogens (Salmonella and Shigella) and the other enteric organisms,making it difficult to inhibit one without reducing the isolation rate ofthe other. The onus is therefore on the end user to decide whichmedium is best for their use. Having found a medium which issuitable, use that as the yardstick to measure the performance of newbatches and to check that the efficiency of all media-makingprocesses are being maintained. It is important to note that theecometric method is a simple technique by which laboratories cancheck the media they are producing. However, it has to be performedwith care, as simple errors such as holding the inoculation loop at adifferent angle may introduce errors. This can be overcome by usinga spiral plater to produce the plates to ensure the plating method isidentical for test and control agars.
14
Figure 7 Schematic diagram of the ecometric method
Reference:Mossel D.A.A. et al (1983) Quality Assurance of Selective CultureMedia for Bacteria, Moulds and Yeasts: An Attempt at Standardisationat the International Level. J. Appl. Bacteriol. 54 313-327
Productivity Ratio (P.R.)Determining the productivity ratio of a medium is another way tocheck its performance related to a control medium, which should bea nutritious agar such as Tryptone Soy Agar (LAB 11). The inoculumused must be the same for both media and the P.R. is calculated bycounting the colonies on the test and control media:
P.R. = No. of colonies on test x dilution factor .No. of colonies on control x dilution factor
A simple method to obtain the P.R. of a medium is using the ModifiedMiles-Misra technique:
1. Prepare tenfold dilutions of an overnight culture of the testorganism in Buffered Peptone Water (LAB 46).
2. Divide the test plates into quadrants and mark each quadrant with thedilution to be used as shown below. Repeat with the control plates.
3. Starting with the highest dilution (10-8 in example below) placeone drop of the dilutions on the relevant quadrant. Repeat forcontrol plate.
4. Spread each drop over the quadrant and incubate the plates at 37˚Cfor 18 hours.
5. Count colonies at the lowest dilution they can be easily counted,for both test and control. Calculate the P.R.
For example: Test = 20 colonies at 10-3 dilution
Control = 25 colonies at 10-3 dilution
P.R. = 20 x 103
25 x 103
P.R. = 2025
P.R. = 0.8
Thus in this example the test medium is 80% as efficient as thecontrol medium.
This technique is not as simple as the ecometric method and isprobably more usefully used to quality control broth media.However, it is less prone to error than the ecometric technique and itsaccuracy can be further increased by using duplicate plates.
Liquid MediaAnother simple technique can be used to quality control fluid mediasuch as Fastidious Anaerobe Broth (LAB 71) and FluidThioglycollate Medium (LAB 25).
1. Prepare tenfold dilutions (to 10-12) of an overnight culture of thetest organism in Tryptone Soy Broth (LAB 4).
2. Add 1ml. of each dilution to 9mls. of test and control broths.Incubate at 37˚C for 18 hours.
3. Examine the broths and note the highest dilution showing growth(turbidity of the broth).
This method can be used in conjunction with the Miles-Misratechnique to demonstrate recovery of known levels of CFU’s in brothmedia.
15
Figure 8 Template for Miles-Misra plates
Mark the bottom of the plate as shown below, using a permanent marker pen.
Once the quadrants are marked, place the plate face-up on
the template below. Charge a lµl loop with a 4-hour broth
culture and spread the plate, going from
Al-Bl-Cl-Dl-A2-B2-etc. without flaming or recharging the
loop. Replace on template after incubation to read result.
16
Templates for the Ecometric Method
17
Suggested Quality ControlRecord Sheet
N.B. The same inoculum should be used for all plates. All plates must be spread at the same time, and incubated under identical conditions.
Quality Control Record (A)DATE: VOLUME OF WATER ADDED:
MEDIUM: CONDUCTIVITY OF WATER ADDED:
LAB CODE: METHOD OF STERILISATION:
BATCH NO: SUPPLEMENTS ADDED:
WEIGHT OF AGAR: EXPIRY DATE OF SUPPLEMENTS:
CONTROL MEDIUM USED:
Q.C. Organism
1
2
3
4
RG1RG1Test Control
End PointTest Control
Q.C. Organism
1
2
3
4
RG1RG1Test Control
End PointTest Control
MeanRG1
Quality Control Record (B)MEDIUM: DATE RECEIVED:
BATCH NO: DATE OPENED:
APPEARANCE OF POWDER WHEN OPENED:
18
Suggested Quality ControlRecord Sheet
N.B. Comparisons of relative growth index are only useful to show a trend in the condition of the medium. Variation between individual figures will occur due toinevitable inaccuracies of the technique.
Date RGI Appearance Comments
1234
1234
1234
1234
1234
1234
1234
1234
Preservation of Stock Cultures
The following method has been used in our laboratory for severalyears. It does not require ultra low temperature equipment, adomestic freezer at -20˚C will suffice.
Preservation of Bacteria using a Modified Glass Bead Technique
1. Grow the organism on an appropriate solid medium until a heavygrowth is obtained. Use several plates with organisms formingsmall colonies.
2. Remove growth from plate using a sterile cotton swab andemulsify in 10% w/v glycerol in Brain Heart Infusion Broth.
3. Carefully pipette the suspension on to sterile glass beads with ahole for threading, then place them in a plastic freezer tube.Replace the cap and tap the bottle to remove the bubbles frombead centres.
4. Pipette off the excess fluid into phenolic disinfectant in a discardjar. Cap the tube, label and place in freezer.
5. To recover the organisms, remove the beads using forceps, roll onan appropriate medium and streak out to obtain single colonies.
6. There are potential problems with this technique which can beavoided:(i) The tubes should be duplicated and then stored in separate pots- one for routine access, the other as a back-up in a separatefreezer. (ii) The frozen beads will soon defrost on the bench. A block ofaluminium or copper drilled out to take the plastic tubes willprevent thawing. The block is kept in the freezer along with thebeads, and removed wearing a glove.
A commercially available form of the above system called ProtectBeads is available from LAB M, please call for further details.Details of recommended QC strains of organisms are contained in theindividual entries for media. QC Assays, which combines certifiedmedia control cultures and Protect storage beads, is also available asa convenient kit.
Organism/Bacteria ATCC NCTC Other
Acinetobacter calcoaceticus 15309 5866
Aerococcus viridans 11563 8251
Aeromonas hydrophila 7966 8049 NCIMB 9240
Bacillus cereus 11778
Bacillus coagulans 7050 10334 NCIMB 9365
Bacillus licheniformis 14580 10341 NCIMB 9375
Bacillus megaterium 14581 10342 NCIMB 9376
Bacillus subtilis 5398
Bacteroides fragilis 25285 9343
Brocothrix thermosphacta 11509 10822 NCIMB 10018
Campylobacter coli 11366 CIP 7080
Campylobacter jejuni 11168
Campylobacter laridis 11352
Citrobacter freundii 6272
Clostridium bifermentans 506
Clostridium perfringens 8237
Clostridium perfringens 8238
Corynebacterium diptheriae 19409 3984
Enterobacter aerogenes 13048 10006
Enterobacter cloacae 13047 10005
Enterococcus faecalis 8043 775
Enterococcus faecium DSM 2918
Organism/Bacteria ATCC NCTC Other
Escherichia coli 11775 9001
Escherichia coli 10090
Escherichia coli NCIMB 9483
Escherichia coli O:111 9111
Haemophilus influenzae 10479
Hafnia alvei 13337 8109
Klebsiella oxytoca CMCC 2703
Lactobacillus acidophilus 19992
Lactobacillus brevis 14869
Lactobacillus bulgaricus 11842
Lactobacillus casei 7469
Lactobacillus plantarium 14917
Lactobacillus sake 15521
Lactobacillus viridescens 12706
Leuconostoc mesenteroides DSM 20241
Microbacterium flavum NCIMB 8707
Microbacterium lacticum NCIMB 8540
Micrococcus luteus 15307 2665
Moraxella sp. NCIMB 10762
Morganella morganii 8076h 235 NCIMB 235
Neisseria gonorrhoeae 19242 8375
Pediococcus damnosus 29258
Proteus mirabilis 25933
Pseudomonas aeruginosa 25668 10662
Pseudomonas fluorescens 10038
Pseudomonas fragi 10689
Pseudomonas putida 10936
Salmonella dublin 9676
Salmonella enteritidis 5188
Salmonella gallinarum 9240
Salmonella saint paul 6022
Salmonella senftenberg 10384
Salmonella typhi 8394
Salmonella typhimurium 13311 74
Salmonella virchow 5742
Serratia liquefaciens NCIMB 9321
Serratia marcescens 13880 10211
Shigella flexneri 29903
Shigella sonnei 29930
Staphylococcus aureus 25923 637
Staphylococcus aureus 6538
Staphylococcus aureus CMCC 2656
Staphylococcus epidermidis 19990 1466
Streptococcus bovis Kiel 27421
Streptococcus lactis 19435 6681 NCIMB 6681
Streptococcus pneumoniae 10319
Streptococcus pyogenes 8198
Streptococcus thermophilus 19258
Vibrio cholerae 11348
Vibrio fluvialis 11327
Vibrio parahaemolyticus 11344
Yersinia enterocolitica 9610 CCUG 11291
Yersinia enterocolitica CCUG 4588
Yersinia enterocolitica CCUG 4586
19
TABLE – 5 Culture collection strains for the quality control ofculture media
Yeasts & Moulds ATTC NCYC CMI
Alternaria alternata 89343
Aspergillus amstelodami 17455
Aspergillus flavus 91856
Aspergillus niger NCIMB 50097
Aurobasidium cladosporoides 45534
Candida albicans 18804
Cladosporum cladosporoides 45534
Debaryomyces kloeckeri 10620
Fusarium moniliforme 61274
Hansenula anomala 432
Mucor racemosus 17364
Penicillium cyclopium 19795
Pichia burtonii 439
Rhizopus stolonifer 61269
Saccharomyces cerevisiae 79
Zygosaccharomyces rouxii 1522
Abbreviation key:
ATCC American Type Culture Collection, 12301 Parklawn DriveRockville, Maryland, USA.
CCUG Culture Collection, University of Goteborg, GuldhedsgatenlOA, 5-413 46, Goteborg, Sweden.
CMCC Colworth Microbiological Culture Collection, ColworthHouse, Sharnbrook, Bedford MK44 1 LQ, U. K. [strainsfrom this source will be deposited with NCIMB].
CMI Commonwealth Mycological Institute, Ferry Lane, Kew,Surrey, UK
CIP Collection de l’Institut Pasteur, Paris, France.
DSM Deutsche Sammlung von Mikroorganismen, (GermanCollection of Micro-organisms), Grisebachstrasse 8, D-3400, Gottingen, FRG.
KIEL Federal Dairy Research Institute, Hermann Weigmanstrasse1, Kiel, FRG.
NCIMB National Collections of Industrial and Marine Bacteria Ltd.,AURIS Business Centre, 23 St. Machar Drive, Aberdeen,AB2 1RY. Scotland.
NCTC National Collection of Type Cultures, Central Public HealthLaboratory, Colindale Avenue, London NW9 5HT, U. K.
NCYC National Collection of Yeast Cultures, Food ResearchInstitute,Colney Lane, Norwich NR4 7UA, U. K.
The above table is adapted from the original published by the IUMS- ICFMH Working party on Culture media. Int. J. Food Microbiol.1987 5 297 -299.
Laboratory Accreditation
The accreditation of a microbial testing facility must be the goal of allinvolved in the production of goods or provision of services toconsumers. Accreditation is the means by which external assessmentensures that the facility, personnel, and methods of a testinglaboratory are appropriate, monitored and challenged within acontinuously improving quality system.
The impetus for laboratory accreditation has, in the past, differed byindustry sector; for example pharmaceutical laboratories have formany years been subjected to Good Laboratory Practice and regularinspection by bodies such as the FDA and MCA. However, morerecently, the benefits of accreditation are being applied in diagnosticand food testing laboratories, and probably the greatest area ofgrowth is the food industry.
The impetus for this growth is essentially the Food Safety Act of1990, which laid down the requirements for food manufacturers withregard to the provision of safe food, and introducing the ‘DueDiligence Defence’ whereby the defendant must show that allreasonable precautions which could have been taken to avoid anincident were indeed in place. It follows from this that ensuring thetesting laboratory is operating to agreed standards could beinterpreted as a reasonable precaution which should be taken.
There are a number of schemes which provide assessment andcertification of laboratory performance; NAMAS, CLAS, MAFF, GLP,ISO9000, CPA. The choice of scheme is bewildering, but narroweddepending upon the nature of testing carried out, e.g. a food laboratorywould not use CPA (Clinical Pathology Accreditation scheme) andmay also consider GLP (Good Laboratory Practice) not targeted to itsspecific needs. In addition, other schemes are available and the choicemay continue to expand. The choice of scheme should not be takenlightly, and should take into consideration the demands of thelaboratory customers (internal and external), and industry practice.
Further consideration might be given to possible future legislation, aschanges between systems will almost certainly involve extra cost if itbecomes necessary. One driving force for any future regulations islikely to be The Official Control of Foodstuffs Directive (89/397)which is an EC Directive concerned with the establishment of theSingle Market in Foodstuffs, and sets out the requirements for officialfood testing laboratories. Part of this sets out the requirements foraccreditation, stating that this must be to the EN 45000 Series ofstandards. Currently only NAMAS in the UK meet the criteria foraccreditation bodies laid down in European Standard EN 45003.Whilst these standards relate specifically to official food testinglaboratories, i.e. those laboratories who will be monitoring food withregard to enforcement of the law, it will reflect the best practice towhich all testing facilities should aspire.
20
Hazard Analysis Critical Control Points (HACCP)
In the same way that European legislation is driving the issue oflaboratory accreditation, it also affects other areas where the expertiseof the food microbiologist is essential, and a prime example of this isin the use of HACCP in the production of food.
The European Directive on the Hygiene of Foodstuffs 93/43/EECincludes the requirement that all businesses shall develop a system inaccordance with the HACCP principles to identify and control foodhazards.
Developed in the USA to ensure the absolute safety of food used inthe NASA space programme (can you imagine suffering foodpoisoning in zero gravity?), it is a system which recognises thelimitations of end-product testing and identifies the points in theproduction process critical to producing a safe product. It does notreplace end product testing; it puts end product testing into the correctcontext as a validation that the control system is functioningcorrectly.
The hazards inherent in any food production process are specific tothat process in that company at that time, and so the HACCP systemused to control it also has to be designed specifically and reviewedregularly.
Like Quality Systems HACCP can only work with the backing of theentire organisation starting from the top and requires a team approachto implement. The team must be multi-disciplinary and typically willinclude personnel from production, engineering, microbiology, andhygiene.
The team will then follow the principles of HACCP to implement asystem which will ensure the safety of the product:
Hazard AnalysisThis is the identification of the hazards associated with theproduction of the product and the ranking of the risk involved witheach and involves the input of relevant members of the team with thenecessary technical background. Once hazards are agreed then theCCP’s can be identified.
Critical Control PointsThis is any process, location, practice, or raw material which, if notcontrolled, will allow the product to pose a threat to consumer of theproduct. For example, if a product must reach 70˚C on cooking,failure to do so would pose a threat to the consumer. This is a criticalcontrol point.
Establish Control CriteriaThe control criteria must be a parameter or practice which can bemonitored, adjusted and verified in a time scale relevant to theproduction of the food. For example, oven temperature can be setaccurately determined limits, monitored by probes and adjusted inreal time. With the advent of rapid technology the hygienic status ofsurfaces can be measured in real time and re-cleaning performed ifnecessary.
Establish Monitoring ProceduresThe parameters which ensure that the critical control point is actuallyin control of the safety of the product, which allow positive feedbackand rapid corrective action. These are generally physical (time,temperature) chemical (pH, biocide levels, aw, ATP) or visual(handling procedures, equipment). Documentation of all monitoringresults is essential.
Microbiology is seldom an effective monitoring tool due to the timeinvolved. However it may be useful for monitoring contaminationlevels of raw materials prior to use.
Establish Corrective Action PlansAgreed actions necessary to regain control if monitoring of a CCPindicates that it is not controlling the safety of the product.Documentation is again essential to show the ‘quality loop’ -monitoring > non-conformance > action > monitoring > control.
Verification ProceduresFurther actions taken to verify the monitoring of CCP’s, to ensure thatthe control is in place, and the monitoring parameters are correct. If aproduct fails verification when the monitoring process shows noproblem, it indicates a failure in the system and a review is necessaryto identify a missing CCP or other source of the problem.
End product testing falls into this category, as could measuring thelevel of microbial contamination of the manufacturing environment.
Auditing and ReviewThe system must be reviewed and audited at regular intervals toensure that nothing has changed which would affect the efficacy ofthe control process, or omission of important detail. This mustinclude audit of the documentation and monitoring records, as well asphysical examination of the whole procedure. As with verification,any areas of concern must be subject to the corrective action anddemonstrate the ‘quality loop’.
Like all systems, HACCP is only effective if implemented properlywith the co-operation of all employees involved. A poorly operatedsystem could make things appear under control, when the reality isthat potentially dangerous products may be produced.
Developing a HACCP system can be a time consuming and complexbusiness, and the above representation is vastly over-simplified. It istherefore advisable to seek professional help via research associationsor expert consultants with many years experience of developingHACCP systems.
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23
Microbiology Methods
We have set out some of the more common methods in use, plus oneor two newer methods, indicating the LAB M products required toperform them. This is not intended to be a comprehensive list, norreplace other sources of information, but as a simple guide only.There are numerous sources of information regarding microbiologymethods, just some of which are listed below:
British Standards Institute, Customer Services, Linford Wood, MiltonKeynes, MK14 6LE. Numerous microbiological standards e.g.BS5763 Methods for Microbiological examination of food andanimal feeding stuffs. Part 11. Enumeration of Bacillus cereus.
Compendium of Methods for the Microbiological Examination ofFoods(1992) Third edition. Edited by Vanderzant, C. andSplittstoesser D.F. American Public Health Association. ISBN 0-87553-173-3
Food and Drug Administration. Bacteriological Analytical Manual7th edition (1992) AOAC International. ISBN 0-935584-49-8
Foodborne Pathogens An Illustrated Text (1991) Varnam, A.H. andEvans, M.G. Wolfe Publishing (now Times Mirror International).ISBN 0-7234-1521-8)
Manual of Microbiological Methods for the Food and Drink Industry.Technical Manual No. 43 2nd edition. (1995) Campden &Chorleywood Food Research Association, Chipping Campden,Goucestershire, GL55 6LD.
Micro organisms in foods - Their significance and methods ofenumeration 2nd edition (1978) ICMSF edited by Thatcher F.S. andClark D.S. ISBN 0-8020-2293-6
Practical Food Microbiology. Methods for the Examination of Foodfor Micro organisms of Public Health Significance 2nd edition (1995)Edited by Roberts D. Hooper W. and Greenwood M. Public HealthLaboratory Service ISBN 0-901144-36-3
Reports on Public Health and Medical Subjects No.71. TheBacteriological Examination of Drinking Water Supplies 1994.Methods for the Examination of Waters and Associated Materials.HMSO. ISBN 0-11-751675-9
Total Viable Count(Total Aerobic Count)
Prepare an appropriate 1:10 dilution of the samplee.g. 10g* sample + 90ml MRD LAB103
||
Homogenise for 2 mins||
Prepare serial dilutions by adding 1ml of homogenate to 9ml of MRD LAB103
||
Inoculate 1ml of appropriate dilutions into a sterile petri dish andadd aseptically 15ml of molten, cooled:
Plate Count Agar APHA LAB010 orPlate Count Agar LAB149 or
Milk Plate Count Agar LAB115 orMilk Agar LAB019 or
Yeast Extract Agar LAB018||
Incubate at the appropriate temperature to count the requiredbacterial population:
30˚C for 48hrs (aerobic mesotroph count)21˚C for 5 days or 6.5˚C for 10 days (aerobic psycrotroph count)
55˚C for 48hrs (aerobic thermotroph count)||
Select plates with between 30 and 300 colonies for enumeration. If the highest dilution has more than this use 300 as the figure to
calculate the original count and express as ‘greater than…’||
Calculate the original count in the sample expressed as CFU’s per gram or ml (The number should be given in standard scientific
notation e.g. 2.4 x 103)This method can be adapted to perform an anaerobic count by
incubating in anaerobic conditions. However incubation times mayvary to those above and specific texts should be consulted.
Remember that facultative organisms will give a count for both theaerobic and anaerobic counts.
*For liquid samples use 10ml to 90ml. No homogenisation is required prior to preparing serial dilutions.
Coliform/Enterobacteriaceae(Enumeration)
Prepare an appropriate 1:10 dilution of the samplee.g. 10g* sample + 90ml MRD LAB103
||
Homogenise for 2 mins||
Prepare serial dilutions by adding 1ml of homogenate to 9ml of MRD LAB103
||
Inoculate 1ml of appropriate dilutions into a sterile petri dish andadd aseptically 15ml of molten, cooled:
VRBA LAB031 (Coliform count)or VRBGA LAB088 (Enterobacteriaceae count)
When set, overlay with more of the same agar to cover surface||
Incubate at 37˚C for 24hr(some USA methods 35˚C, coliforms for dairy purposes 30˚C)
||
Count all colonies >0.5mm diameter present on the plate. Selectplates with between 15 and 150 colonies for enumeration. If the
highest dilution has more than this use 150 as the figure to calculatethe original count and express as ‘greater than…’
||
Calculate the original count in the sample expressed as CFU’s pergram or ml (The number should be given in standard scientific
notation e.g. 2.4 x 103)*For liquid samples use 10ml to 90ml.
No homogenisation is required prior to preparing serial dilutions.
Escherichia coli (Presence or Absence)
Prepare an appropriate 1:10 dilution of the samplee.g. 10g* sample + 90ml MRD LAB103
||
Homogenise for 2 mins||
To detect presence in 0.1g of product, inoculate 1ml of homogenateinto 10ml of single strength MacConkey Broth Purple LAB005 orBrilliant Green Bile Broth LAB051 (with inverted Durham tubes).
To detect presence in 1g of product, inoculate 10ml of doublestrength medium with 10ml of homogenate
||
Incubate at 37˚C for 24hr and examine for acid and gas(LAB005) or turbidity and gas (LAB051)
(30˚C is used in the dairy industry)||
If negative incubate for a further 24hrs, and examine again||
Subculture 0.1ml of positive broth into 10ml single strengthBrilliant Green Bile Broth (LAB051), and
Tryptone Water (LAB129)||
Incubate at 44 ± 0.5˚C for 24hrs and examine for turbidity and gas production
||
If negative incubate for a further 24hrs and examine again||
For all positive tubes add 2-4 drops of indole reagent to thematching Tryptone Water. Wait one minute and examine for a red
colour in the alcohol layer at the top of the broth, indicating apositive indole reaction
||
Record result as presumptive presence of E.coli in0.1g or 1g of product as appropriate
*For liquid samples use 10ml to 90ml. No homogenisation is required prior to preparing serial dilutions.
E.coli(Enumeration Using Membranes)
Prepare an appropriate 1:10 dilution of the samplee.g. 10g* sample + 90ml MRD LAB103
||
Homogenise for 2 mins||
Prepare serial dilutions by adding 1ml of homogenate to 9ml ofMRD LAB103
||
Inoculate membrane on the surface of MineralsModified Glutamate Agar (LAB080A + LAB080B
+ 12g/litre of Agar No.2 MC006)||
Incubate at 37˚C for 4hrs then transfer membraneto a Tryptone Bile Agar plate (LAB072)
||
Incubate with membrane uppermost at 44 ± 0.5˚C for 18-24hrs||
Put 2ml of Vracko-Sherris reagent into lid of dish, and placemembrane on top. Allow to soak in reagent for 5 min.
||
Dry membrane in natural daylight or expose to UV for 5-30 min.||
Indole positive colonies will turn pink. Count all pink colonies aspresumptive E.coli and calculate the original count in the sample,expressed as CFU’s per gram or ml. (The number should be given
in standard scientific notation e.g. 2.4 x 103
*For liquid samples use 10ml to 90ml. No homogenisation is required prior to preparing serial dilutions.
E.coli (Enumeration Without Membranes)
Prepare an appropriate 1:10 dilution of the samplee.g. 10g* sample + 90ml MRD LAB103
||
Homogenise for 2 mins||
Prepare serial dilutions by adding 1ml of homogenate to 9ml ofMRD LAB103
||
Inoculate the surface of Tryptone Bile Glucuronide Agar (LAB162)||
Incubate at 30˚C for 4hrs then transfer to 44 ± 0.5˚C for 18-24hrs||
*For liquid samples use 10ml to 90ml. No homogenisation is required prior to preparing serial dilutions.
E.coli O157:H7(Presence or Absence)
Add 25g sample to 225ml of Modified Tryptone Soy Broth(MTSB) LAB165
||
Homogenise for 2 mins||
Incubate at 42˚C for 6 and 24hrs||
Perform Captivate™
immunomagnetic separationat 6 and 24hrs
||
Inoculate 50µl of Captivate™ beads onto SMAC(LAB161) and CT SMAC (LAB161 + X161).
Incubate plates at 37˚C for 24hrs.||
Examine plates for sorbitol negative colonies||
Confirm as E.coli O157 by serology and biochemistry (commercial ID kits)
It should be noted that not all O157 isolates produce theverocytotxin which is the mode of pathogenesis of the organism.
Furthermore, other serovars of E.coli can produce the verocytotxinand therefore be pathogenic for humans.
24
Yeasts and Moulds (Enumeration)
Prepare an appropriate 1:10 dilution of the samplee.g. 10g* sample + 90ml MRD LAB103
||
Homogenise for 2 mins||
Prepare serial dilutions by adding 1ml of homogenate to 9ml ofMRD LAB103
||
Inoculate 1ml of appropriate dilutions onto the surface of :Rose Bengal Chloramphenicol Agar (LAB036), or
Malt Extract Agar (LAB037), orOxytetracycline Glucose Yeast Extract Agar (LAB089)
||
Incubate at 25˚C for 5 days||
Select plates with between 15 and 150 colonies for enumeration. If the highest dilution has more than this use 150 as the figure to
calculate the original count and express as ‘greater than…’||
Calculate the original count in the sample expressed as CFU’s per gram or ml (The number should be given in standard
scientific notation e.g. 2.49 x 103)It is always prudent to perform a simple wet preparation of suspectyeast colonies by emulsifying a colony in a drop of saline on a slideand placing a coverslip on top. Examine microscopically to confirmlarge, rounded, usually budding yeast cells as opposed to the much
smaller bacterial rods and cocci).*For liquid samples use 10ml to 90ml.
No homogenisation is required prior to preparing serial dilutions.
Staphylococcus aureus (Enumeration)
Prepare an appropriate 1:10 dilution of the samplee.g. 10g* sample + 90ml MRD LAB103
||
Homogenise for 2 mins||
Prepare serial dilutions by adding 1ml of homogenate to 9ml ofMRD LAB103
||
Inoculate 0.1ml or 0.5ml of appropriate dilutions by spreading overthe entire surface of Baird Parker medium (LAB085)
||
Incubate at 37˚C for 48hr||
Select plates with between 15 and 150 black colonies forenumeration. If the highest dilution has more than this use
150 as the figure to calculate the original count and express as ‘greater than…’
||
Confirm at least 5 colonies of each colony type present using slideor tube coagulase tests, or a proprietary kit
||
Calculate the original count in the sample expressed as CFU’s pergram or ml (The number should be given in standard scientific
notation e.g. 2.4 x 103), taking into account the ratio of coagulasepositive and negative colonies.
The advice to count all black colonies is a reflection of the variationof appearance of S.aureus (and other staphylococci) on Baird Parker
Medium. The production of the ‘typical’ lecithinase and lipasereactions is not a stable characteristic, and in particular damagedorganisms may lose the ability to produce them. Moreover, other
staphylococci can mimic these reactions and so confirmation of allcolony types is necessary to reduce the risk of reporting false counts.
*For liquid samples use 10ml to 90ml. No homogenisation is required prior to preparing serial dilutions.
Pseudomonas species (Enumeration)
Prepare an appropriate 1:10 dilution of the samplee.g. 10g* sample + 90ml MRD LAB103
||
Homogenise for 2 mins||
Prepare serial dilutions by adding 1ml of homogenate to 9ml ofMRD LAB103
||
Inoculate 0.1ml or 0.5ml by spreading over the surface ofPseudomonas Agar (LAB108) supplemented with CFC (X108)
||
Incubate at 25˚C for 48hrs||
Select plates with between 15 and 150 colonies for enumeration. If the highest dilution has more than this use 150 as the figure to
calculate the original count and express as ‘greater than…’||
Calculate the original count in the sample expressed as CFU’s pergram or ml (The number should be given in standard scientific
notation e.g. 2.4 x 103)It is advisable to confirm growth on the plate as Pseudomonas sppby performing an oxidase test. Yeasts and moulds may grow on the
medium, and these may be differentiated by performing a wetpreparation and examining under the microscope for typical cells.
*For liquid samples use 10ml to 90ml. No homogenisation is required prior to preparing serial dilutions.
Bacillus cereus (Enumeration)
Prepare an appropriate 1:10 dilution of the samplee.g. 10g* sample + 90ml MRD LAB103
||
Homogenise for 2 mins||
Prepare serial dilutions by adding 1ml of homogenate to 9ml ofMRD LAB103
||
Inoculate 0.1ml or 0.5ml by spreading over the surface of BacillusCereus Medium (PREP, LAB073)
||
Incubate at 30˚C for 24hrs||
Examine the plate for colonies. If not clearly visible incubate for afurther 24hrs.
||
Count typical colonies (large, 2-4mm, rough, pink in colour, with or without precipitate in surrounding medium).
Due to the large size of the colonies, count the dilution with lessthan 15 colonies on the plate if possible.
||
Calculate the original count in the sample expressed as CFU’s pergram or ml (The number should be given in standard scientific
notation e.g. 2.4 x 103)This is only a presumptive count, as other Bacillus spp. will grow
on PREP agar (also known as MYP agar). Confirmation can bedone by biochemical methods, although this is not straight forward.
Use reference schemes such as Bergey’s DeterminativeBacteriology Volume 2 or Cowan and Steele. Alternatively a
proprietary kit for detection of specific enterotoxin may be used,and strains of other Bacillus spp. have been reported to produce
enterotoxin.*For liquid samples use 10ml to 90ml.
No homogenisation is required prior to preparing serial dilutions.
25
Clostridium perfringens (Enumeration)
Prepare an appropriate 1:10 dilution of the samplee.g. 10g* sample + 90ml MRD LAB103
||
Homogenise for 2 mins||
Prepare serial dilutions by adding 1ml of homogenate to 9ml of MRD LAB103
||
Inoculate 1ml of appropriate dilutions into a sterile petri dish andadd aseptically 15ml of molten, cooled, Perfringens Agar (OPSP,LAB109).When set, overlay with more of the same agar to cover
surface||
Incubate anaerobically at 37˚C for 24hrs||
Select plates with between 15 and 150 colonies for enumeration. If the highest dilution has more than this use 150 as the figure to
calculate the original count and express as ‘greater than…’||
Calculate the original count in the sample expressed as CFU’s pergram or ml (The number should be given in standard scientific
notation e.g. 2.4 x 103)*For liquid samples use 10ml to 90ml.
No homogenisation is required prior to preparing serial dilutions.
Isolation of Listeria species (the ‘FDA’ method)
Add 25g sample to 225ml of Listeria Enrichment Broth (LEB, LAB138) or Buffered LEB (LAB139)
||
Homogenise for 2 mins||
Incubate at 30˚C for 24 and 48hrs||
Streak a loopful (10 µl) onto Oxford Agar (LAB122) or PalcamAgar (LAB148). Incubate plates at 30˚C for 24 and 48hrs.
||
Examine plates for typical colonies||
Confirm colonies as Listeria spp. and speciate using biochemical tests
Colonies can be speciated using Listeriazym (T500), and a blood agar plate to determine haemolytic activity.
Some workers have reported improved isolation by using bothOxford and Palcam agars together, similar to the use of different
plating media for the isolation of Salmonella.
Isolation of Listeria species (the ‘modified USDA’ method)
Add 25g sample to 225ml of UVM I (LAB155)||
Homogenise for 2 mins||
Incubate at 30˚C for 24hrs||
Subculture 0.1ml of enriched UVM I into 10ml Fraser Broth(LAB164) and incubate at 35˚C for 24 and 48hrs
||
Streak a loopful of Fraser Broth (10 µl) onto Oxford Agar(LAB122) or Palcam Agar (LAB148).
Incubate plates at 30˚C for 24 and 48hrs.||
Examine plates for typical colonies||
Confirm colonies as Listeria spp. and speciate using biochemical tests
Colonies can be speciated using Listeriazym (T500), and a blood agar plate to determine haemolytic activity.
Some workers have reported improved isolation by using bothOxford and Palcam agars together, similar to the use of different
plating media for the isolation of Salmonella.
Isolation of Salmonella species (Semi-solid technology)
Add 25g sample to 225ml of Buffered Peptone Water (LAB046)||
Homogenise for 2 minutes and incubate at 37˚C for 24hrs||
Subculture three drops of enriched BPW centrally onto the surfaceof MSRV (LAB150) or Diassalm (LAB537) plates in duplicate.
||
Incubate with the lid uppermost at 37˚C or 42˚C for 24hrs||
Examine for a spreading zone of growth. If no growth incubate fora further 24hrs
||
Subculture spreading growth to XLD (LAB032) and BrilliantGreen Agar (LAB034) to obtain pure growth for confirmation
by biochemical and serological methodsUse of a filter paper disc soaked in polyvalent H Salmonella
antiserum can increase the specificity of the media. Place the discmid way between the inoculation point and the edge of the dishbefore incubation. The motility of salmonellas will be inhibited
around the disc, whereas non-salmonellas will grow up to the disc.Use of the disc with Diassalm enhances the H2S reaction on this
medium. The area where the motility is inhibited by the disc has ahigh concentration of Salmonella and gives a good H2S reaction
indicated by blackening of the growth around the disc.
26
Subculture 1ml of enriched BPW into 9ml of Selenite Cystine Broth(LAB055A + LAB044B). Incubate at 37˚C for 24 and 48hrs
||
Inoculate selective agars: Brilliant Green Agar (LAB034) and XLD Agar (LAB032)
||
Incubate at 37˚C for 24 hrs and examine for typical colonies||
Confirm suspect colonies by serological and biochemical methods
Subculture 0.1ml of enriched BPW into 10ml of RappaportVassiliadis (Soy) Broth (LAB086). Incubate at 41.5 ± 0.5˚C* for 24hrs
||
Inoculate selective agars: Brilliant Green Agar (LAB034) and XLD Agar (LAB032)
||
Incubate at 37˚C for 24 hrs and examine for typical colonies||
Confirm suspect colonies by serological and biochemical methods
27
Isolation of Salmonella species (conventional method)
Add 25g sample to 225ml of Buffered Peptone Water (LAB046)||
Homogenise for 2 minutes and incubate at 37˚C for 24hrs
If Selenite cystine broth is not acceptable because of the use of sodium biselenite, then an alternative medium may be substituted, such as Mueller Kauffman Tetrathionate Broth (LAB042) or Tetrathionate Broth (LAB097). Both these formulations require the addition of chemicals not supplied by LAB M.
Typical colony descriptions for Brilliant Green Agar and XLD agar can be found under the individual listings in the main body of the catalogue,and are also available as colony cards which show typical colonies in full colour.
*It is critical that the incubation temperature does not exceed 43˚C as this can inhibit some salmonellas. To ensure this lower temperatures arerecommended: 41.5 ± 0.5˚C for incubators, 42 ± 0.1˚C for water baths. (Reference: Peterz M., Wiberg C., and Norberg P. 1989. The effect ofincubation temperature and magnesium chloride concentration on growth of Salmonella in home-made and in commercially available dehydratedRappaport-Vassiliadis broths. J. Appl. Bact. 66 523-528).
||
||
Isolation of Campylobacter species Add 25g sample to 75ml (or 1:4 w/v) of
Campylobacter Enrichment Broth (LAB135)||
Homogenise for 2 mins||
Incubate at 37˚C for 2-4 hrs followed by a further 14-44 hrs at 42˚C||
Streak a loopful (10µl) onto Campylobacter Blood Free Medium(LAB112) and incubate at 37˚C for 40-48 hrs
||
Examine plates for typical colonies||
Confirm colonies as Campylobacter spp. using biochemical tests
28 This page left intentionally blank
Format and abbreviation guide
Product Name(Alternative name or commonly used abbreviation)
PRODUCT CODE
Description:A brief outline which may include any of the following informationon the medium:
● HISTORY
● MECHANISMS
● APPLICATIONS
● RECOGNITION BY REGULATORY/ADVISORY BODIES
● ADVANTAGES
Formula: The product composition in grams per litre; minoradjustments to the published formula may be made to meetperformance criteria.
Method for reconstitutionDistilled water can be substituted for deionised water. “Allow to soak times” are not critical. If agar media are to be dispensed prior to sterilising, first bring to the boil to dissolve the agar.
Appearance: – of the finished cooled medium.
pH: at 20˚C. For agars, pour a small quantity into a universal bottle,allow to set and plunge the probe into the medium.
Minimum Q.C. organisms – for use every time a new batch ofprepared medium is reconstituted. This short form check should notbe confused with a full Q.C. of the medium. Where an organismshould show inhibition this could be complete or partial. Recordsshould be kept of these results to help recognise changes inperformance over a period of time.
Storage of Prepared Media – All prepared media should be storedin the dark. If a medium is to be used beyond the suggested shelf life,appropriate quality control should be performed to demonstrate thatthere has been no detectable fall off in performance.
Growth characteristicsAbbreviation key for colonial descriptions:
CV = convex CR = crenated
F = flat Rz = rhizoid
E = entire G = glossy
P.P. = pinpoint D = dull
( ) brackets are used to denote occasional variations.
References A list of related publications and sources of information.
N.B. The formulae in this manual and on the product label areadhered to wherever possible. However it is occasionally necessaryto make minor adjustments to meet performance criteria.
Dehydrated culture mediaselection guide
Agars, Peptones and other culture media ingredients
MC7 Acid Hydrolysed CaseinMC2 Agar No. 1 Bacteriological-High ClarityMC6 Agar No. 2 Bacteriological-General PurposeMC29 Agar 4-Plant Tissue CultureMC24 Bacteriological PeptoneMC4 Balanced PeptoneMC19 Beef ExtractMC25 Bile Salts No. 3MC23 Malt Extract PowderMC9 Mycological PeptoneMC11 Proteose Peptone AMC27 Skim Milk PowderMC26 Sodium DesoxycholateMC3 Soy PeptoneMC5 TryptoneMC8 TryptoseMC1 Yeast Extract Powder
Anaerobes
LAB 160 Brazier’s CCEY AgarLAB 24 Cooked Meat GranulesLAB 90 Fastidious Anaerobe Agar (F.A.A.)LAB 71 Fastidious Anaerobe Broth (F.A.B.)LAB 109 Perfingens Agar (O.P.S.P.)LAB 22 Reinforced Clostridial Medium (BrothLAB 23 Reinforced Clostridial AgarLAB 64 Thioglycollate Medium (Brewer)LAB 25 Thioglycollate Medium (Fluid)
Blood Agar Bases
LAB 1 Columbia Agar BaseLAB 28 Blood Agar BaseLAB 15 Blood Agar Base No. 2LAB 90 Fastidious Anaerobe Agar
Blood Culture Media
LAB 49 Brain Heart Infusion BrothLAB 71 Fastidious Anaerobe Broth (F.A.B.)LAB 4 Tryptone Soy Broth
Coliform Media
LAB 51 Brilliant Green Bile 2% BrothLAB 91 E.E. BrothLAB 61 Eosin Methylene Blue Agar LAB 60 Endo Agar BaseLAB 126 Lactose BrothLAB 5 MacConkey Broth (Purple)LAB 72 Tryptone Bile AgarLAB 162 Tryptone Bile Glucuronide Agar (TBGA)LAB 31 Violet Red Bile AgarLAB 88 Violet Red Bile Glucose Agar
Diagnostic Medical Microbiology
LAB 121 Bromocresol Purple Lactose AgarLAB 41 C.L.E.D. (Mackey & Sandys)-single indicatorLAB 6 C.L.E.D. (Bevis) – double indicatorLAB 67 G.C. Agar BaseLAB 123 Kirschners-T.B. enrichmentLAB 35 T.Y.C Medium-investigation of dental caries
29
Enteric Pathogens
LAB 13 Bismuth Sulphite AgarLAB 34 Brilliant Green AgarLAB 46 Buffered Peptone Water – pre enrichment brothLAB 112 Campylobacter Agar (Blood Free – Improved)LAB 135 Campylobacter Enrichment BrothLAB 161 CT-SMACLAB 29 Desoxycholate Citrate Agar (DCA)LAB 65 Desoxycholate Citrate Agar (Hynes)LAB 3 D.C.L.S.LAB 537 DiassalmLAB 16 Fluorescence Agar – Pseudomonas spp.LAB 110 Hektoen Enteric Agar – Shigella spp.LAB 2 MacConkey Agar (without salt)LAB 30 MacConkey Agar No 3 (crystal violet)LAB 116 M.L.C.B. AgarLAB 150 M.S.R.V.LAB 42 Muller Kauffman Tetrathionate BrothLAB 165 0157 Broth (MTSB)LAB 86 Rappaport Vassiliadis Medium (broth)LAB 44 Selenite BrothLAB 55 Selenite Cystine BrothLAB 161 Sorbitol MacConkey Agar. (SMAC)LAB 97 Tetrathionate Broth BaseLAB 96 T.C.B.S. Cholera MediumLAB 32 XLD AgarLAB 120 Yersinia CIN Agar
Food Microbiology
LAB 85 Baird Parker MediumLAB 34 Brilliant Green AgarLAB 105 China Blue Lactose AgarLAB 135 Campylobacter Enrichment BrothLAB 537 DiassalmLAB 136 Easter and Gibson Pre-enrichmentLAB 137 Easter and Gibson SalmonellaLAB 164 Fraser BrothLAB 122 Listeria Isolation MediumLAB 138 Listeria Enrichment BrothLAB 139 Listeria Enrichment Broth (Buffered)LAB 158 Liquid Baird Parker MediumLAB 93 M.R.S. AgarLAB 94 M.R.S. BrothLAB 150 M.S.R.V.LAB 109 Perfringens Agar (O.P.S.P.)LAB 149 Plate Count AgarLAB 10 Plate Count Agar A.P.H.A.LAB 73 P.R.E.P. Agar-cultivation of Bacillus cereusLAB 108 Pseudomonas AgarLAB 23 Reinforced Clostridial Agar-enumeration of anaerobesLAB 87 Sugar Free Agar-enumeration of organisms in
butter and similar productsLAB 162 T.B.G.A.LAB 155 U.V.M. BrothLAB 31 V.R.B.A.LAB 88 V.R.B.G.A.LAB 79 W.L. Agar-examination of worts,
beers and yeast culturesLAB 38 Wort Agar-yeasts in dairy and sugar productsLAB 99 Wort Broth
Identification Media
LAB 104 Peptone WaterLAB 69 Simmons Citrate AgarLAB 53 Triple Sugar Iron AgarLAB 129 Tryptone WaterLAB 130 Urea AgarLAB 131 Urea Broth
Nutrient Media for general use
LAB 48 Brain Heart Infusion AgarLAB 49 Brain Heart Infusion BrothLAB 8 Nutrient AgarLAB 68 Nutrient Broth ‘E’LAB 62 Tryptose Phosphate BrothLAB 18 Yeast Extract Agar
Sensitivity Test Media
LAB 39 Mueller Hinton Agar (II)LAB 114 Mueller Hinton Broth (II)LAB 74 Nu-Sens AgarLAB 12 Sensitivity Test Agar
Staphylococci Media
LAB 85 Baird Parker MediumLAB 95 DN’ase Test AgarLAB 158 Liquid Baird Parker MediumLAB 7 Mannitol Salt AgarLAB 84 Single Step Staph Selective Agar
(4S)-rapid method for S. aureusLAB 113Z Salt Meat Broth (tablets)
Streptococci Media
LAB 106 Kanamycin Aesculin Azide AgarLAB 107 Kanamycin Aesculin Azide Broth
Sterility Test Media
LAB 25 Fluid Thioglycollate U.S.P.LAB 14 Nutrient Broth No. 2 B.P.LAB 33 Sabouraud Liquid Medium U.S.P.LAB 11 Tryptone Soy AgarLAB 4 Tryptone Soy Broth U.S.P.
Transport Media
LAB 124 Amies with charcoalLAB 125 Amies without charcoal
Total Viable Counts
LAB 19 Milk AgarLAB 115 Milk Plate Count AgarLAB 10 Plate Count Agar A.P.H.A.-standard methodsLAB 149 Plate Count Agar-for spiral platers or pour platesLAB 63 Tryptone Glucose Extract Agar A.P.H.A.LAB 11 Typtone Soy Agar
Veterinary
LAB 78 C.E.M.O. Agar Base
Water Testing
LAB 5 MacConkey Broth PurpleLAB 82 Membrane Lauryl Sulphate BrothLAB 80 Minerals Modified Glutamate BrothLAB 126 Lactose BrothLAB 163 R2A Medium
Yeasts and Moulds
LAB 117 Dermatophyte Test MediumLAB 37 Malt Extract AgarLAB 89 O.G.Y.E. AgarLAB 98 Potato Dextrose AgarLAB 36 Rose Bengal Chloramphenicol AgarLAB 9 Sabouraud Dextrose AgarLAB 33 Sabouraud Liquid MediumLAB 111 Sabouraud Maltose AgarLAB 38 Wort AgarLAB 99 Wort BrothLAB 119 Yeast Extract Dextrose Chloramphenicol Agar
30
Aeromonas AgarBile Salt Irgasan Brilliant Green Agar
LAB 167DescriptionAeromonas Agar is a highly selective medium for the isolation ofAeromonas spp. from food, clinical and environmental samples.Based on the selective agents, brilliant green and irgasan, thismedium will not inhibit those strains of Aeromonas sensitive toampicillin used in other media.
Formulation g/litre
Beef Extract 5.0
Meat Peptone 5.0
Xylose 10.0
Bile Salts No.3 8.5
Sodium thiosulphate 5.44
Irgasan 0.005
Brilliant green 0.005
Neutral red 0.025
Agar 11.5
Appearance: Purple, Clear gel
pH: 7.0 ± 0.2
Method for reconstitutionWeigh 45.5 grams of powder and disperse in 1 litre of deionisedwater. Allow to soak for 10 minutes, swirl to mix and sterilise bybringing to the boil. Cool to 47˚C, mix well and dispense into petridishes.
Inoculation: Faecal specimens: Inoculate surface of mediumdirectly, spreading for single colonies. Samples requiring enrichment:Inoculate alkaline peptone water and incubate at 37˚C for 18-24 hr.Subculture onto Aeromonas Agar, surface spreading for singlecolonies.
Incubation: Incubate plates aerobically at 37˚C for 18-24 hr.Examine for typical colonies and confirm as Aeromonas spp.
Storage: Poured plates: 7 days at 2-8˚C in the dark (may be extendedif moisture tight packaging used).
Minimum Q.C. organisms: Aeromonas hydrophiliaNCIMB 9240E.coliNCIMB 50034 (inhibited)
ConfirmationTypical colonies (translucent pink colonies 0.5-3.0mm diameter)should be confirmed as presumptive Aeromonas spp. by performingan oxidase test and inoculating into Hugh & Leifsons O/F medium.Aeromonas spp. will give a positive oxidase reaction and demonstrateboth oxidative and fermentative metabolism. Pseudomonas spp. willalso be oxidase positive, but do not possess fermentative metabolism.An alternative method is to inoculate triple sugar iron tubes.Aeromonas will typically produce an acid butt (yellow) and analkaline or unchanged slant (red) whilst Pseudomonas spp. willremain unchanged in both the butt and slant. To fully identifycolonies as Aeromonas spp. the above tests should be supported usinga proprietary kit such as API 2ONE or Microbact 24E (other productsmay be available).
InterpretationOrganism Size Shape Colour
Aeromonas spp.* 0.5-3.0 CV.E.G Translucent pink
Pseudomonas spp. 0.5-1.0 CV.E.G Translucent pink
S.aureus No growth
E.coli No growth
*The selective nature of the medium may mean occasional strains do not grow,or grow poorly.
Amies Transport Medium without Charcoal
LAB 125
DescriptionThis medium is as LAB 124 without the charcoal. It is used whenmicroscopic examination of a film is an important part of theprocedure and the charcoal may interfere with interpretation.
Formula g/litre
Sodium chloride 3.0
Potassium chloride 0.2
Disodium hydrogen phosphate 1.15
Sodium thioglycollate 1.0
Calcium chloride 0.1
Magnesium chloride 0.1
Potassium phosphate 0.2
Agar No. 1 4.0
Method for reconstitutionWeigh 9.75 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then bring to boil todissolve agar. Distribute into bijou bottles filling to shoulder.Constantly mix whilst distributing. Sterilise by autoclaving at 121˚C for 15 minutes. Screw caps down after autoclaving.
Appearance: Soft translucent gel.
pH: 7.1 ± 0.2
Minimum Q.C. organisms: N. gonorrhoeaeS. pyogenes
Storage of Prepared Medium: Capped containers up to 6 months at 15-20˚C dark.
Inoculation: As LAB 124.
Amies Transport Medium with Charcoal
LAB 124
DescriptionAmies introduced his modification of Stuarts transport medium toovercome a number of problems. Stuarts transport medium sufferedfrom overgrowth by coliforms that were capable of utilising sodiumglycerophosphate. Amies replaced the problem component with aninorganic phosphate buffer system. Calcium and magnesium salts areadded to control the permeability of the bacterial cell wall and thus prolong their survival. The addition of charcoal to the medium extended the survival time of Neisseria gonorrhoeae from 24 to 72 hours.
Formula g/litre
Charcoal-activated 10.0
Sodium chloride 3.0
Potassium chloride 0.2
Disodium hydrogen phosphate 1.15
Sodium thioglycollate 1.0
Calcium chloride 0.1
Magnesium chloride 0.1
Potassium phosphate 0.2
Agar No. 1 4.0
31
Method for reconstitutionWeigh 19.75 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then bring to boil todissolve agar. Distribute into bijou bottles filling to shoulder.Constantly mix whilst distributing. Sterilise by autoclaving at 121˚Cfor 15 minutes. Screw caps down after autoclaving.
Appearance: Soft gel with heavy concentration of evenly suspendedcharcoal.
Minimum Q.C. organisms: N. gonorrhoeaeS. pyogenes
Storage of Prepared Medium: Capped containers – up to 6 monthsat 15-20˚C dark.
pH: 7.2 ± 0.2
Inoculation: Push the swab containing the sample into the gel toapproximately one third of the gels depth. Cut off the unwanted swabstick then screw on the cap pushing the swab further down into thegel. Cap tightly and keep cool during transport to the laboratory.
ReferencesAmies, C.R. 1967. Can. J. Pub. Health. 58: 296-300. A modifiedformula for the preparation of Stuarts Transport Medium.
Anaerobe Identification Medium BaseLAB 66
DescriptionA medium introduced by Phillips in 1976 for testing the fermentationcapabilities of non-sporing anaerobes. The base is carbohydrate freeand nutritious.
Formula g/litre
Beef Extract 4.0
Peptone mixture 16.0
Sodium chloride 5.0
Agar No. 2 15.0
Method for reconstitutionWeigh 40 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Cool to 48˚C and asepticallyadd 50-70mls of sterile defibrinated horse blood. Mix well and pour.Before use, flood the surface with 1ml of a sterile solution of thesubstrate under test.
Appearance: Blood agar plate.
pH: 7.2 ± 0.2
Minimum Q.C. organisms: B. fragilis
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark. Capped Container – up to 3 months at 15-20˚C in the dark.
Inoculation: Heavily inoculate a small area of the plate with aloopful of a fresh culture of the test organism. Up to 4 organisms perplate can be tested.
Incubation: 37˚C anaerobically for 24-48 hours. Recognition offermentation: Remove a small plug of agar from below the growth.Cover the plug with bromothymol blue indicator (0.04%). Colourchanges due to production of acidity will develop in a few secondsand should be viewed against a white background. Comparison withcontrols is useful, a plug taken from an area well away from anygrowth can be used as a negative control.
ReferencesPhillips K.D. 1976. A Simple and sensitive technique for determiningthe fermentation reactions of non-sporing anaerobes. J. Appl. Bact.41: 325-328.
Aseptic Commissioning BrothLAB157
DescriptionA general growth medium specifically designed for thecommissioning of aseptic filling machines in the pharmaceutical andallied industries. It is a simple formulation incorporating peptone,yeast extract and sucrose as energy sources, and phenol red toindicate growth of organisms producing acid. Gas production andturbidity also indicate growth and are indicated by bubbles inDurham’s tubes or distortion of plastic packaging. It is suitable forsterilisation by filtration, passing quickly through the filters, andreducing blockage.
Formula g/litre
Tryptone 5.0
Yeast Extract 2.5
Sucrose 5.0
Sodium chloride 5.0
Phenol red 0.005
Method for reconstitutionWeigh 17.5 grams of powder and disperse in 1 litre of deionisedwater. Allow to soak for 10 minutes, swirl to mix and heat gently todissolve. Sterilise at 121˚C for 15 minutes, or alternatively, byfiltration.
Appearance: Pale brown/straw, clear broth.
pH: 7.2 + 0.2
Minimum Q.C. organisms: Escherichia coli (acid and gasproduction) NCIMB 50034Proteus spp. (turbidity, no acid or gas)
Inoculation: Dispense medium through filling line into the finalpackaging, or suitable container, as appropriate.
Incubation: As per routine sterility testing laid down inPharmacopoeias or in house methodology. Typically 20-25˚C for up to 14 days.
32
Bacillus Cereus MediumPhenol Red Egg Yolk Polymixin Agar (P.R.E.P.)Mannitol Egg Yolk Polymixin Agar
LAB 73
DescriptionIntroduced by Mossel and his co-workers in 1967 for the enumerationof Bacillus cereus in foods, this formula was shown to be the mosteffective for this purpose by Inal in 1972. Two reactions on thismedium differentiate B. cereus from other members of the Bacillusgroup, these are mannitol fermentation and lecithinase production.Mannitol fermentation on this medium produces a yellow colour, B. cereus is mannitol negative and produces red colonies. The lecithinase production of B. cereus is indicated by a whiteprecipitate around the colonies. Polymixin is added to suppresscoliforms but some Proteus spp and Gram positive cocci may grow through.
Formula g/litre
Beef Extract 1.0
Balanced Peptone No. 1 10.0
D-Mannitol 10.0
Sodium chloride 10.0
Phenol red 0.025
Agar No. 1 15.0
Method for reconstitutionWeigh 46 grams of powder, disperse in 900ml of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C and asepticallyadd 100mls of X073 egg yolk emulsion and 2 vials of X074Polymixin.
Appearance: Pink, opaque gel.
pH: 7.2 ± 0.2
Minimum Q.C. organisms: B. cereus NCIMB 50014E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: – up to 7 days at 2-8˚C in the dark.
Inoculation: Surface, spreading or streaking for single colonies.
Incubation: 30˚C aerobically for 24-48 hours.
Growth Characteristicscolony size shape &
organism (mm) surface colour
B. cereus 3.0-4.0 F.CR.D. Pink, white halo
B. subtilis 2.0-3.0 F.CR.D. Yellow
B. coagulans 2.0 F.CR.D. Yellow
B. licheniformis 2.0 F.Rz.D. Yellow
Proteus spp. 1.0 CV.E.G. Pink (swarms)
E. faecalis 0.5 CV.E.G. Yellow
E. coli no growth
S. aureus 1.0 CV.E.G. Yellow (white halo)
ReferencesInal, T.: Vergleictiende Untersuchungen über die Selektivmedien zumqualitativen und quantitativen Nachweis von Vacillus cereus inLebensmitteln.
I. Mitteilung: Fleischwritsch, 51: 1629-1632 (1971). IV. Mitteilung:Fleischwritsch, 52: 1160-1162 (1972).
Mossel, D. A. A., Koopman, M. J. and Jongerius, E. 1967.Enumeration of Bacillus cereus in foods. Appl. Microbiol. 15: 650-653. Thatcher, F. S., Clarke, D. S. 1978 Micro-organisms in foods.Volume 1 second edition. University of Toronto.
BS5763 Part 1L:1994. ISO7932:1993 3/100
Baird-Parker Medium BaseLAB 85
DescriptionBaird-Parker introduced this complex medium in 1962 to overcomethe problems of recovering damaged Staphylococcus aureus fromfoodstuffs. The medium is highly selective due to potassium telluriteand lithium chloride. The tellurite inhibits most coliforms and is alsoreduced by S. aureus to telluride giving typical black colonies.Glycine and sodium pyruvate are both utilised by staphylococci asgrowth factors, pyruvate also neutralises toxic peroxides which maybe formed in the medium. Sulphamethazine may be added to inhibitProteus spp. Two reactions typical of S. aureus can be detected by theegg yolk. (1) lecithinase production – an opaque zone round thecolony; (2) lipase production – a zone of clearing outside the opaquezone.
Colonies suspected of being S. aureus should be confirmed by thecoagulase test or by a latex agglutination kit.
Formula g/litre
Tryptone 10.0
Beef Extract 7.5
Yeast Extract 1.0
Lithium chloride 5.0
Glycine 12.0
Sodium pyruvate 10.0
Agar No. 2 20.0
Method for reconstitutionWeigh 65.5 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C and add 50mls ofX085 sterile egg yolk tellurite emulsion. Mix well and dispense intopetri dishes.
Appearance: Cream/pale fawn, opaque.
pH: 6.8 ± 0.2
Minimum Q.C. organisms: S. aureus NCIMB 50080S. epidermidis NCIMB 50082E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Plates – up to 3 days at 2-8˚C in the dark.
Inoculation: Surface spread.
Incubation: 37˚C aerobically for 48 hours.
Growth characteristicscolony size shape &
organism (mm) surface colour other
S. aureus 1.0-3.0 CV.E.G. Black Narrow opaquemargin surroundedby a 2-5 mm zone
of clearing
S. saprophyticus 0.5-2.0 CV.E.G. Black (poor growth)
Other coagulase 0.5-1.0 CV.E.G. Black (no growth)negativestaphylococci
Proteus spp. 0.5-2.0 F.Rz.G Brown (no growth)Black
Bacillus spp. 0.5-1.0 F.Rz.D. Brown (no growth)
Entero- no growthbacteriaceae
33
ReferencesBaird-Parker, A. C. 1962. An improved diagnostic and selectivemedium for isolating coagulase positive staphylococci. J. Appl. Bact.25(1): 12-19.
Baird-Parker, A. C. and Davenport, E. 1965. The effect of Recoverymedium on the isolation of Staph. aureus after heat treatment and after storage of frozen or dried cells. J. Appl. Bact28: 390-402.
Ten Broeke, R. 1976. The Staphylococcus medium of Baird-Parker inpractical use. The occurrence of coagulase-positive, egg yolk non-clearing staphylococci. Antonie van Leeuwenhoek 33: 220-236.
Smith, B. A. and Baird-Parker, A. C. 1964. The use ofsulphamethazine for inhibiting Proteus spp. on Baird-Parker’sisolation medium for Staphylococcus aureus. J. Appl. Bact 27(1): 78-82.
Bismuth Sulphite Agar(Wilson and Blair Medium)
LAB 13A + LAB 13B
DescriptionA modification of Wilson and Blair’s original medium for theisolation of Salmonella typhi and other Salmonella from clinicalsamples, sewage and other materials. The presence of bismuthsulphite and brilliant green make this medium highly selective. As the medium contains neither lactose nor sucrose it can be used todetect lactose and sucrose fermenting Salmonella.
Formula g/litre
Bismuth Sulphite Agar Base ‘A’ LAB 13a
Beef Extract 6.0
Balanced Peptone No. 1 10.0
Ferric citrate BPC 0.4
Brilliant Green 0.01
Agar No. 2 20.0
Bismuth Chemical Mixture ‘B’ LAB 13b
Bismuth ammonium citrate 3.0
Sodium sulphite 5.0
Disodium phosphate 5.0
Glucose 5.0
Method for reconstitutionAgar Base ‘A’: Weigh 36.4 grams of powder and mix with 1 litre ofdeionised water. Sterilise for 15 minutes at 121˚C. Cool to 50˚Capprox. and add 100ml of Chemical Mixture ‘B’. Mix well and pourthin plates. Store at 4˚C for 3 days to mature, before use.
Chemical Mixture ‘B’: Suspend 18 grams of powder in 100ml. of deionised water. Bring to boil over a tripod and gauze, and coolquickly in cold water. Add to 1 litre of Agar Base ‘A’ prepared as above.
Appearance: Pale green, opaque gel.
pH: 7.6 ± 0.2
Minimum Q.C. organisms: Salmonella sp. NCIMB 50076E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Plates – store 3 days before use. Use within 7 days. Store at 2-8˚C in the dark.
Inoculation: Surface, streak out to single colonies.
Incubation: 37˚C for 24 hours aerobically.
Growth characteristicscolony size shape &
organism (mm) surface colour other
S. typhi 1.5-2.0 CV.E.G. Black Metallic sheenblack deposit in
medium. (H2S-vestrains green)
Other Salmonella spp. 1.0-2.5 CV.E.G. Black/ Metallic sheen
Green especially in heavy growth,
single colonies maygive rabbit eye
appearance
E. coli P.P.-1.0 CV.E.G. Green
Klebsiella spp. P.P-2.0 CV.E.G. Green
Citrobacter spp. 1.0-2.5 CV.E.G. Green (black centre)
Proteus spp. 1.0-2.5 CV.E.G. Green/ (black centre)
Brown
ReferencesWilson, W. J. and Blair, E. M. M’V 1926. A combination of bismuthand sodium sulphites affording an enrichment and selective mediumfor the typhoid-paratyphoid groups of bacteria. J. Pathol. Bacteriol.,29: 310-311.
International Journal of Food Microbiology 1987 5:3:200-202.
I.C.M.S.F. 1978 Micro organisms in Foods I. Their significance andenumeration. 2nd edition Univ of Toronto Press. Speck M. L. 1984.Compendium of methods for microbiological examination of foods.2nd edition. American Public Health Association, Washington. 3/102
Blood Agar baseLAB 28
DescriptionAn inexpensive general purpose agar base which, with the addition of5% sterile blood, can be used to cultivate a wide range of microorganisms of clinical significance. Typical haemolysis patterns areobtained with this medium.
Formula g/litre
Beef Extract 10.0
Balanced Peptone No. 1 10.0
Sodium chloride 5.0
Agar No. 2 12.0
Method for reconstitutionWeigh 37 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving for 15 minutes at 121˚C. Cool to 47˚C and add 5-7%sterile defibrinated blood. Mix by swirling the flask and pour intopetri dishes.
Appearance: Dependent upon blood additive.
pH: 7.4 ± 0.2
Minimum Q.C. organisms: S. aureus. NCIMB 50080S. pyogenes ATCC 19615
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation: Surface, streaking to single colonies.
Incubation: 37˚C aerobically, anaerobically or microaerobically for 24 hours.
34
Growth characteristicscolony size shape &
organism (mm) surface colour other
S. aureus 0.5-1.5 CV.E.G. White- haemolyticGolden
S. pyogenes P.P.-1.0 CV.E.G. Grey beta haemolyticalpha haemolyticnon-haemolytic
S. pneumoniae P.P.-1.0 F.E.G. Grey alpha haemolyticdraughtsman
N. meningitidis P.P.-1.5 CV.E.G. Grey mucoid
E. coli 1.5-2.5 CV.E.G. Grey haemolytic
Ps. aeruginosa 0.5-3.0 F.CR.D. Grey many colonialforms
green pigment
C. perfringens 0.5-1.5 CV.CR.G. Grey Target haemolysisnon-haemolytic
B. fragilis 0.5-1.5 CV.E.G. Grey mucoid
P. anaerobius P.P.-0.5 CV.E.G. Grey-White
F. necrophorum P.P. CV.E.G. Trans- haemolyticparent
ReferencesCruikshank, R. 1972. Medical Microbiology. 11th edn. Livingstone,London
Blood Agar Base No. 2LAB 15
DescriptionA very rich agar base which, with the addition of blood, is capable ofgrowing delicate clinical pathogens. The medium gives colonialappearances, haemolysis patterns and pigment production ofdiagnostic value. When the blood is ‘chocolated’ the medium givesgood recovery of Haemophilus spp. The medium can be madeselective for various groups by the addition of appropriate antibioticmixtures eg:
Streptococci – Colistin/Oxolinic acid (XO13)Gardnerella spp. – Colistin/Oxolinic acid (XO11)C. perfringens – Neomycin (XO15) (XO16)Staphylococci/streptococci – Colistin/Naladixic acid (XO12)
Formula g/litre
Tryptose 15.0
Soy Peptone 2.5
Yeast Extract 5.0
Sodium chloride 5.0
Agar No. 2 12.0
Method for reconstitutionWeigh 39.5 grams of powder, disperse in 1 litre of deionised water.Soak for 10 minutes, swirl to mix then sterilise for 15 minutes at 121˚C. Cool to 47˚C then aseptically add 5-7% sterile, defibrinatedhorse or sheep blood. Mix well before pouring.
Appearance: Dependent upon blood additive.
pH: 7.4 ± 0.2
Minimum Q.C. organisms: S. aureus NCIMB 50080S. pyogenes ATCC 19615
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation: Surface, streaking out to single colonies.
Incubation: 37˚C aerobically or microaerobically for 24 hours,anaerobically for 24 and 48 hours.
Growth characteristicscolony size shape &
organism (mm) surface colour other
S. aureus 1.5-2.0 CV.E.G. White/ (haemolytic)Golden
S. pyogenes 1.0-1.5 CV.E.G. Grey beta haemolytic)(alpha or nonhaemolytic)
S. pneumoniae 0.5-1.0 F.E.G. Grey (draughtsman(alpha haemolytic)
(mucoid)(require CO2)
N. meningitidis 0.5-1.0 CV.E.G. Grey (May requireCO2)
E. coli 2.0-3.0 CV.E.G. Grey (haemolytic)
Ps. aeruginosa 1.0-3.0 F.CR.D. Grey (green pigment (haemolytic)
C. perfringens 1.0-2.5 CV.CR.-(E)G Grey “Target”-haemolysis
(non-haemolytic)
B. fragilis 1.0-1.5 CV.E.G. Grey non haemolytic
P. anaerobius 0.5-1.0 CV.E.G. White non haemolytic
Brain Heart Infusion AgarLAB 48
DescriptionA general purpose nutritious agar base. This medium was first usedfor the isolation of dental pathogens. The mixture of brain and heartinfusions is particularly useful in the isolation of Actinomyces israeliand Histoplasma capsulatum. With the addition of 7% defibrinatedblood the medium will support the growth of a wide range offastidious organisms, the phosphate buffer will help neutralise theacids produced from the utilisation of glucose and thus maintainviability. The medium is not recommended for the determination ofhaemolytic reactions because of the glucose content.
The use of porcine material in this product ensures there are noSpecified Risk Materials (SRM’S) with respect to TransmissibleSpongeform Encephalopathies (TSE’S).
Formula g/litre
Brain-Heart Infusion Solids (porcine) 17.5
Tryptose 10.0
Glucose 2.0
Sodium chloride 5.0
Disodium phosphate 2.5
Agar No. 2 12.0
Method for reconstitutionWeigh 49 grams of powder, disperse in 1 litre of deionised water.Allow to stand for 10 minutes then swirl to mix. Sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C then pour intopetri dishes.
Appearance: Pale Straw colour, clear gel.
pH: 7.4 ± 0.2
Minimum Q.C. organisms: S. aureus NCIMB 50080E. coli NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark. Capped container – up to 3 months at 15-20˚C in the dark.
Inoculation: Surface, streaking out to single colonies.
Incubation: Time and temperature to suit specimen/organisms.
35
Growth characteristics (with horse blood)colony size shape &
organism (mm) surface colour
S. aureus 1.0-1.5 CV.E.G. White/Golden
other staphylococci 0.5-1.5 CV.E.G. White/Yellow
S. pyogenes 0.5-1.0 CV.E.G. White
S. milleri P.P.-0.1 CV.E.G. Transp.(White)
E. faecalis 1.0-1.25 CV.E.G. Grey/Green
S. pneumoniae 0.5-1.0 F.E.G. Grey/Green
E. coli 2.0-3.0 CV.E.G. Grey
Pseudomonas 2.0-4.0 F.CR.D. Greyaeruginosa
ReferencesRoseburg. T., Epps, L. J. and Clarke, A. R. 1944. A study of theisolation, cultivation and pathogenicity of Actinomyces israelirecovered from the human mouth and from actinomycosis in man. J. inf. Dis., 74: 131-149.
Howell, E. 1948 Efficiency of methods of isolation of Histoplasmacapsulatum. Pbl. Hlth. Rep. 63: 173-178. 3/108
Brain Heart Infusion BrothLAB 49
DescriptionA rich isotonic infusion medium with tryptose (a mixture of meat andmilk peptones) providing a wide range of substrates. A lowconcentration of glucose is used to stimulate early growth. Themedium is lightly buffered to prevent the early death of some speciesdue to acid production. Organisms which produce significantamounts of acid may well overwhelm the buffering system and auto-sterilise. The medium is suitable for use as a blood culture medium oras an enrichment broth for fastidious organisms.
The use of porcine material in this product ensures there are noSpecified Risk Materials (SRM’S) with respect to TransmissibleSpongeform Encephalopathies (TSE’S).
Formula g/litre
Brain-Heart Infusion solids (porcine) 17.5
Tryptose 10.0
Glucose 2.0
Sodium chloride 5.0
Disodium hydrogen phosphate 2.5
Method for reconstitutionWeigh 37 grams of powder then disperse in 1 litre of deionised water.Allow to stand for 10 minutes then dissolve with gentle heat beforedispensing into tubes or bottles. Sterilise at 121˚C for 15 minutes.Overheating will cause caramelisation and darkening of the medium.
Appearance: Straw colour, clear liquid.
pH: 7.4 ± 0.2
Minimum Q.C. organisms: S. aureus NCIMB 50080E. coli NCIMB 50034
Storage of Prepared Medium: Capped container – up to 3 monthsat 15-20˚C in the dark.
Inoculation: (as a blood culture medium). Using a minimum volumeof 50mls of medium add the blood to a dilution of from 1:10 to 1:20.Use in conjunction with an anaerobic culture medium eg FastidiousAnaerobe Broth LAB 71.
Incubation: 37˚C aerobically for 7 to 15 days.
Interpretation: Observe daily, subculture after 1, 2, 3, 7 and 15 daysor immediately on showing signs of growth.
ReferencesRosenow. E.C. 1919. Studies on selective localisation; focal infectionwith special reference to oral sepsis. J. Dent. Res. 1:205-267.
Brazier’s CCEY AgarLAB 160
DescriptionBrazier’s CCEY agar is the formulation currently used by theAnaerobe Reference Unit for the isolation of C.difficile, resultingfrom work initiated by Ken Phillips and Paul Levett, and completedby Jon Brazier.
Based upon the market leading anaerobe medium, FastidiousAnaerobe Agar, it incorporates additional ingredients to improve theisolation and differentiation of C.difficile from clinical specimens.
Cholic acid is present to promote spore germination followingalcohol shock treatment, and p-hydroxyphenylacetic acid to enhancethe production of p-cresol, a distinctive metabolite of C.difficile
Selectivity is achieved by addition of supplement X093 (cefoxitincycloserine) and egg yolk emulsion X073 is added to helpdifferentiate C.difficile from lecithinase positive clostridia. Finallythe addition of lysed horse blood optimises the recognition of colonyfluorescence when cultures are examined using UV light.
36
Formula g/litre
Peptone Mix 23.0
Sodium chloride 5.0
Soluble Starch 1.0
Agar 12.0
Sodium bicarbonate 0.4
Glucose 1.0
Sodium pyruvate 1.0
Cysteine HCl 0.5
Haemin 0.01
Vitamin K 0.001
L-arginine 1.0
Soluble pyrophosphate 0.25
Sodium succinate 0.5
Cholic acid 1.0
p-Hydroxyphenylacetic acid 1.0
Method for reconstitutionWeigh 48 grams of powder and add to 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix, and sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C and asepticallyadd the following: 2 vials of X093, 40ml of Egg Yolk Emulsion X073and 10ml lysed horse blood. Mix well and pour into petri dishes.
Appearance: Tan opaque gel.
pH: 7.0 + 0.2
Minimum Q.C. organisms: C.difficile E.coli (inhibition) NCIMB 50034
Storage of prepared medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation: Surface streak untreated or alcohol shocked specimensfor single colonies.
Incubation: 37˚C for 24-48hrs under anaerobic conditions
Characteristics of C.difficile: Gray opaque flat colonies, raisedelevation, 2-3mm diameter, generally circular but tending to elongatein the direction of spreading, ground glass appearance and a rough,fimbriate edge. Lecithinase negative. Incubation longer than 48hrsmay result in a lighter gray or white centre to the colony. Phenolicodour due to the production of p-cresol. Colonies fluoresce yellow-green under UV light. Confirm by latex agglutination.
ReferencesBrazier J S (1993) Role of the Laboratory in Investigations ofClostridium difficile Diarrhoea. Clinical Infectious Diseases 16 (4)S228-33.
Brilliant Green Agar (modified)Phenol Red Brilliant Green Agar
LAB 34
DescriptionFirst introduced by Kristensen et al in 1925 as a selective medium forthe isolation of salmonellae (except S. typhi). The medium wasmodified by the Netherlands Institute for Public Health, Utrecht. Themodification was to increase the selectivity of the medium byincreasing the dye concentration. This formulation is quoted by theInternational Standards Organisation, standard European CommunityMethods, the American Public Health Association and theAssociation of Official Analytical Chemists. The medium is suitablefor subcultures from selective enrichment media. However becausethis medium is highly selective, small numbers of salmonellae maybe missed. This medium is definitely not recommended for S. typhi
and Shigella spp. Less inhibitory media such as X.L.D. and HektoenEnteric will be useful in detecting salmonellae and shigellae inhibitedby Brilliant Green Agar.
Formula g/litre
Beef Extract 5.0
Balanced Peptone No. 1 10.0
Yeast Extract 3.0
Disodium hydrogen phosphate 1.0
Sodium dihydrogen phosphate 0.6
Lactose 10.0
Sucrose 10.0
Phenol red 0.09
Brilliant green 0.0047
Agar No. 2 12.0
Method for reconstitutionWeigh 52 grams of powder and disperse in 1 litre of deionised water.Allow to soak for ten minutes and then bring to the boil with frequentswirling to dissolve the solids and cool to 47˚C in a water bath. Pourplates and dry the surface before inoculation. DO NOT remelt orautoclave: overheating causes precipitation of the medium. Storeplates away from light.
Appearance: Tan, clear gel.
pH: 6.9 ± 0.2
Minimum Q.C. organisms: Salmonella sp. NCIMB 50076E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation: Surface streaking for single colonies, a heavy inoculumcan be used.
Incubation: 37˚C for 18-24 hours aerobically.
Growth characteristicscolony size shape &
organism (mm) surface colour other
Salmonella spp. 1-1.5 CV.E.G. Pink (red zone incolonies medium)
S. typhi 1.0 CV.E.G. Pink/Red (may not grow)
E. coli no growth (0.5-1.0 yellowcolony)
Proteus spp no growth
Ps aeruginosa no growth (crenated small redcolonies)
Klebsiella spp. 1-1.5 CV.E.G. Green (yellow) (NG)colonies
Enterococcus spp. no growth
S. sonnei no growth (0.5 mm red)
ReferencesEdel, W. and Kamplemacher, E.H. 1968. Comparative studies onSalmonella isolation in eight European laboratories. Bull. Wld. Hlth.Org. 39: 487-491.
Edel, W. and Kamplemacher, E.H. 1969. Salmonella infections innine European laboratories using a standard technique. Bull Wld.Hlth. Org. 41: 297-306.
American Public Health Association 1966. Recommended Methodsfor the Microbiological Examination of Foods, 2nd end. (ed. J.M.Sharf) A.P.H.A. Washington.
Association of Official Analytical Chemists (AOAC) 1978Bacteriological Analytical Manual, 5th edn., Washington D.C.
Pharmacopoeia of culture media for food microbiology. 1987. Int. J.Food Microbiol. 513: 245-247. 3/112
37
Brilliant Green Bile 2% BrothLAB 51
DescriptionA modification of MacConkey’s medium, formulated in 1926 byDunham and Schoenlein, for the recovery of coliform bacteria infoodstuffs and water. The brilliant green and bile inhibit most Grampositive organisms thus overcoming the problem of someClostridium spp. fermenting lactose and giving false positive results.
Formula g/litre
Balanced Peptone No. 1 10.0
Lactose 10.0
Ox Bile 20.0
Brilliant green 0.0133
Method for reconstitutionWeigh 40 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then warm to dissolve.Dispense into tubes or bottles with inverted Durham tubes. Steriliseby autoclaving at 115˚C for 15 minutes.
Appearance: Green, clear.
pH: 7.4 ± 0.2
Minimum Q.C. organisms: Salmonella sp. NCIMB 50076E. coli NCIMB 50034
Storage of Prepared Medium: Capped containers – up to 1 monthat 2-8˚C in the dark.
Inoculation: Serial 1:10 dilutions of homogenised sample areinoculated into the broth in the proportion of 1ml sample to 9mlsbroth. Ensure the Durham tube is free from gas bubbles beforecommencing inoculation. B.G.B. broth can be used at double strengthif required but cannot be sterilised by autoclaving, pasteurisationmust be used instead.
Incubation: E. coli and thermotrophs 44˚C for 18 hours aerobically.Mesopholic coliforms 32˚C for 24-48 hours aerobically.Psychrotrophic coliforms 4˚C for 10 days aerobically.
Interpretation: Turbidity, colour changes (to yellow or yellowishgreen) and production of gas are all presumptive evidence of thegrowth of organisms of the coli-aerogenes group. Confirmation byindole production in Tryptone Water LAB 129 (44˚C for E. coli).
ReferencesPharmacopoeia of Culture Media for Food Microbiology 1987. Int. J.Food Microbiol. 5:3:206-207.
American Public Health Association, American Water WorksAssociation and Water Pollution Control Federation, 1975, StandardMethods for the Examination of Water and Wastewater, 14th ed.,Washington D.C.
Association of Official Analytical Chemists (AOAC). BacteriologicalAnalytical Manual, 5th ed., Washington, D.C. Association of OfficialAnalytical Chemists. 1978.
Hausler, W. J. (ED) 1972. Standard Methods for the Examination ofDairy Products. 13th ed., Washington. D.C. American Public HealthAssociation.
Shane, M.S. 1947. Studies on false confirmed test using B.G.B. andcomparison studies on Lauryl Sulfate Tryptose Broth as presumptivemedium. J. Am. Water Works Assoc., 39: (4), 337.
Bromocresol Purple Lactose Agar(Drigalski agar)
LAB 121
DescriptionA non selective differential medium for the isolation and enumerationof Enterobacteriaceae from urine, water and food products. Lactosefermenting organisms produce yellow colonies, non lactosefermenters produce purple colonies.
Formula g/litre
Peptone mixture 7.4
Lactose 8.5
Bromocresol purple 0.025
Agar No. 1 12.0
Method for reconstitutionWeigh 28 grams of powder and disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Allow to cool to 47˚C then pourinto petri dishes.
Appearance: Purple, clear agar.
pH: 6.8 ± 0.2
Minimum Q.C. organisms: E. coli. NCIMB 50034S. aureus NCIMB 50080
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation: Surface, plating either over entire surface for colonycount or streak out to single colonies.
Incubation: 37˚C aerobically for 18-24 hours.
Growth Characteristicscolony size shape &
organism (mm) surface colour other
E. coli 1.5-2.0 CV.E.G. Yellow (N.L.F. – purple)
Klebsiella spp. 4.0-6.0 CV.E.G. Yellow (mucoid)
Citrobacter spp. 2.0-2.5 CV.E.G. Yellow
Proteus spp. 1.0-1.5 CV.E.G. Purple
Salmonella spp. 1.0-2.0 CV.E.G. Purple
S. aureus 0.5 CV.E.G. Cream (purple if N.L.F.)
E. faecalis 0.5 CV.E.G. Yellow
ReferencesDrigalski, Conrad. 1902. Uber ein Verfahren zum Nachweis derTyphusbacillen. Z. Hyg. Infekt. 39:283-300.
38
Buffered Listeria Enrichment BrothLAB 139
DescriptionA medium for the selective enrichment of food and environmentalsamples for Listeria spp, LAB 139 is a buffered version of the ‘FDA’broth LAB 138. The extra buffering capacity maintains the pH of theenrichment culture during incubation, ensuring optimum conditionsfor the recovery of Listeria spp.
Formula g/litre
Tryptone 17.0
Soy peptone 3.0
Sodium chloride 5.0
Dipotassium hydrogen phosphate 2.5
Glucose 2.5
Yeast Extract 6.0
Potassium dihydrogen phosphate 1.35
Disodium hydrogen phosphate 9.6
Method for reconstitutionWeigh 47 grams of powder and add to 1 litre of deionised water.Allow to soak for 10 minutes then swirl to mix and sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C and add 2 vials ofX138 reconstituted in 50% alcohol. Aseptically dispense into steriletubes or bottles.
Appearance: Yellow, clear.
pH: 7.2 ± 0.2
Minimum Q.C. organisms: L. monocytogenes NCIMB50007E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Capped containers – up to 14 days at2-8˚C in the dark.
Inoculation: Homogenised samples of food, 25 grams ofhomogenate to 225mls of broth.
Incubation: 30˚C aerobically for up to 48 hours.
Subculture: After 24 and 48 hours onto Listeria Isolation Medium –LAB 122.
Buffered Peptone WaterLAB 46
DescriptionA pre-enrichment medium design to help sublethally damagedsalmonellae recover before introducing them into a selectivemedium. This nutrient medium is free from inhibitors and is wellbuffered to maintain the pH at 7.2 for the incubation period. Sublethalinjury to salmonellae occurs in many food processes and this pre-enrichment step greatly increases recovery of these organisms.
Formula g/litre
Peptone 10.0
Sodium chloride 5.0
Disodium hydrogen phosphate 3.7
Potassium dihydrogen phosphate 1.5
Method for reconstitutionWeigh 20 grams of powder and disperse in 1 litre of deionised water.Mix to dissolve then distribute into tubes or bottles. Sterilise byautoclaving at 121˚C for 15 minutes.
Appearance: Pale straw, clear liquid.
pH: 7.2 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034
Storage of Prepared Medium: Capped containers – up to 3 monthsat 15-20˚C in the dark.
Inoculation: Add 25 grams of sample to 225ml of Buffered PeptoneWater and homogenise.
Incubation: Aerobically at 37˚C for 18-24 hours.
Subculture: 10ml aliquots in 100mls of Selenite Cystine Broth LAB 55 and 0.1ml into 10ml Rappaport Vassiliadis Medium LAB 86.
ReferencesEdel W. and Kampelmacher E. H. 1973. Bull. Wld Hlth Org. 48: 167-174.
Poemla P. K. and Silliker J. H. 1976 Salmonella in Compendium ofMethods for microbiological examination of foods. Am. Pub. HealthAss., Washington.
Campylobacter Blood Free Selective Medium(Modified CCDA-Improved)
LAB 112
DescriptionA blood free medium which will support the growth of most entericcampylobacters. The selective cocktail X112 makes the mediumselective for C. jejuni. C. coli and C. laridis when incubated at 37˚C.With this product incubation at 42˚C is no longer necessary andhigher recovery rates have been reported at 37˚C than at 42˚C.
The antibiotic sensitive strains of campylobacter e.g. C. cinaedi andC. fennelliae can be isolated on this medium by the membrane filtermethod, but it is essential the antibiotic supplement is omitted toallow these organisms to grow. The supplement X112 consists ofcefoperazone and amphoteracin and is superior to the selectivecocktails of Skirrow, Butzler and Blazer-Wang all of which containantibiotics shown to be inhibitors to C. coli. The colonialmorphologies of Campylobacter spp. on this medium are distinctive.
Formula g/litre
Peptone blend 25.0
Bacteriological Charcoal 4.0
Sodium chloride 3.0
Sodium desoxycholate 1.0
Ferrous sulphate 0.25
Sodium pyruvate 0.25
Agar No. 2 12.0
Method for reconstitutionWeigh 45.5 grams of powder, disperse in 1 litre of deionised waterand allow to soak for 10 minutes. Swirl to mix, then sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C then add 2 vials ofX112 supplement, mix well and pour into petri dishes. Continuouslymix whilst pouring to prevent the charcoal settling.
Appearance: Black agar.
pH: 7.4 ± 0.2
Minimum Q.C. organisms: C. jejuniE. coli (inhibition) NCIMB 50034Candida albicans (inhibition)NCIMB 50010
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in thedark.
Inoculation: C. jejuni, C. coli. C. laridis surface streaking to singlecolonies. C. cinaedi, C. fennelliae use the membrane filtrationmethod of Goosens et al.
Incubation: 37˚C for 48 hours in an atmosphere of 5% oxygen, 10%carbon dioxide and 85% nitrogen. C. cinaedi and C. fennelliaerequire up to 7 days. 39
Growth Characteristicscolony size shape &
organism (mm) surface colour other
C. jejuni 2.0-3.0 F.E.G. Grey/White Efflorescent
(spreading moist)
C. coli 1.0-2.5 CV.E.G. Creamy MoistGrey
C. laridis 1.5-3.0 F.E.G. Grey (C.V. moist)
C. cinaedi 2.0-3.0 F.E.G. Pale Grey requires 7 days in
absence of X112
C. fenneliae 2.0-3.0 F.E.G. Pale Grey requires 7 days in
absence of X112
Other Enterobacteriacae – No growth if sensitive to cefoperazone.
ReferencesBolton F. J. Hutchinson D.N., Parker G. Reassessment of SelectiveAgars and Filtration Techniques for Isolation of CampylobacterSpecies from Feces. Eur.J. Clin. Microbiol. Infects. Dis. 1988 7 p155-160.
Bolton F. J. 1988 Personal Communication.
Bolton F. J. Hutchinson D. N., Parker G. Isolation of Campylobacter:What are we missing? J.Clin.Path. 1987 40 p 702-703.
Goosens H., De. Boeck M., Coignau H.. Vlaes L., Van Den Borre C.,Butzler J. P. Modified Selective Medium for Isolation ofCampylobacter spp from Feces : Comparison with Preston Medium,a Blood Free Medium, and a Filtration System. J.Clin. Micro. 198624 p 840-843.
Gun-Munro J., Rennie R. P., Thornley J. H. Richardson H. L., HodgeD., Lynch J. Laboratory and Clinical Evaluation of Isolation Mediafor Campylobacter jejuni J. Clin Micro. 1987. 25 p 2274-2277.
Herbert G. A., Hollis D. G., Weaver R. E., Karmali M. A., Simor A.E., Roscoe M., Fleming P. C., Smith, S. S. Lane J. Evaluation of aBlood-Free, Charcoal-Based, Selective Medium for the Isolation ofCampylobacter organisms from Faeces. J. Clin. Micro. 1986 23 p456-459.
Campylobacter Enrichment BrothLAB 135
DescriptionA selective enrichment broth for the isolation of Campylobacter spp.from food, environmental samples and faeces. The use of a selectiveenrichment broth enhances the recovery of sub-lethally damagedorganisms due to processing of foods, or if small numbers ofcampylobacters are present in heavily contaminated specimens. This broth has been shown to give appreciably better results thanPreston Broth.
Formula g/litre
Meat Peptone 10.0
Lactalbumin Hydrolsates 5.0
Yeast Extract 5.0
Sodium chloride 5.0
Haemin 10.0mg
Sodium pyruvate 0.5
α – ketoglutaric acid 1.0
Sodium metabisulphite 0.5
Sodium carbonate 0.6
Method for reconstitutionWeigh 27.6 grams of powder, disperse in 1 litre of deionised waterand allow to soak for 10 minutes. Swirl to mix and autoclave at 121˚Cfor 15 minutes. Cool to 47˚C, add 2 vials of selective supplementX131 (reconstituted with 5mls of 50% alcohol) and 50mls of saponinlysed horse blood, mix well and dispense into sterile containers.
Appearance: Translucent, wine-red with a fine black suspension.
pH: 7.4 ± 0.2
Minimum Q.C. organisms: Campylobacter jejuniE. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Capped containers: 7 days at 2-8˚Cin the dark.
Inoculation: Food homogenate is added to broth in a ratio of 1:4(w/v) in screw cap containers leaving a head space of 1.5 cm. Forfaeces 1ml of a 10% suspension in Buffered Peptone Water LAB 46is added to 5ml of broth.
Incubation: Aerobically at 37˚C for 2-4 hours, followed by a further16-44 hours at 42˚C.
Subculture: Onto Campylobacter Blood Free Selective MediumLAB 112.
ReferencesBolton, F. J. Personal Communication.
Hunt J. M., Abeyta C., and Tran T. (1998) Chapter 7 Campylobacterin FDA Bacteriological Analytical Manual 8th Edition.
C.E.M.O. Agar Base(Contagious Equine Metritis Organism)
LAB 78
DescriptionThis medium is a selective isolation medium for Taylorellaequigenitalis the causative organism of contagious equine metritis.The medium is a sugar free base with a mixture of high grade caseinand soy peptones as nutrients and with L-cystine and sodium sulphiteas supplements and reducing agents. The medium is made selectivewith the addition of amphoteracin (5 mg/L) and trimethoprim (10mg/L). Streptomycin (200 mg/L) can also be used but sensitivevariants of T. equigenitalis have been described.
Formula g/litre
Tryptone 15.0
Soy Peptone 5.0
Sodium chloride 5.0
Agar No. 2 12.0
L-Cystine 0.3
Sodium sulphite 0.2
Method for reconstitutionWeigh 37.5 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise by autoclavingat 121˚C for 15 minutes. Allow to cool to 80˚C, add 50ml of sterilehorse blood and allow to ‘chocolate’. Further cool to 47˚C beforeadding antibiotic selective agents. Mix well and pour into petri dishes.
Appearance: Chocolated Blood Agar.
pH: 7.3 ± 0.2
Minimum Q.C. organisms: T. equigenitalisE. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation: Surface, streaking out for single colonies.
Incubation: 37˚C in 10% CO2 for 2-3 days.
40
Growth Characteristicscolony size shape &
organism (mm) surface colour other
T. equigenitalis 0.1-1.0 CV.E.G. Cream colony sizevariation iscommon
ReferencesAtherton, J. G. 1978. Inhibition of the C.E.M. organism in mixedcultures. Vet. Rec. 432.
Mackintosh, M. E. 1981. Bacteriological techniques in the diagnosisof equine genital infections. Vet. Rec. 108, 52-55. Atherton, J. G.Personal Communication.
Fleming, M. P. Tribe. G. W. 1977. Vet. Rec. 101, 1470.
Cetrimide AgarU.S.P.
LAB 133
DescriptionA medium recommended by the United States Pharmacopoeia for theisolation of Pseudomonas aeruginosa from pharmacologicalpreparations. Subculture is carried out onto the medium afterenrichment in LAB 4 Tryptone Soy Broth. Cetrimide inhibits thegrowth of many micro organisms whilst allowing Ps. aeruginosa todevelop typical colonies which will fluoresce in ultraviolet light andproduce green pigment.
Formula g/litre
Pancreatic Digest of Gelatin 20.0
Magnesium chloride 1.4
Potassium sulphate 10.0
Cetyl trimethylammonium bromide (cetrimide) 0.3
Agar 13.6
Method for reconstitutionWeigh 45.3 grams of powder, disperse in 1 litre of deionised water.Add 10mls of glycerol, allow to soak for 10 minutes then swirl tomix. Sterilise at 121˚C for 10 minutes.
Appearance: Opalescent, pale yellow agar.
pH: 7.2 ± 0.2
Minimum Q.C. organisms: Ps. aeruginosa NCIMB 50067E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in thedark.
Inoculation: Subculture from enrichment broth, streak out for singlecolonies.
Incubation: 30-35˚C aerobically for 24-48 hours.
Growth Characteristicscolony size shape &
organism (mm) surface colour
Ps. aeruginosa 0.5-1.0 F.CR.D. green pigment (non pigment)green/yellowfluorescence
Ps. fluorescens 0.5 CV.R.E.G. green/yellow fluorescence
E. coli N.G.
S. aureus N.G.
Proteus spp. N.G.
ReferencesUnited States Pharmacopoeia XXI. 1985.
Brown V. I., Lowbury E. J. L. 1965. Use of an improved CetrimideAgar Medium and other culture methods for Pseudomonasaeruginosa. J. Clin. Pathol. 18, 752-756.
China Blue Lactose AgarLAB 105
DescriptionChina Blue Lactose Agar is a non-inhibitory medium for thedifferentiation and enumeration of bacteria in milk. The formulationis the same as that described by the Methodenkomission fürMilchivirtschaft. Lactose fermenters in milk form blue colonies. Themedium is non-inhibitory to the growth of cocci, making it a usefulmedium for the detection of streptococci and staphylococci as well ascoliform organisms.
Formula g/litre
Balanced Peptone No. 1 5.0
Beef Extract 3.0
Lactose 10.0
Sodium chloride 5.0
Aniline blue 0.325
Agar No. 2 12.0
Method for reconstitutionWeigh 35 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C and pour into petridishes.
Appearance: Blue clear agar.
pH: 7.0 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034Staph. aureus NCIMB 50080
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation: Surface, spread inoculum evenly over entire surface.
Incubation: 37˚C aerobically for 18-24 hours.
Interpretation: Count all colonies
Growth Characteristicscolony size shape &
organism (mm) surface colour other
E. coli 1.5-2.0 CV.E.G. Blue (colourless if NLF)
Klebsiella spp. 2.0-2.5 CV.E.G. Blue (mucoid)
Proteus spp. 0.5-2.5 F.Rz.G. colourless (spreading)
Salmonella spp. 2.0 CV.E.G. colourless
Shigella spp. 1.0 CV.E.G. colourless
Pseudomonas spp. 2.5-3.0 F.CR.D. colourless
S. aureus 0.5-1.0 CV.E.G. Pale Blue
S. epidermidis 0.5-1.0 CV.E.G. Blue
ReferencesMethodenbuch Band VI. Vergand Deutscher LandwirtschaftlicherUntersuchungs – und Forschugsanstallen.
41
C.L.E.D. (Bevis modification)(Cystine Lactose Electrolyte Deficient – Double Indicator)
LAB 6
DescriptionBevis modified Mackey and Sandys original medium by introducinga double indicator to improve the differentiation of lactose and nonlactose fermenting coliforms, staphylococci and streptococci. Theswarming of Proteus spp. is inhibited. LAB M C.L.E.D. will growmany of the more demanding streptococci of Lancefield groups A, B,C, G and F. This medium may not grow Pasteurella spp. or halophilicorganisms.
Formula g/litre
Balanced Peptone No. 1 4.0
Beef Extract 3.0
Tryptone 4.0
Lactose 10.0
L-Cystine 0.128
Bromothymol blue indicator 0.02
Andrade’s indicator 0.08
Agar No. 1 15.0
Method for reconstitutionWeigh 36 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving for 15 minutes at 121˚C. Cool to 47˚C and mix beforepouring.
Appearance: Green/blue, clear gel.
pH: 7.5 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034S. aureus NCIMB 50080
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation method: Surface inoculation, either streaking for singlecolonies or spread evenly over entire surface for colony counts.
Incubation: 37˚C for 24 hours aerobically.
Growth Characteristicscolony size shape &
organism (mm) surface colour other
E. coli 2.0-3.0 CV.E.G. Yellow/ (Blue if Orange non-lactose
fermenter)
K. aerogenes 3.0-4.0 CV.E.G. Yellow/ mucoidOrange
Proteus spp. 2.0-3.0 CV.E.G. Blue
Ps. aeruginosa 1.0-4.0 F.CR.D. Blue (Green pigment & odour)
Shigella spp. 1.5-2.5 CV.E.G. Blue
Salmonella spp. 2.0-3.0 CV.E.G. Blue (Yellow-orange if lactose +ve)
S. aureus 1.0-1.5 CV.E.G. Yellow/ (Blue if Orange non-lactose
fermenting)
Other (Yellow ifStaphyloccocci 0.5-1.5 CV.E.G. Blue-White lactose
fermenting)
Enterococcus spp. 0.5 CV.E.G. Yellow-Orange
ReferencesBevis, T. D. 1968. A modified electrolyte-deficient culture medium.J. Med. Lab. Tech., 25: 38-41.
Mackey, J. P. and Sandys, G. H. 1966. Diagnosis of urinaryinfections, Brit.Med. J., 1: 1173.
Sandys, G.H. 1960. A new medium for preventing swarming ofProteus spp. with a description of a new medium suitable for use inroutine laboratory practice. J. Med.Lab. Tech., 17: 224-233.
C.L.E.D. Medium(Mackey and Sandys) (Cystine Lactose Electrolyte Deficient-Single Indicator)
LAB 41
DescriptionA medium for urine culture first described by Mackey and Sandys in1960. The absence of electrolytes inhibits the swarming of Proteusspp. Cystine is added for the benefit of those organisms which havea specific cystine requirement. Differentiation of lactose and nonlactose fermenters is achieved using bromothymol blue as pHindicator. This medium supports the growth of Streptococcuspyogenes and most other fastidious organisms that do not requireblood.
Formula g/litre
Balanced Peptone No. 1 4.0
Beef Extract 3.0
Tryptone 4.0
Lactose 10.0
L-Cystine 0.128
Bromothymol blue indicator 0.02
Agar No. 1 15.0
Method for reconstitutionWeigh 36 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving for 15 minutes at 121˚C. Cool to 47˚C mix and distributeinto petri dishes.
Appearance: Green/blue clear gel.
pH: 7.3 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034S. aureus NCIMB 50080
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation: Surface inoculation either spreading for single coloniesor spread evenly over entire surface for colony counts.
Incubation: 37˚C aerobically for 18-24 hours.
42
Growth Characteristicscolony size shape &
organism (mm) surface colour other
E. coli 2.0-3.0 CV.E.G. Yellow (blue if nonlactose fermenters)
K. aerogenes 3.0-4.0 CV.E.G. Yellow (mucoid)
Proteus spp. 2.0-3.0 CV.E.G. Blue
Ps. aeruginosa 1.0-4.0 F.CR.D. Blue (green pigment& odour)
Shigella spp. 1.5-2.5 CV.E.G. Blue
Salmonella spp. 2.0-3.0 CV.E.G. Blue (yellow iflactose +ve)
Staph. aureus 1.0-1.5 CV.E.G. Yellow (blue if non-lactose
fermenting)
other Staphylococcus (yellow if spp. 0.5-1.5 CV.E.G. Blue-white lactose
fermenting)
Enterococcus spp. 0.5 CV.E.G. Yellow
ReferencesMackey, J. P. and Sandys, G. H. 1966. Diagnosis of urinaryinfections. Brit.Med.J. 1: 1173.
Guttman, D and Naylor, G.R.E. 1967. Dip-slide: an aid toquantitative urine culture in general practice. Brit.Med. J. 3: 343-345.
Columbia Agar BaseLAB 1
DescriptionA general purpose nutritious agar base formulated by Ellner et al.When further enriched by the addition of sterile blood, Columbiaagar can be used for the isolation of most clinically significantpathogens. The blood can be ‘chocolated’ if required. The mediumcan be made selective for various groups by the addition ofappropriate antibiotic mixtures eg:
Streptococci – Colistin/Oxolinic acid (X013)Gardnerella spp. – Colistin/Nalidixic acid (X011)C. perfringens – Neomycin (XO15) (X016)Staphylococci/streptococci – Colistin/Naladixic acid (X012)
Formula g/litre
Columbia Peptone Mixture 23.0
Corn Starch 1.0
Sodium chloride 5.0
Agar No. 2 12.0
Method for reconstitutionWeigh 41 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving for 15 minutes at 121˚C. Cool to 48˚C and add 5-7%sterile, defibrinated horse or sheep blood. Mix well before pouring.
Appearance: Cherry red if blood is fresh and well oxygenated.
pH: 7.3 ± 0.2
Minimum Q.C. organisms: S. aureus NCIMB 50080S. pyogenes ATCC 19615
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation: Surface plating, streaking out for single colonies.
Incubation: 37˚C aerobically or microaerobically for 24 hours.Anaerobically for 24 and 48 hours.
Growth Characteristics
colony size shape &organism (mm) surface colour other
S. aureus 1.5-2.0 CV.E.G. White-Yellow Haemolytic
S. pyogenes 0.5-1.0 CV.E.G.(D) White α, β-haemolyticdependent on
strain
S. pneumoniae 0.5-1.5 F.E.G. Grey greenish discolouration
in medium, mucoidin H2/C02
Neisseria meningitidis 1.0-2.0 CV.E.G. trans/ (mucoid)
Grey
E. coli 2.0-3.0 CV.E.G. Opaque/ (haemolytic)Grey
Ps. aeruginosa 0.5-4.0 F.CR.D. Opaque many colonialGrey forms
(green pigment)(haemolytic)
(mucoid)
C. perfringens 1.5-2.0 CV.CR.G. Grey usually targethaemolysis
(non haemolytic)
B. fragilis 1.0-1.5 CV.E.G. Grey (mucoid)
P. anaerobius P.P.-0.5 CV.E.G. White/Grey
ReferencesEllner, P. D., Stoessel, C. J., Drakeford, E and Vasi, F. 1966. A newculture medium for medical bacteriology. Amer. J. Clin Pathol.,45:502-504.
Goldberg, R. L., and Washington, J. A., 1976. Comparison ofisolation of Haemophilus vaginalis (Corynebacterium vaginale) fromPeptone-Starch-Dextrose Agar and Columbia Colistin-Nalidixic AcidAgar. J. Clin. Microbiol., 4:245-247.
Thayer, D. D. and Martin, H. E. 1966. An improved medium for thecultivation of N. gonorrhoeae and N. meningitidis. Publ. Hlth.Report, 81:559-562.
Cooked Meat Granules(for Cooked Meat Medium)
LAB 24
DescriptionDried minced ox heart which has been trimmed of excess fat andprepared according to the Martin and Lekpers modification ofRobertsons original formulation. The medium gives good growth of mostorganisms and is especially useful in the recovery of fastidiousanaerobes. The medium is also suitable for prolonged storage of cultures.
Method for reconstitutionTo make Cooked Meat Medium the granules should be added toNutrient Broth (LAB 14) or Fastidious Anaerobe Broth (LAB 71) inthe ratio of 15 grams of granules to 200ml. of broth. Cooked MeatMedium should be dispensed into containers allowing at least 20-25mm depth of meat particles and a broth supernatant of at least 10-15mm. To sterilise autoclave at 121˚C for 15 minutes in capped tubeswhich should be tightened after autoclaving to prevent re-oxygenation.
Medium prepared with Nutrient Broth LAB 14 should be re-steamed when used after a period of storage. Medium made withFastidious Anaerobe Broth LAB 71 will not require re-steaming after storage.
Storage of Prepared Medium: – up to 6 months at 15-20˚C in the dark.
ReferencesCruickshank, R. 1972. Medical Microbiology. 11th Edn. Livingstone,London.
43
Cooked Meat MediumLAB 127
DescriptionThis is a complete version of Robertson’s Cooked Meat Broth whichwas designed to grow all anaerobes found in battlefield woundsduring the First World War. The medium is based on LAB M CookedMeat Granules – LAB 24 and Nutrient Broth – LAB 14.
Formulation g/litre
Cooked meat granules 75.0
Beef Extract 10.0
Balanced Peptone No. 1 10.0
Sodium chloride 5.0
Method for reconstitutionUse a calibrated scoop to distribute 0.9 gram amounts of granules intotubes or bottles. Add 10ml deionised water. Sterilise at 121˚C for 10minutes. Use proportionately more granules and water if greaterdepths of medium are required.
Appearance: Granules covered in slightly opalescent pale yellow liquid.
pH: 7.0 ± 0.2
Inoculation: Samples or swabs directly into the medium.
Incubation: 37˚C for mesophiles, appropriate temperature for thermophiles.
Interpretation: Reddening of meat – saccharolytic organism –Blackening and digestion – proteolytic organism.
ReferencesRobertson, M., (1916) J. Path. & Bact. 20 327-349.
Cooked Meat Medium TabletsLAB 24Z
DescriptionAs Cooked Meat Granules (LAB 24) with Nutrient Broth (LAB 14)incorporated and presented in tablet form.
Method for reconstitutionAdd 2 tablets to 10mls of deionised water in a narrow container.Allow to soak for 15 minutes then sterilise by autoclaving at 121˚Cfor 15 minutes.
D.C.A.(Desoxycholate Citrate Agar)
LAB 29
DescriptionThis is Leifson’s original formulation of this selective medium for theisolation of Salmonella spp. and Shigella spp. from faeces andenvironmental samples. It has approximately half the quantity ofinhibitors used in the Hynes modification. The medium uses sodiumcitrate and sodium desoxycholate as inhibitors. Sodium thiosulphateis the substrate for the enzyme thiosulphate reductase being brokendown to form sulphite and hydrogen sulphide. The hydrogen sulphidereacts with the ferric ions to produce a black precipitate of ferroussulphide. This gives a typical black centre to the colonies of mostspecies of Salmonella.
Formula g/litre
Beef Extract 5.0
Balanced Peptone No. 1 5.0
Lactose 10.0
Sodium citrate 5.0
Sodium thiosulphate 5.0
Ferric citrate 1.0
Sodium desoxycholate 2.5
Neutral Red 0.025
Agar No. 2 12.0
Method for reconstitutionWeigh 45.5 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix, then bring to the boil withfrequent stirring. When the medium boils up into the neck of theflask, quickly remove from the source of heat and allow the froth tosubside. Return to the heat and allow the foam to boil up into the neckof the flask once more. Remove at once and cool to 47˚C approx.before pouring plates. Dry the surface before inoculation. DO NOTREMELT OR AUTOCLAVE THIS MEDIUM.
Appearance: Pale pink, translucent, a fine precipitate ofdesoxycholate may be present which may clear if the pH is increasedby the growth of organisms.
pH: 7.0 ± 0.2
Minimum Q.C. organisms: Salmonella sp. NCIMB 50076E. coli NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation: Surface, streaking for single colonies.
Incubation: 37˚C for 18-24 hours aerobically.
Growth Characteristicscolony size shape &
organism (mm) surface colour other
S. typhi 0.5. 1.5 CV.E.G. transp (black centre)Yellow
Other Salmonella spp. 1.5-2.0 CV.E.G. transp (black centre)
(Opaque) (clearing Yellow around colony)
S. sonnei 1.5-2.0 CV.E.G. transp (more opaque(pinkish) centre)
S. flexneri group 1.0-1.5 CV.E.G. transp-(pinkish)
S. dysenteriae 0.5-1.0 CV.E.G. transp
E. coli P.P.-1.5 CV.E.D. Red/Pink ppt in medium(uninhibited) (G)
Citrobacter spp. P.P.-2.0 CV.E.D. Red/Pink ppt in medium(G) (black centre)
Proteus spp. 1.0-2.0 CV.E.G. Yellow (black/grey centre)
fishy odour(clearing around
colony)
Pseudomonas spp. 0.5-1.5 CV.E.D. Yellow/ (green pigment)
(G) Pink
ReferencesHynes. M. 1942. The isolation of intestinal pathogens by selectivemedia. J. Path. Bact. 54. 193-207.
Liefson. E. 1935. New culture media based on Sodium desoxycholatefor the isolation of intestinal pathogens and for the enumeration ofcolon bacilli in milk and water. J. Path. Bact. 40: 581-589.
44
D.C.A. Hynes(Desoxycholate Citrate Agar -Hyne’s modification)
LAB 65
DescriptionThis modification of Leifson’s D.C.A. medium was introduced in1942. The medium was designed to be more inhibitory to commensalflora whilst allowing for adequate growth of Salmonella spp andShigella spp. The citrate and desoxycholate levels are significantlyincreased. To keep the desoxycholate in solution the pH also had tobe increased. The medium still uses lactose fermentation andhydrogen sulphide production as differential indicators.
Formula g/litre
Beef Extract 5.0
Balanced Peptone No. 1 5.0
Lactose 10.0
Sodium thiosulphate 5.4
Sodium citrate 8.5
Ferric citrate 1.0
Sodium desoxycholate 5.0
Neutral red 0.02
Agar No. 2 12.0
Method for reconstitutionWeigh 52 grams of powder, disperse in 1 litre of deionised water in atwo litre flask. Bring to the boil over a gauze, swirling frequently toprevent burning. Simmer for 30 seconds to dissolve. Cool to 47˚Cbefore pouring plates. Dry the surface before inoculation. DO NOTREMELT OR AUTOCLAVE THIS MEDIUM.
Appearance: Pink, clear, bile aggregates may appear on the surfaceon refrigeration.
pH: 7.4 ± 0.2
Minimum Q.C. organisms: Salmonella sp. NCIMB 50076E. coli NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation: Surface, streaking out for single colonies.
Incubation: 37˚C aerobically for 24 hours.
Growth Characteristicscolony size shape &
organism (mm) surface colour other
S. sonnei 1.0-2.0 CV.E.G.(D) Colourless- pale pink
S. flexneri 1.0-2.0 CV.E.G. Colourless
Salmonella spp. 1.0-4.0 CV.E.G. Colourless (black centre)
S. typhi 0.5-1.5 CV.E.G. Colourless (Black/greycentre)
E. coli P.P.-1.5 CV.CR.D. Red (No growth)
K. aerogenes 1.0-2.5 CV.E.G. Pink (mucoid)
Proteus spp 0.5-2.0 CV.E.G. Colourless (Yellow) fishy odour
P. aeruginosa 0.5-1.0 CV.CR.D. Colourless (green)
ReferencesHynes, M. 1942. The isolation of intestinal pathogens by selectivemedia. J. Path. Bact, 54: 193-207
D.C.L.S. Agar(Desoxycholate Citrate Lactose Sucrose Agar)
LAB 3
DescriptionA modification of Leifson’s D.C.A. medium which incorporatessucrose as an additional fermentable substrate to differentiate lactosenegative sucrose positive coliforms from Salmonella spp. Thismedium is unsuitable for the isolation of Yersinia spp. which aresucrose positive.
Formula g/litre
Balanced Peptone No. 1 7.0
Beef Extract 3.0
Lactose 5.0
Sucrose 5.0
Sodium citrate 10.5
Sodium thiosulphate 5.0
Sodium desoxycholate 2.5
Agar No. 2 12.0
Neutral Red 0.03
Method for reconstitutionWeigh 50 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes then heat gently with frequent mixingand bring to the boil. Simmer for 1 minute to complete dissolution ofthe solids. Cool to 47˚C then distribute 20ml into 90mm Petri dishes.Dry the surface by partial exposure, before use. DO NOT REMELTOR AUTOCLAVE THIS MEDIUM.
Appearance: Pale Pink, clear.
pH: 7.2 ± 0.2
Minimum Q.C. organisms: Salmonella sp. NCIMB 50076E. coli NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation: Surface plating, streaking out to single colonies.
Incubation: 37˚C aerobically for 24 hours.
Growth Characteristicscolony size shape &
organism (mm) surface colour other
S. typhi 0.5-1.0 CV.E.G. Trans.colourless
Other Salmonella spp. 1.5-2.0 CV.E.G. Slight
cloudycolourless
S. sonnei 1.5-2.0 CV.E.G. Trans. (More opaquePinkish centre)
S. flexneri 0.5-1.0 CV.E.G. Trans.Pinkish
S. dysenteriae 0.5 CV.E.G. Trans.colourless
E. coli P.P.-1.5 CV.E.G.(D) Red (ppt around(inhibited) colonies)
Citrobacter spp. P.P.-2.0 CV.E.D.(G) Red (ppt around(inhibited) colonies)
Proteus spp. 1.0-2.0 CV.E.G. Yellow (Fishy odour)
Pseudomonas spp. 0.5-1.0 CV.E.D. Yellow (Green pigment)
Pink
45
ReferencesHynes, M. 1942. The isolation of intestinal pathogens by selectivemedia. J. Path. Bact. 54: 193-207.
Leifson, E. 1935. New culture media based on sodium desoxycholatefor the isolation of colon bacilli in milk and water. J. Path. Bact. 40: 581-589.
Dermatophyte Test Medium (D.T.M.)LAB 117
DescriptionA modification of the formulation of Taplin, Zaias, Rebell and Blankfor the detection of dermatophytic fungi. This medium helps in thedifferentiation between saprophytic and environmental fungi.
Formula g/litre
Balanced Peptone No. 1 10.0
Glucose 40.0
Agar No. 2 12.0
Phenol Red 0.2
Method for reconstitutionWeigh 62 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes then bring to the boil with frequentstirring. Dissolve 2 vials of Chloramphenicol X009 in ethanol andadd these to the agar, mix well and distribute into tubes or universalcontainers. Sterilise at 121˚C for 15 minutes, allow to cool in thesloped position.
Note: Do not exceed the times stated for sterilisation, overheatedacidified agar loses gel strength and the sugars are caramelised.
Appearance: Yellow, clear gel.
pH: 5.5 ± 0.2
Minimum Q.C. organisms: Aspergillus sp. NCIMB 50097Trichophyton sp.
Storage of Prepared Medium: Slopes – up to 1 month at 2-8˚C inthe dark.
Inoculation: Surface plating or stab inoculation.
Incubation: 22-25˚C aerobically for 10-14 days.
Interpretation: Dermatophytes appear as fluffy colonies, colourvaries with species, the medium is reddened. Fungi other thandermatophytes cause the medium to become yellow due to acidproduction. If incubation is prolonged the medium may becomereddened. Yeasts appear as white creamy colonies. Blastomyces,Histoplasma and Coccidiodes may also turn the medium red, thoughthese are rarely encountered in lesions associated with ring worm.
ReferencesTaplin, D., Zaias, N., Rebell, G., Blank, H. 1969. Isolation andrecognition of dermatophytes on a new medium. (DTM) Arch.Dermatol. 99: 203-209.
Dextrose Tryptone AgarLAB 20
DescriptionA medium for the enumeration of thermophilic spore bearers infoods. The medium was designed to detect the thermophilic bacteriacausing ‘flat sour’ spoilage of canned foods. The medium also detectsthe ‘flat sour’ organism Bacillus stearothermophilus in sugar andother sweetening agents used in the preparation of frozen dairy foods,cereals and other food products.
Formula g/litre
Tryptone 10.0
Glucose 5.0
Bromocresol purple 0.04
Agar No. 2 12.0
Method for reconstitutionWeigh 27 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then bring to the boil todissolve agar before dispensing in 20ml amounts for poured platetechnique. Sterilise by autoclaving at 121˚C for 15 minutes.
Appearance: Purple clear agar.
pH: 6.9 ± 0.2
Minimum Q.C. organisms: B. stearothermophilus
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark. Capped container – up to 3 months at 15-20˚C in the dark.
Inoculation: Pour plate technique, pre-heat sample by steaming for20 minutes if a spore count is required.
Incubation: For Thermophiles – Aerobically for 48 hours at 55˚C.For Mesophiles – Aerobically for 48-72 hours at 30-32˚C.
Interpretation: Count all colonies for total counts, count yellowcolonies for differential acid producer count. Non acid producingcolonies are grey to colourless.
Growth Characteristicsorganism colony shape & colour
size (mm) surface
B. stearothermophilus 2.0 Rz.D Yellow zone mauve centre
Bacillus spp. 1.5-3.0 Rz.D Mauve (Yellow halo)
S. aureus 0.5-1.5 CV.E.G. Yellow
E. coli 1.0-1.5 CV.E.G. Yellow
Klebsiella spp. 1.5-2.5 CV.E.G. Yellow (mucoid)
Enterococci 0.5 CV.E.G. Yellow
Proteus spp. 2.0-3.0 RzD Yellow (spreads)
ReferencesWilliams, O. B. 1963. Tryptone Medium for the Detection of FlatSour Spores. Food Research 1, (3): 217-221.
American Public Health Association. 1972. Standard Methods for theExamination of Dairy Products. 13th Edn. Ed. W. J. Hausler A.P.H.A.Washington.
Tanner, F. W. 1946. The Microbiology of Food 2nd edn., GarrardPress, Champners.
Baumgartner, J. G. and Hersom, A. C. 1956. Canned Foods. 4th Edn.Churchill, London.
46
Diagnostic Semi-Solid SalmonellaAgar (Diassalm)According to Van Netten and Van der Zee et al
LAB 537
DescriptionDiassalm, as developed by Van Netten et al (1991), is a semi-soliddifferential medium for the isolation of Salmonella spp. from foodand water. It is an improved modification of MSRV (De Smedt andBolderdijk 1988) and SR (Perales and Audicana 1989) with regard tothe composition of the basal medium, selective system and theintroduction of a differential system.
The original basal medium was a commercially available sulphidemobility-indole medium (SIM BBL) (Blazevic 1968). LAB M havesubstituted their raw materials into Blazevic’s formula to create aricher base for Diassalm. Selectivity is achieved by the use ofmalachite green oxalate, magnesium chloride and novobiocin. Thediagnostic properties of Diassalm are based on the use of twoindicator systems; saccharose combined with bromocresol purple;and ferro-iron in combination with thiosulphate.
The efficiency of Diassalm is due to the ability of salmonellae tomove through the highly selective mobility medium in a petri dish,whilst the double diagnostic system allows visualisation of motileand non-motile suspected salmonellae due to blacking zones againstthe turquoise background. Diassalm can be seeded after pre-enrichment or after 8hr enrichment in selective broth (De Smedt andBolderdijk 1987).
Formula g/litre
Tryptone 20.0
Meat Peptone 6.1
Ferrous ammonium sulphate 0.2
Sodium thiosulphate 5.0
Sucrose 7.5
Lactose 0.5
Bromocresol purple 0.08
Malachite green oxalate 0.037
Magnesium chloride anhyd. 11.0
Agar No.1 2.8
PreparationWeigh 53.0 grams of powder, disperse in 1 litre of deionised water.Mix well, bring quickly to the boil. Allow to cool to 47˚C and add 1vial of Novobiocin supplement – X150 (10mg/vial). Mix well andpour plates. Nitrofurantoin may be used instead of Novobiocin toimprove the isolation of S. enteritidis.
Physical propertiesAppearance: - Green transparent, soft gel
pH - 5.5 ± 0.2
Shelf life: Ready-to-use medium – 7 days at 2-8˚C.
Inoculation method for samples3 drops (0.1ml) of 8 to 20hr. incubated pre-enrichment broth areinoculated in one spot in the centre of one plate of Diassalm.
Incubation methodAt 42 ± 0.5 or 37˚C for 18-24 hours. Keep the lid uppermost at alltimes.
InterpretationAfter incubation the plates are examined for a mobility zone with apurple/black colour change. When the mobility zone is absent, but thecentre is blackened, non-motile salmonellae may be present. A loopfull of the motile zone which is the farthest from the sampleinoculum (or the blackened centre if non-motile) is sub-cultured ontobrilliant green agar and XLD agar. Futher biochemical and serologicalidentification are performed according to recognised procedure.
Direct latex agglutination may also be carried out from the edge ofthe mobility zone.
Minimum QC organisms: Salmonella typhimuriumNCIMB 50076Proteus mirabilis (Inhibition)
ReferencesBlazevics, D.J. 1968 Appl. Microbiol. 16, 688
De Smedt J.M. et al 1987 J. Food Protection 50, 658
Perales, I and Audicana. Evaluation of semi-solid Rappaport mediumfor detection of Salmonellae in meat products. J. Food Protection 52,
Van Netten, P., Van de Moosdijk, A., Perales, I. and Mossel, D.A.A.Letters in Applied Microbiology
Van Netten, P., Van der Zee, H., and Van der Moosdijk, A., 1991. Theuse of diagnostic selective semi-solid medium for the isolation ofSalmonella enteritidis from poultry. Proceedings of the 10thSymposium on the quality of poultry meat, Spelderholt Beckbergen,pp. 59-67.
Van der Zee, H., and Van Netten, P., 1992. Diagnostic semi-solidmedia based on Rappaport-Vassiliadis Broth for the detection ofSalmonella spp. and S. enteritidis in foods. Proceedings of theInternational Symposium of Salmonella and Salmonellosis.
Van der Zee, H., 1992. Detection of Salmonella spp. with the use ofa standard method, diagnostic semi-solid agars and immunocapturekit. Proceedings Third World Congress Foodborne infections andintoxications, Berlin.
D.N.’ase AGARLAB 95
DescriptionDN’ase agar provides a convenient means of identifying potentiallypathogenic staphylococci, based on the ability of coagulase-positivespecies to split DNA. DN’ases produced by the organisms hydrolysethe DNA molecule to a mixture of smaller mono and polynucleotides. DiSalvo observed perfect correlation between coagulaseactivity and DN’ase production using S. aureus strains from clinicalspecimens. Other publications have also reported a close correlation.
Formula g/litre
Tryptone 20.0
Deoxyribonucleic acid (DNA) 2.0
Sodium chloride 5.0
Agar No. 2 12.0
Method for reconstitutionWeigh 39 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Allow to cool to 47˚C then pourinto petri dishes.
Appearance: Pale cream, clear.
pH: 7.3 ± 0.2
Minimum Q.C. organisms: S. aureus NCIMB 50080S. epidermidis NCIMB 50082
Storage of Prepared Medium: Plates – up to 7 days: at 2-8˚C in the dark. Capped container – up to 1 month at 4˚C in the dark.
Inoculation: Use a heavy inoculum on a small area. Four or moreorganisms can be tested on one 90mm petri dish.
Incubation: 37˚C aerobically for 18-24 hours.
Interpretation:Having obtained good growth flood the plate with 1N hydrochloricacid. This will precipitate the DNA in the medium. DN’ase producingorganisms will be surrounded by a clear area where the DNA hasbeen broken down into fractions which are not precipitated by theHydrochloric acid. Gram positive, catalase positive cocci that
47
produce DN’ase can be provisionally classified as S. aureus, andconfirmed by tube coagluase or thermostable DN’ase tests. DN.’aseis also produced by some Gram negative bacilli such as Serratiamarcescens, Pseudomonas aeruginosa. Some corynebacteria andstreptococci may also produce DN’ase.
ReferencesBaird-Parker, A. C. 1965. The classification of staphylococci andmicrococci from world-wide sources. J. Gen. Microbiol. 38, 363-387.Black, W. A., Hodgson, R. and McKechnie, A. 1971.
DiSalvo, J. W. 1958 Deoxyribunuclease and coagulase activity ofmicrococci. Med. Tech. Bull. U.S. Armed Forces Med. J. 9, 191.
Martin, W. J and Ewing, W. H. 1967. The deoxryibonuclease test asapplied to certain gram-negative bacteria. Can. J. Microbiol. 13, 616-618.
Messinova, O. V., Yusupova, D. V. and Shamsutdinov, N. S. 1963.Deoxyribonuclease activity of Corynebacterium and its relation tovirulence. Fed. Proc. 22, T1033.
Streitfeld, M. M., Hoffmann, E. M. and Janklow, H. M. 1962.Evaluation of extracellular deoxyribonuclease activity inPseudomonas. J. Bacteriol. 84, 77. Wannamaker, L. W. 1964.Streptococcal deoxryribonuclease, pp. 140-165. J. W. Uhr (ed.). TheStreptococcus, Rheumatic Fever, Glomerulophritis. Baltimore:Williams & Williams.
Weckman, B. G. and Catlin, B. W. 1957 Deoxryribonuclease activityof micrococci from clinical sources. J. Bacteriol. 73, 747-753.
Zierdt, C. H. and Golde, D. W. 1970. Deoxryribonuclease-positiveStaphylococcus epidermidis strains. Appl. Microbiol. 20(1), 54-57.
Easter and Gibson Pre-enrichment Broth
LAB 136
DescriptionA pre-enrichment broth for use in detection procedures utilisingconductance/impedance techniques. This broth is developed fromLAB 46 Buffered Peptone Water and will enhance the recovery ofsub-lethally damaged bacteria. Salmonella reduce T.M.A.O. toproduce a significant change in the medium that can be detected bythe conductance/impedance equipment.
Best results will be obtained by those customers using Pre-Enrichment Broth (LAB 136) and Salmonella Medium (LAB 137)bought from LAB M. These media are both manufactured with thesame peptones, so that organisms “switched on” to utilising thesubstrates in LAB 136 can carry on metabolising the same substratesin LAB 137, significantly shortening the lag time.
Formula g/litre
Tryptone 7.5
Meat Peptone 2.5
Sodium chloride 5.0
Disodium hydrogen phosphate 3.56
Potassium di-hydrogen phosphate 1.5
Mannitol 5.0
Method for reconstitutionWeigh 25 grams of powder, disperse in 1 litre of deionised water.Heat gently to dissolve supplement, dispense into appropriatecontainers, then sterilise by autoclaving at 121˚C for 15 minutes.
Appearance: Very pale yellow clear solution.
pH: 7.2 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034
Storage of Prepared Medium: Capped container – up to 1 month at15-20˚C in dark.
Inoculation: Homogenised food samples.
Incubation: 37˚C for 18 hours aerobically.
Sub-culture: Into LAB 137 Easter and Gibson Salmonella Medium.
ReferencesEaster M. C. Gibson D. M. 1985. Rapid and automated detection ofSalmonella by electrical measurements. J. Hyg. 94, 245-262.
Gibson D. M. 1987. Some modifications to the media for rapid,automated detection of salmonellas by conductance. J. Appl.Bacteriol. 63, 299-304.
Ogden I. D., Cann P. C. 1987. A modified conductance medium forthe detection of Salmonella spp. J. Appl. Bacteriol. 63, 459-464.
Easter and Gibson SalmonellaMedium
LAB 137
DescriptionA medium for the rapid detection of Salmonella spp byconductance/impedance techniques. This medium is based on LAB55 Selenite Cystine Broth and is supplemented with dulcitol andT.M.A.O. (trimethylamine-N-oxide). Salmonella reduce T.M.A.O. toT.M.A. (trimethylamine) and, in so doing, increase the conductivityof the medium which can be detected and measured by monitoringequipment such as Malthus.
Best results will be obtained by those customers using Pre-Enrichment Broth (LAB 136) and Salmonella Medium (LAB 137)bought from LAB M. These media are both manufactured with thesame peptones, so that organisms ‘switched on’ to utilising thesubstrates in LAB 136 can carry on metabolising the same substratesin LAB 137 significantly shortening the lag time.
Formula g/litre
Meat Peptone 2.5
Tryptone 2.5
Sodium dihydrogen phosphate 10.0
Dulcitol 5.0
Sodium carbonate 5.0
Method for reconstitutionWeigh 2.5 grams of powder, disperse in 100mls of deionised water.Add 1 vial of X137 T.M.A.O./Selenite supplement. Swirl to mix, heatto boiling to effect sterilisation. When cool add 1ml of stock solutionof L-cystine. Distribute into sterile conductance tubes or bottles. DO NOT AUTOCLAVE.
Stock solution of L-cystine0.1gm L-cystine dissolved in 15mls of normal NaOH – add to 100mlssterile distilled water. Keep refrigerated. Discard after 1 month.
Appearance: Clear reddish/orange solution. A slight precipitate may form.
pH: 7.2 ± 0.2
Minimum Q.C. organisms: Salmonella sp. NCIMB 50076E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: capped container – up to 1 month at15-20˚C in dark.
Inoculation: From LAB 136 Easter and Gibson Pre-EnrichmentBroth.
Incubation: 37˚C aerobically connected to monitoring equipment.
ReferencesEaster M. C. Gibson D. M. 1985. Rapid and automated detection ofSalmonella by electrical measurements. J. Hyg. 94, 245-262.
48
Gibson D.M. 1987. Some modifications to the media for rapidautomated detection of salmonellas by conductance. J.Appl.Bacteriol. 63, 299-304.
Ogden I.D., Cann P.C. 1987. A modified conductance medium for thedetection of Salmonella spp. J. Appl. Bacteriol. 63, 359-464.
E.E. Broth(Enterobacteriaceae Enrichment Broth)
LAB 91
DescriptionE.E. Broth is recommended as an enrichment medium whenexamining food and feedstuffs for Enterobacteriaceae. It is amodification of LAB 51 Brilliant Green Bile Broth, with an improvedbuffering capacity to encourage early growth and preventautosterilization. E.E. Broth uses glucose instead of lactose to makethe medium a test for all enterobacteria including non lactosefermenting organisms.
Formula g/litre
Balanced Peptone No. 1 10.0
Dextrose 5.0
Disodium hydrogen phosphate 6.45
Potassium dihydrogen phosphate 2.0
Bile Salts 20.0
Brilliant green 0.0135
Method for reconstitutionWeigh 43.5 grams of powder and add to 1 litre of deionised water.Swirl to dissolve, warm gently if necessary, then distribute intobottles or tubes and heat at 100˚C for 30 minutes only. Cool rapidly.OVERHEATING THIS MEDIUM WILL ADVERSELY AFFECTITS PERFORMANCE.
Appearance: Green, clear.
pH: 7.2 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034B. subtilis (inhibition)
Storage of Prepared Medium: capped containers – up to 3 monthsat 15-20˚C in the dark.
Inoculation: Add 1 part of sample suspension or dilution to 10 partsof medium.
Incubation: 44˚C for 18 hours for thermotrophs. 32˚C for 24-48hours for mesotrophs. 4˚C for 10 days for psychrotrophs.
Interpretation: Turbidity and a colour change to yellow-green ispresumptive evidence of Enterobacteriaceae. Subculture ontoconfirmatory media e.g. LAB 88 V.R.B.G.A. must be carried out.
ReferencesMossel, D. A. A., Visser, M. and Cornelissen, A. M. R. 1963. Theexamination of foods for Enterobacteriaceae using a test of the typegenerally adopted for the detection of salmonellae. J.Appl. Bacteriol.26, 444-452.
Mossel, D. A. E., Harrewijn, G. A. and Nesselrooy-van Zadelhoff, C.F. M. 1974. Standardisation of the selective inhibitory effect ofsurface active compounds used in media for the detection ofEnterobacteriaceae in food and water. Health Lab. Sci. 11, 260-267.Richard, N. 1982. Monitoring the quality of selective liquid media bythe official French dilution technique used for the bacteriologicalexamination of foods. In: Quality assurance and quality control ofmicrobiological culture media, edited by J. E. L. Corry, G.I.T.-VerlagDarmstadt, pp. 51-57.
Endo AgarLAB 60
DescriptionThis medium was developed in 1914 for the isolation of Salmonellatyphi; other media have since proved superior for this purpose, butEndo agar has a role as a coliform medium. It is recommended by theAmerican Public Health Association as a standard medium for theenumeration of coliforms in water and dairy products. In this mediumacetaldehyde is produced by coliforms and then fixed by the sulphiteto produce a metallic sheen with the basic fuchsin dye. Most entericGram negative organisms will grow well, whilst Gram positiveorganisms are mostly inhibited.
Formula g/litre
Balanced Peptone No. 1 10.0
Lactose 10.0
Dipotassium phosphate 3.5
Sodium sulphite 2.5
Agar No. 1 15.0
Method for reconstitutionWeigh 41 grams of powder, disperse in 1 litre of deionised water. Add4ml of a 10% w/v alcoholic solution of basic fuchsins (95% ethylalcohol). Bring to the boil with frequent swirling to dissolve thesolids. Sterilise by autoclaving at 121˚C for 15 minutes. Cool to 47˚Cin a water bath before pouring. The precipitate typically associatedwith this medium should be dispersed by gentle swirling prior topouring the plates.
This medium is light sensitive and should therefore be stored in thedark, preferably under refrigeration. The medium will become darkred in colour if exposed to light.
Basic Fuchsin is a potential Carcinogen and care should be takenwhen handling it to avoid inhalation of the powdered dye andcontamination of the skin.
Appearance: Pale pink/orange
pH: 7.5 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation: Surface, streaking out for single colonies.
Incubation: 37˚C for 18-48 hours aerobically.
Growth Characteristicscolony size shape &
organism (mm) surface colour other
E. coli 1.0-2.0 CV.E.G. Deep Red (Metallic sheen)
K. aerogenes 1.0-2.5 CV.E.G. Red (mucoid)
Proteus spp 2.0-3.0 CV.E.G. Pale Pinkcolourless
Ps. aeruginosa 0.5-1.0 F.CR.D. Pale Pink
Shigella spp 0.5-1.0 CV.E.G. Pale Pink
Salmonella spp 1.0-1.5
Gram positive no growth.organisms
ReferencesEndo, 1914, Centr. Bakt., Abt 1, Orig., 35: 109.
American Public Health Association, 1975. Standard Methods for theExamination of Water and Wastewater, 14th Edn. American PublicHealth Association, Inc. Washington D.C.
American Public Health Association, 1972. Standard Methods for theExamination of Dairy Products, 13th End., American Public HealthAssociation, Inc., Washington, D.C.
49
Eosin Methylene Blue Agar (Levine)LAB 61
DescriptionThis medium was introduced in 1916 by Holt-Harris and Teague todifferentiate Escherichia spp. and Aerobacter spp. It was modified byLevine in 1918 who removed sucrose from the formula and increasedthe lactose content. The distinctive metallic sheen produced by E. colion this medium is due to acid production resulting in an amidebonding between the eosin and methylene blue, other coliforms donot produce enough acid to cause this reaction. Eosin inhibits mostGram positive organisms. The prepared medium is sensitive to light.
Formula g/litre
Balanced Peptone No. 1 10.0
Lactose 10.0
Dipotassium phosphate 0.7
Monopotassium phosphate 1.3
Eosin Y 0.4
Methylene Blue 0.065
Agar No. 2 15.0
Method for reconstitutionWeigh 37.5 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Cool to 50˚C and agitate gentlyto ensure uniform distribution of the flocculant precipitate which is afeature of this medium before pouring into petri dishes. STORE IN THE DARK.
Appearance: Blue/purple with a light precipitate.
pH: 6.8 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation: Surface, streaking for single colonies.
Incubation: 37˚C aerobically for 24 hours.
Growth Characteristicscolony shape &
organism size (mm) surface colour other
E. coli 2.0-3.0 CV.E.G. Blue Black (Metallic sheen)
Klebsiella spp. 3.0-4.0 CV.E.G. BrownBlue (mucoid)
Salmonella spp. 2.0-3.0 CV.E.G. Colourless
Shigella spp. 1.0-2.0 CV.E.G. Colourless
Candida spp. 0.5-1.5 CV.Rz.G.(D) White
S. aureus P.P. CV.E.G. Colourless
E. faecalis P.P. CV.E.G. Colourless
ReferencesAmerican Public Health Association, American Water WorksAssociation and Water Pollution Control Federation, 1975. StandardMethods for the Examination of Water and Wastewater, 14th Edn.,Washington, D.C. American Public Health Association.
Girolami, R. L. and Stamm, J. M. 1976. Inhibitory effect of light ongrowth supporting properties of Eosin Methylene Blue Agar. Appl.Environ. Microbiol., 31:1 141-142.
Haesler, W. J. (ed) 1972. Standard Methods for the Examination ofDairy Products, 13th edn., Washington, D.C., American PublicHealth Association.
Levine, M. 1918. Differentiation of E. coli and B. aerogenes on asimplified Eosin-Methylene Blue agar. J. Infect. Dis., 23: 43-47.
Fastidious Anaerobe Agar (F.A.A.)LAB 90
DescriptionA primary isolation medium capable of growing most clinicallysignificant anaerobes. Developed by LAB M, comparisons haveshown this medium to be superior to other formulations as a primaryisolation medium for fastidious organisms. The peptones includedhave been chosen for maximum growth stimulation. Starch andsodium bicarbonate act as de-toxification agents whilst haeminencourages pigment production in Porphyromonas melaninogenicus.Specific growth promoting agents are Cysteine for Fusobacteriumnecrophorum, Propionibacterium acne and Bacteriodes fragilis,arginine for Eubacterium spp. soluble pyrophosphate for Porph.gingivalis and Porph. assacchrolyticus. Pyruvate helps neutralisehydrogen peroxide and is also utilised by Veillionella spp. as anenergy source. Vitamin K and sodium succinate provide essentialgrowth factors for some anaerobes as does the 0.1% glucose. The lowlevel of glucose prevents the production of high levels of acids andalcohols which would inhibit colonial development.
Formula g/litre
Peptone mix 23.0
Sodium chloride 5.0
Soluble starch 1.0
Agar No. 2 12.0
Sodium bicarbonate 0.4
Glucose 1.0
Sodium pyruvate 1.0
Cysteine HCl monohydrate 0.5
Haemin 0.01
Vitamin K 0.001
L-Arginine 1.0
Soluble pyrophosphate 0.25
Sodium succinate 0.5
Method for reconstitutionWeigh 46 grams of powder and add to 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C then asepticallyadd 5-10% of sterile defibrinated horse blood, mix well and pour intopetri dishes. This medium can be made selective for various speciesof anaerobes by the addition of appropriate selective cocktails e.g.
Gram negative anaerobes X090
Non-sporing anaerobes X091
Actinomyces spp. X092
Clostridium difficile X093
Appearance: Red due to addition of blood. The blood will darken(reduce) because of the presence of reducing agents.
pH: 7.2 ± 0.2
Minimum Q.C. organisms: B. fragilisP. anaerobious
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation: Surface plating, streaking out to single colonies.
Incubation: 37˚C anaerobically with 10% CO2 for 48 hours to 5 days.
50
Growth Characteristics (48 hours)colony shape &
organism size (mm) surface colour other
B. fragilis 1.0-2.0 CV.E.G. Grey
C. perfringens 1.0-2.0 CV.E.G. Grey ‘Target’haemolysis
(non haemolytic)
F. necrophorum 1.0-2.0 CV.E.G.(D) trans- (grey)parent (haemolytic)
Porphyromonas assachrolyticus1.0-2.0 CV.E.G. Grey/
Brown (clearing)
B. ureolyticus 0.5 F.E.D. translucent pitting
Prop acne 0.5 CV.E.G. White
Pept. anaerobius 0.5-2.0 CV.E.G. White/Grey
A. israeli 0.5-1.0 CV.E.G. White (‘molar tooth’)(smooth)
ReferencesBrazier, J. S. (1986). Yellow fluorescence of Fusobacteria Letters inApplied Microbiol. 2: 124-126.
Brazier, J. S. 1986. A note on ultra violet red fluorescence ofanaerobic bacteria in vitro. J. Appl. Bact. 60: 121-126.
Eley, A., Clarry, T., Bennett, K. W. 1989. Selective and differentialmedium for isolation of Bacteriodes ureolyticus from clinicalspecimens. European Journal of Clinical Microbiology, InfectiousDiseases. 8: 83-85.
Wade W. Griffiths, M. 1987. Comparison of Media for cultivation ofsubgingival bacteria. J. Dent. Res. 66: no. 4 abstract 334.
Heginbotham M., Fitzgerald T. C., and Wade W. G. (1990).Comparison of solid media for the culture of anaerobes. J. Clin. Path.43: 253-256.
Fastidious Anaerobe Broth (F.A.B.)LAB 71
DescriptionF.A.B. was developed by LAB M working in conjunction with themicrobiology department of a University of Manchester teachinghospital. The medium was designed to give optimum growth offastidious anaerobes and has found applications as a blood culturemedium and an enrichment broth for the isolation of anaerobes. Themedium is very rich in nutrients from the specially selected peptonemixture. Vitamin K. haemin and L-cysteine are all growth factorsrequired by some anaerobes. L-cysteine together with sodiumthioglycollate reduce the Eh of the medium and the agar contentinhibits absorption of oxygen and convection currents. Resazurin is aredox indicator. Several published evaluations show F.A.B. to be theliquid medium of choice for fastidious anaerobes.
Formula g/litre
Peptone mixture 15.0
Yeast Extract 10.0
Sodium thioglycollate 0.5
Sodium chloride 2.5
Agar No. 1 0.75
L-Cysteine HCl 0.5
Resazurin 0.001
Sodium bicarbonate 0.4
Haemin 0.005
Vitamin K 0.0005
Method for reconstitutionWeigh 29.7 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix. Boil to dissolve the agarthen dispense into screw cap containers. Sterilise by autoclaving at121˚C for 15 minutes. Tighten the caps as soon as possible afterautoclaving.
Appearance: Pale straw, clear, viscous. May have a narrow band ofred/purple at the surface due to action of oxygen on the resazurin. Ifthe medium is reddish this indicates too much oxygen has beenabsorbed, the medium should be reheated to deoxygenate. Do notreheat more than once.
pH: 7.2 ± 0.2
Minimum Q.C. organisms: B. fragilis
Storage of Prepared Medium: Capped containers – up to 3 monthsat 15-20˚C in the dark.
Inoculation: If used as a blood culture medium a minimum dilutionof 1:10 should be used.
Incubation: 37˚C for 24-72 hours. Keep the container airtight.
Growth indicators: The broth may become turbid or individualcolonies may form suspended in the medium.
ReferencesGould, J. H., Duerden, B. I. 1983. Blood culture – current state andfuture prospects. J. Clin. Pathol. 36: 963-977.
Ganguli, G. A., O’Hare, W., Hyde, W. A. 1984. Rapid Detection ofBacteraemia by early subculture. J. Med. Microbiol. 17: 311-315.
Ganguli, L. A., Keaney, M. G. L., Hyde, W. A., Fraser, B. J. 1985.More Rapid identification of bacteraemia by manual rather thanradiometric methods. J. Clin. Pathol. 38: 1146-1149.
Junt, G. H., Price, E. H. 1982. Comparison of a home made bloodculture broth containing a papain digest of liver, with fourcommercially available media, for the isolation of anaerobes fromsimulated paediatoic blood cultures. J. Clin. Pathol. 35: 1142-1149.
Ganguli, L. A., Turton, L. J., Tillotson, G. S. 1982. Evaluation ofFastidious Anaerobe Broth as a blood culture medium. J. Clin. Pathol.35: 458-461.
Tillotson, G. S. 1981. Evaluation of ten commercial blood culturesystems to isolate a pryridoxal dependent streptococcus. J. Clin.Pathol. 34: 930-934.
Fluid Thioglycollate Medium (U.S.P.)LAB 25
DescriptionA medium for sterility tests, prepared according to the specificationof the United States Pharmacopeia. Aerobic and anaerobic organismsgrow well in this medium even from small inocula. In appropriatetubes or bottles the thioglycollate ensures adequate anaerobicconditions. The low level of agar reduces oxygen diffusion into themedium. The thioglycollate will also serve to inactivate anymercurial compounds used as preservatives.
Formula g/litre
Tryptone 15.0
L-Cystine 0.5
Glucose 5.5
Yeast Extract 5.0
Sodium chloride 2.5
Sodium thioglycollate 0.5
Resazurin 0.001
Agar 0.75
51
Method for reconstitutionWeigh 29.75 grams, disperse in 1 litre of deionised water. Soak for 10minutes, swirl to mix, then bring to the boil to dissolve and dispense15ml into 6mm x 150mm tubes. Sterilise by autoclaving for 15minutes at 121˚C. Store at ambient temperature in the dark, but not inthe refrigerator. If more than 30% of the medium turns pink(oxidised) the Eh may be restored (once only) by heating in a boilingwater bath or by free-steaming.
Appearance: Pale straw colour, clear. Surface may be pink/blue dueto oxidation of Resazurin.
pH: 7.1 ± 0.2
Minimum Q.C. organisms: C. sporogenesS. aureus NCIMB 50080
Storage of Prepared Medium: Capped container – up to 3 monthsat 15-20˚C in the dark.
Incubation: 30-35˚C aerobically for 14 days.
Growth indicators: Turbidity, colonies in medium.
ReferencesThe Pharmacopeia of the United States of America. 21st End. 1985.
Fluorescence AgarLAB 16
DescriptionThis medium is a modification of King, Ward and Raney’s mediumformulated for the demonstration of the fluorescein pigmentproduced by many strains of Pseudomonas. The pyocyanin pigmentproduced by most strains of Pseudomonas aeruginosa is alsoproduced. Can be made selective by the addition of selective agentssuch as X108 Cetrimide Fucidin Cephaloridine.
Formula g/litre
Balanced Peptone No. 1 20.0
Dipotassium phosphate 1.5
Magnesium sulphate 1.5
Agar No. 2 12.0
Method for reconstitutionWeigh 35 grams of powder, disperse in 1 litre of deionised watercontaining 10ml Glycerin B.P. Allow to soak for 10 minutes, swirl tomix then sterilise for 15 minutes at 121˚C. Mix well before pouring.Slant over a generous butt if required.
Appearance: Straw coloured, clear gel.
pH: 7.2 ± 0.2
Minimum Q.C. organisms: Ps. aeruginosa NCIMB 50067
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation: Surface spreading.
Incubation: 30-37˚C for 24 and 48 hours aerobically.
Growth Characteristicscolony size shape &
organism (mm) surface colour other
Ps. aeruginosa 0.5-2.5 F.CR.D. Grey (colony size variesGreen- with strain)
fluorescent-(non pigmented)pigment (mucoid)
Ps. fluorescens 1.0-2.5 F.CR.D. Greyfluorescent
pigment
ReferencesKing, E. O., Ward, M. K. and Raney, D. E. 1954. 2 simple media fordemonstration of pyocyanin and fluorescein, J. Lab. Clin Med., 44:301-307.
Brown, V. I. and Lowbury, E. J. L. 1965. Use of an improvedcetrimide agar medium and other culture methods for Pseudomonasaeruginosa. J. Clin. Path. 18: 752-756.
Fraser BrothLAB 164
DescriptionDeveloped as a modification of UVM II medium, Fraser broth is asecondary enrichment broth for the isolation of Listeria spp., and issimilar to Palcam broth in that it contains aesculin to indicate thepresence of a potential Listeria isolate. It also contains lithiumchloride in an attempt to suppress the growth of enterococci in themedium (as does Palcam). Fraser broth may also be used as a primaryenrichment medium by incorporating 1/2 strength supplement intothe broth base (X164 or X564).
Formula g/litre
Peptone mixture 15.0
Yeast extract 5.0
Aesculin 1.0
Disodium hydrogen phosphate 9.6
Potassium dihydrogen phosphate 1.35
Sodium chloride 20.0
Lithium chloride 3.0
Method for reconstitutionWeigh 55 grams of power and add to 1 litre of deionised water (addto 900ml if preparing 1/2 Fraser). Allow to soak for 10 minutes, swirlto mix and sterilise at 121˚C for 15 minutes. Cool to 47˚C and add 2vials of Fraser supplement X165 (or 2 vials of 1/2 Fraser supplementX164), mix well and aseptically dispense into sterile tubes or bottles.
Appearance: Straw opalescent broth with precipitate (clears onstorage)
pH 7.2 + 0.2
Minimum Q.C. organisms: Listeria sp NCIMB 50007E.coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Bottles – up to 14 days at 2-8˚C.
Inoculation: 1/2 Fraser – Add 25g sample to 225ml of 1/2 Fraserbroth and homogenise Fraser – Subculture 0.1ml of primaryenrichment broth (UVM I or 1/2 Fraser) into 10ml of Fraser broth.
Incubation: 1/2 Fraser – 30˚C aerobically for 24hrs
Fraser – 35˚C aerobically for 24hrs and 48hrs. Subculture ontoselective agars at 24 and 48hrs.
InterpretationBlackening of the broth indicates the presence of a potential Listeriaand should be subcultured onto Listeria isolation medium (Oxford)LAB122 or Palcam agar LAB148. All broths should be subculturedbefore discarding, irrespective of colour change.
ReferencesFraser J.A., and Sperber W.H., (1988) Rapid detection of Listeria sppin food and environmental samples by esculin hydrolysis. J.FoodProtection 51 (10) 762-765
McClain D., and Lee W.H. (1989) FSIS method for isolation ofL.monocytogenes from processed meat and poultry products.Lab.Comm.No.57, Revised May 24, 1989. US Dept of Agric.FSIS,Microbiol. Div.
52
G.C. Agar BaseLAB 67
DescriptionA nutritious agar base described by Thayer and Martin for theisolation of Neisseria gonorrhoeae. The right peptone mixture isenhanced by the use of corn starch to absorb toxic metabolites and abuffering system is used to maintain neutral pH. The medium is madeselective by the use of various antibiotic cocktails. Thayer and Martinoriginally recommended the use of vancomycin, colistin and nystatin(X067 V.C.N.) but the addition of trimethoprim (X068 V.C.N.T.) isuseful in preventing the swarming of proteus. More recently theemergence of vancomycin sensitive gonococci has made the NewYork City selective agents (lincomycin, colistin, amphoteracin,trimethoprim X070 LCAT) the combination of choice. Enrichment ofthe base is usually by the addition of lysed blood. Alternativelychocolated blood or haemoglobin powder and Thayer and Martin’smixture of vitamins, amino acids and coenzymes can be used.
Formula g/litre
Special Peptone 15.0
Corn Starch 1.0
Sodium chloride 5.0
Dipotassium hydrogen phosphate 4.0
Potassium dihydrogen phosphate 1.0
Agar No. 2 10.0
Method for reconstitutionWeigh 36 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Cool to 48˚C and add 50-70mlof lysed blood and 2 vials of X070 selective agent. Mix well and pourinto petri dishes.
Appearance: Dependent on blood supplement used.
pH: 7.2 ± 0.2
Minimum Q.C. organisms: N. gonorrhoeae ATCC CDC98E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation: Surface, streaking out for single colonies.
Incubation: 37˚C microaerobically for 24-48 hours.
Growth Characteristicscolony size shape &
organism (mm) surface colour other
N. gonorrhoeae 1.0-2.0 CV.E.G. Transp variations inarent colony size
N. lactamica 1.0-2.0 CV.E.G. Grey
N. meningitidis 2.0-3.0 CV.E.G. Grey
B. catarrhalis 2.0-3.0 CV.E.G. Cream
Other organisms should not grow with the exception of antibioticresistant variants.
ReferencesYoung, H. 1978. Cultural diagnoses of gonorrhoea with modifiedNew York City (MNYC) medium. Brit. Journ. Ven. Dis. 54: 36-40:Thayer, J. D. and Martin, J. E. 1966. Improved medium selective forthe cultivation of N. gonorrhoeae and N. Meningitidis: Public Healthrep. 81: 559-562.
Hektoen Enteric AgarLAB 110
DescriptionA medium developed at the Hektoen Institute in Chicago for theenhanced recovery of shigellae from clinical specimens. Thismedium has high levels of peptones and sugar which counteract someof the toxic effects of bile salts used to make the medium selective.This allows the shigellae to grow as well as the salmonellae. Salicinis fermented by many coliforms including those that do not fermentlactose and sucrose. The medium employs a double indicator systemsimilar to that used in LAB 6 C.L.E.D., (Bevis) and an H2S indicatorsystem similar to that used in LAB 32 XLD. Although intendedprimarily for clinical use this medium is quoted in B.S. 4285 assuitable for the examination of dairy products for salmonellae.
Formula g/litre
Meat Peptone 12.0
Yeast Extract 3.0
Lactose 12.0
Sucrose 12.0
Salicin 2.0
Bile Salts No. 3 7.0
Sodium desoxycholate 2.4
Sodium chloride 5.0
Sodium thiosulphate 5.0
Ammonium ferric citrate 1.5
Acid fuchsin 0.1
Bromothymol blue 0.065
Agar No. 1 14.0
Method for reconstitutionWeigh 76 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then heat gently and bringto the boil. Cool to 47˚C and pour plates. DO NOT AUTOCLAVEOR OVERHEAT THIS MEDIUM.
Appearance: Green, clear.
pH: 7.5 ± 0.2
Minimum Q.C. organisms: Salmonella sp. NCIMB 50076Shigella sp.E. coli (some inhibition)NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation: Surface plating, streak out to single colonies.
Incubation: 37˚C aerobically for 18-24 hours.
53
Growth Characteristicscolony size shape &
organism (mm) surface colour other
H2S +ve 2-3 CV.E.G. Green+Salmonella Black
H2S -ve 2-3 CV.E.G. GreenSalmonella
S. sonnei 2-2.5 CV.E.G. Green (Rough)
S. flexneri 1.0-2.5 CV.E.G. Green
S. dysenteriae 1-2 CV.E.G. Green
E. coli 0.5-2 CV.E.G. Salmon ppt. (Rough)around (No growth)
colonies
Citrobacter spp. 1.0-2.0 CV.E.G. Salmon (Rough)
Klebsiella spp. 0.5-2.0 CV.E.G. Salmon (Mucoid)
Proteus spp. 1.0-2.0 CV.E.G. Green/ (No growthBlack brownish centre)centre
Pseudomonas spp. 0.5-1.5 F.Rz.D. Green (No growth)
ReferencesKing, S. and Metzger, W. I. 1967. A new medium for the isolation ofSalmonella and Shigella species. Bact. Proc. Am. Soc. Microbiol. 77.King, S. and Metzger, W. I. 1968. A new plating medium for theisolation of enteric pathogens. Hektoen Enteric Agar, Appl.Microbiol., 16(4), 577.
King, S. and Metzger, W. I. 1968. A new plating medium for theisolation of enteric pathogens. II. Comparison of Hektoen Agar withSS and EMB agar. Appl. Microbiol., 16(4), 579.
Speck, M. L. (ed.). 1976. Compendium of Methods for theMicrobiological Examination of Food. Washington, D.C.: AmericanPublic Health Association.
Helicobacter Pylori MediumLAB 140
DescriptionA selective medium for the isolation of Helicobacter Pylori, thecausative agent of chronic gastritis.
This campylobacter-like organism was described in 1983 colonisingthe gastric mucosa, a site previously thought to be sterile due to thelow PH. The high level of Urease production by this organismappears to be the major pathogenicity factor enabling it to withstandthe strongly acidic environment.
Helicobacter Pylori medium is a modification of CCDA Medium forthe isolation of Campylobacter spp. Formulated by Bolton et al atPreston Public Health Laboratory, it incorporates a rich agar basesupplemented with horse serum to promote optimum growth, andVancomycin, Cefsulodin, and Amphoteracin as selective agents.
Formula g/litre
Beef Extract 10.0
Meat Peptone 5.0
Tryptone 5.0
Sodium chloride 5.0
Charcoal 4.0
Acid Hydrolysed Casein 3.0
Ferrous sulphate 0.25
Sodium pyruvate 0.25
Sodium carbonate 0.4
Agar no.2 12.0
Method for reconstitutionWeigh 45.0 grams of powder and disperse in 1 litre of deionisedwater. Soak for 10 minutes, swirl to mix and sterilise at 121˚C for 15minutes. Cool to 47˚C and add 100ml of Horse Serum BP 50, and 2vials of VCA supplement X040. Mix well and pour, continuing tomix whilst pouring to keep the charcoal in suspension.
Appearance: Black Agar
pH: 7.4 ± 0.2
Minimum Q.C. organisms: Helicobacter Pylori S.aureus (inhibition) NCIMB 50080
Storage of prepared medium: Plates – up to 7 days at 2-8˚C the dark.
Inoculation: Surface streaking for single colonies.
Incubation: 37 ˚C for 72 hours.
Growth CharacteristicsOrganisms Colony Size Shape and Colour
(mm) Surface
H. Pylori 1.0-1.5 CVEG Grey
B. Catarrhalis 1.0-2.0 CVEG White/Cream
ReferencesBolton et al. Public Health Laboratory, Preston. Personalcommunication.
Hoyle’s MediumLAB027
DescriptionA highly selective culture medium for the isolation anddifferentiation of Corynebacterium diphtheriae types gravis, mitisand intermedius. Hoyle’s medium gives rapid growth of all types ofC. diphtheriae, which results in most specimens giving adequategrowth with overnight incubation.
Formula g/litre
Beef Extract 10.0
Peptone 10.0
Sodium chloride 5.0
Agar 12.0
Method of ReconstitutionWeigh 37 grams of powder and disperse in 1 litre of deionised water.Allow to soak for 10 minutes, and sterilise by autoclaving at 121˚Cfor 15 minutes. Cool to 47˚C, add 50ml of lysed horse or sheep bloodand 10ml of X027 potassium tellurite solution. Mix well beforepouring.
Appearance: Dark Red, clear gel.
pH: 7.8 ± 0.2
InoculationSpread the entire surface with the swab or sample underinvestigation. Hoyle’s medium is very selective and spreading forsingle colonies using a wire loop is not necessary. Use of a non-selective blood agar alongside Hoyle’s is recommended.
Incubation: 37˚C for 18-48 hrs, aerobically
54
Interpretationcolony size shape &
organism (mm) surface colour other
C.diphtheriae 1.5-2.5 CV.CR.D Grey Colonies difficult var gravis (daisy head) to emulsify
C.diphtheriae 0.5-2.0 CV.E.G. Grey Easily var mitis (dark centre) emulsified
C.diphtheriae 0.5-1.0 CV.E.G. Greyvar intermedius (dark centre)
C. Ulcerans 1.0-1.5 CV.E.G. Grey Streptococcal (dark centre) appearance
in Gram stain
C.hoffmanii 0.5-1.0 CV.E.G. Black White/grey(confluent growth)colonies
C.xerosis 0.5-2.5 CV.E.G. Black
Streptococcus pp - 1.5 CV.E.G. Black Enterococci mayspp. be larger
H.influenzae pp - 1.5 CV.E.G. Grey/black Some strains no growth
Storage: Plates – up to 7 days at 2-8˚C
Minimum Q.C. Organisms C.diphtheriae var mitis(non-toxigenic) E. coli NCTC 10418 (inhibition)
Reference:Hoyle L. (1941) A Tellurite Blood Agar Medium for the RapidDiagnosis of Diphtheria. Lancet 1 175-
176. Elek S.D. (1948) The Recognition of Toxigenic Bacterial Strainsin vitro. Brit. Med. J. 1 493-496.
Kanamycin Aesculin Azide Agar(K.A.A. Agar)
LAB 106
DescriptionA selective isolation and enumeration medium for enterococci(Lancefield group D streptococci) in food. Sodium azide andkanamycin provide the selective inhibition required whilst aesculinand iron salts form an indicator system for the presumptiveidentification of enterococci. Incubation at 42˚C will increase themedium’s selectivity.
Formula g/litre
Tryptone 20.0
Yeast Extract 5.0
Sodium chloride 5.0
Sodium citrate 1.0
Aesculin 1.0
Ferric ammonium citrate 0.5
Sodium azide 0.15
Kanamycin sulphate 0.02
Agar No. 1 10.0
Method for reconstitutionWeigh 43 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C, then dispense intopetri dishes.
Appearance: Pale straw, clear.
pH: 7.0 ± 0.2
Minimum Q.C. organisms: E. faecalis NCIMB 50030E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation: Surface, spread 0.1ml to 0.5ml over entire surface ofplate.
Incubation: 37˚C or 42˚C aerobically for 18-24 hours.
Interpretation: Count all white/grey colonies, approx 2mmdiameter, surrounded by a black halo to give presumptiveenterococcus/faecal streptococcus count.
ReferencesMossel, D. A. A., P. H. G. Bijken, I. Eelderink, and K. A. vanSpreekens, 1978. Streptococci, edited by F. A. Skinner and L. B.Quesnel, SAB Symposium Series No. 7 Academic Press, London.
Kanamycin Aesculin Azide Broth(K.A.A. Broth)
LAB 107
DescriptionAn enrichment and isolation medium for enterococci. The mediumcan be used with the M.P.N. technique to enumerate enterococci infood. This broth is identical to LAB 106 K.A.A. agar with theomission of the agar.
Formula g/litre
Tryptone 20.0
Yeast Extract 5.0
Sodium chloride 5.0
Sodium citrate 1.0
Aesculin 1.0
Ferric ammonium citrate 0.5
Sodium azide 0.15
Kanamycin sulphate 0.02
Method for reconstitutionWeigh 33 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, warm gently to dissolve completelythen disperse into tubes or bottles. Sterilise by autoclaving at 121˚Cfor 15 minutes.
Appearance: Light straw, clear.
pH: 7.0 ± 0.2
Minimum Q.C. organisms: E. faecalis NCIMB 50030E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Capped containers – up to 3 monthsat 15-20˚C in the dark.
Inoculation: Inoculate tubes with decimal dilutions of foodsuspension.
Incubation: 37˚C or 42˚C aerobically for 18-24 hours.
Interpretation: Blackening of the medium suggests the presence ofenterococci/faecal streptococci.
ReferencesMossel, D. A. A., P. H. G. Bijken, I. Eelderink, and K. A. vanSpreekens. 1978. Streptococci, edited by F. A. Skinner and L. B.Quesnel, SAB Symposium Series No. 7 Academic Press, London.
55
Kirchner’s T.B. MediumLAB 123
DescriptionThe cultivation of non-sputum specimens for mycobacteria requiresspecial attention. Specimens such as C.S.F., tissue or body fluidsobtained by surgical procedures are not easily repeated and maycontain small numbers of mycobacteria. It is important, therefore,that as large a portion of specimen as possible be inoculated intosuitable growth media.
Historically, such specimens were inoculated into guinea pigs;however improvements in culture methods have led to a situationwhere comparable results, can be obtained from the use of liquidculture media.
Selective Kirchner’s Medium is a liquid culture media which is madeselective by the addition of an antibiotic cocktail, and has been shownto be sufficient in the recovery of mycobacteria from non-sputumspecimens.
Formula g/litre
Sodium dihydrogen phosphate 7.54
Potassium dihydrogen phosphate 2.0
Magnesium sulphate 0.34
Trisodium citrate 2.5
L-Asparagine 5.0
Phenol red 0.012
Method for reconstitutionWeigh 17.4 grams disperse in 1 litre deionised water, add 20ml.Glycerol A.R. Mix well and dissolve by gentle heat. Dispense 9ml.volumes into Macartney bottles. Sterilise at 121˚C for 10 minutes.Before use add 1ml. sterile heat in activated horse serum andselective agents, if required.
Selective AgentsPolymixin B 200 units/ml
Carbenicillin 100 µg/ml
Trimethoprin 10 µg/ml
Amphoterocin 10 µg/ml
Appearance: Pale red, clear fluid.
pH: 7.0 ± 0.2
Minimum Q.C. organisms: M.tuberculosis (H37RV strain)E. coli (inhibition)NCIMB 50034Candida sp. (inhibition)NCIMB 50010
Storage of Prepared Medium: up to 3 months at 2-8˚C in the dark.
Inoculation: Kirchner’s Medium is suitable for the cultivation ofmycobacteria from the following specimens:
Pre-treatment Required
Sputum, Urine & Faeces Concentration and acid or
alkali decontamination.
C.S.F., Pus with no other bacteria None.
Tissue Disintegration followed byacid or alkali decontamination, if necessary.
The acidified or alkaline de-contaminated material may be addeddirectly to the Kirchner. The pH is then adjusted using the appropriatenormal solution observing colour or indicator.
Although Kirchner’s Medium has been shown to give higherisolation rates than solid media, it is recommended that specimensshould also be inoculated onto Lowenstein Jensens slopes for thefollowing reasons: – Growth suitable for further characterisation maybe obtained more rapidly – Some Mycobacterium species includingM.intracellulare, M.kansasii and M.scrofulaceum may be inhibitedby the antibiotic cocktail.
Incubation: 37˚C aerobically for up to six weeks.
Growth Characteristicsorganism
M. tuberculosis white floccular ‘snowflake’ colonies
M. scrofulaceum- ‘ropey’ deposit
M. kansasii- fine granular deposit.
ReferencesKirchner, O., 1932. Erfahrungen bei diagnosticher Verwendung derTiefenkulter. Sbl fur Bact. Abt 1. Originale 124, 409-412.
Marks, J. 1972, Ending the routine guinea pig test. Tubercle 1972. 53:31-34.
Mitchison, D. A. Allen, B. W., Manickavasagar, D. 1983. SelectiveKirchner’s medium in the culture of specimens other than sputum formycobacteria. J.Clin. Path. 36: 1357-1361.
Kligler Iron AgarLAB059
DescriptionA differential medium for the recognition of enteric pathogens bytheir ability to ferment glucose and/or lactose, and liberate sulphides.Fermentation liberates acid, with or without gas, turning phenol redindicator yellow. Fermentation of glucose only, is followed byreversion in pH on the slope, from initial acidity to final alkalinity(red colour), but not in the anaerobic conditions of the butt, whichremains acid (yellow). Fermentation of lactose as well as glucose,produces acidity in both slope and butt (yellow). Liberation ofsulphide results in the formation of iron sulphide (blackening ofeither slope or butt).
Formula g/litre
Peptone 20.0
Lactose 10.0
Glucose 1.0
Sodium chloride 5.0
Ferric ammonium citrate 0.5
Sodium thiosulphate 0.3
Phenol red 0.025
Method of reconstitutionWeigh 49 grams of powder and mix with 1 litre of distilled water.Bring to the boil with frequent stirring to dissolve completely.Dispense into tubes and sterilise for 15 minutes at 121˚C. Cool in aslanted position such that slopes are formed over deep butts approx.3cm in depth.
Appearance: Reddish brown agar.
pH: 7.4±0.2
Minimum Q.C. Organisms Salmonella typhimuriumNCIMB 50076Pseudomonas aeruginosaNCIMB 50067
InoculationSubcultures for further identification are picked from the centre ofisolated colonies on selective media and streaked across the slant andstabbed deep into the butt of tubes of Kligler Iron Agar.
Incubation: 37˚C aerobically for 18-24 hours.
56
InterpretationOrganism Butt Slope Sulphide
Salmonella typhi Acid Alkaline +
S. paratyhi A + B Acid Alkaline -
Other Salmonella Acid/gas Alkaline +
E. coli Acid/gas Acid -
Proteus spp Acid/gas Alkaline +
Shigella sonnei Acid Alkaline -
S. flexneri Acid Alkaline -
Storage: Tightly capped containers - up to 3 months at 15-20˚C in thedark.
References:Kligler, I.J. 1917. A Simple Medium for the Differentiation ofMembers of the Typhoid - Paratyphoid Group. Am. J. Publ. Hlth,7:1042-1044.
Bailey, S.F. and Lacey, G.R. 1927. A modification of the Kligler LeadAcetate Medium. J. Bact. 13:182-189.
Lactose BrothLAB 126
DescriptionA medium for the performance and confirmation of the PresumptiveTest for members of the coliform group in water and dairy products,recommended by the U.S.P.
Formula g/litre
Beef Extract 3.0
Gelatin Peptone 5.0
Lactose 5.0
Method for reconstitutionWeigh 13 grams of powder, disperse in 1 litre of deionised water, heatto dissolve then distribute into bottles with Durham tubes. Sterilise byautoclaving at 121˚C for 15 minutes.
Appearance: Straw coloured, clear.
pH: 6.9 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034
Storage of Prepared Medium: Capped containers – up to 3 monthsat 15-20˚C in the dark.
Inoculation: See methods for standard techniques.
Incubation: 35˚C aerobically for 48 hours.
Interpretation: Coliforms are presumptively identified by theirability to ferment lactose and produce gas within 48 hours at 35˚C.
ReferencesAmerican Public Health Association. 1975. Standard Methods for theexamination of water and waste water, 892. Washington. UnitedStates Pharmocopeia, XXI, 1985.
Liquid Baird Parker MediumLAB 158
DescriptionDeveloped by Van Doorne et al in 1981, this medium is essentiallyBaird Parker Agar LAB 085 without the agar and egg yolkcomponents. This medium was chosen for development to overcomethe problems of other selective enrichments and non-selectiveenrichments, in the isolation of Staphlococcus aureus.
Other selective broth media suffer from the potential of inhibiting sublethally damaged S. aureus cells where the salt content is greater than40g/l. Even Giolitti Cantoni Medium may be inhibitory to somestrains of S. aureus.
Non selective enrichments have been suggested, but are not ideal dueto potential inhibition of S. aureus by microbial antagonism in amixed bacterial population.
Liquid Baird Parker Medium is the ideal solution for detecting lownumbers (<20/g) of S. aureus as it has been shown to give acceptableselectivity whilst being non-inhibitory to injured cells.
Formulation g/litre
Meat Peptone 8.0
Tryptone 2.0
Beef Extract 5.0
Yeast Extract 1.0
Sodium pyruvate 10.0
Glycine 12.0
Lithium chloride 5.0
Method for ReconstitutionDouble Strength Medium: Weigh 86 grams of powder and dispersein 1 litre of deionised water. Soak for 10 minutes, mix to dissolve anddispense 10ml amounts in tubes. Sterilise at 121˚C for 15 minutes.Before use, heat to 100˚C in a water bath or steamer for 15 minutesto remove oxygen, cool and add 0.2ml of 1% potassium telluritesolution to each tube.
Single Strength Medium: Weigh 43 grams of powder and proceedas above. When cool, add 0.1ml of 1% potassium tellurite solution toeach tube.
Appearance: Clear straw broth.
pH: 6.8 ± 0.2.
Minimum Q.C. organisms: S. aureus NCIMB 50080E. coli (inhibition) NCIMB 50034
Storage of prepared medium: Up to 1 month at 2-8 ˚C (withouttellurite added).
Inoculation: Double strength; 10ml of sample homogenate to eachtube. Single strength; 1ml of sample homogenate to each tube.
Incubation: 37˚C anaerobically for 48 hours. Anaerobic conditionscan be achieved using an anerobic jar (container caps must be loosefitting) or by overlaying the surface of the broth with 1cm of moltenagar or liquid parafin.
Interpretation: Culture tubes showing growth (blackening of themedium and turbidity) onto Baird Parker Agar LAB 085.
ReferencesVan Doorne, H., Baird, R.M., Hendriksz, D.T., Van der Kreek, D.M.,Pauwels, H.P. (1981) Liquid Modification of Baird Parker’s Mediumfor the Selective Enrichment of Staphylococcus aureus. Antonie VanLeeuwenhoek 47 267-278.
57
Listeria Enrichment BrothLAB 138
DescriptionA medium for the selective enrichment of food and environmentalsamples for Listeria spp. first described in 1987 by J. Lovett. Themedium offers more rapid enrichment than the low temperatureenrichment techniques. This medium is now recommended by theCommission of European Communities and the International DairyFederation for the examination of soft cheeses for Listeriamonocytogenes. The medium is incubated at 30˚C and utilisesacriflavine, nalidixic acid and cyclohexamide as selective agents.
Formula g/litre
Tryptone 17.0
Soy Peptone 3.0
Sodium chloride 5.0
Dipotassium hydrogen phosphate 2.5
Glucose 2.5
Yeast Extract 6.0
Method for reconstitutionWeigh 36 grams of powder and add to 1 litre of deionised water.Allow to soak for 10 minutes then swirl to mix and sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C and add 2 vials ofX138 reconstituted in 50% alcohol. Aseptically dispense into steriletubes or bottles.
Appearance: Yellow, clear.
pH: 7.3 ± 0.2
Minimum Q.C. organisms: L. monocytogenes NCIMB 50007E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Capped containers – up to 14 days at2-8˚C in the dark.
Inoculation: Homogenised samples of food, 25 grams ofhomogenate to 225mls of broth.
Incubation: 30˚C aerobically for up to 48 hours.
Subculture: After 24 and 48 hours onto Listeria Isolation Medium –LAB 122.
ReferencesLovett, J. Frances, D. W. Hunt, J. M. J. Food Protect. 50: 188-192.Bolton, F. J. Personal Communication Public Health Laboratory,Preston U.K.
Listeria Isolation Medium(Oxford Formulation)
LAB 122
DescriptionA selective identification medium for the isolation of Listeriamonocytogenes from food and clinical material. Columbia agar is thenutrient base to which selective inhibitors have been added. Lithiumchloride is used to inhibit enterococci and acriflavine to inhibit someGram negative and Gram positive species. Further selective agentsmay be added after autoclaving to increase the selectivity; these arecolistin, fosfomycin, cefotetan and cyclohexamide. Aesculin isincluded in the formula as a differential indicator. L. monocytogeneswill hydrolyse aesculin to aesculutin which reacts with the iron salt togive a black precipitate around the colonies.
LAB M’s formulation has been used to successfully isolate Listeriafrom such diverse products as chicken giblets and dairy cheeses. Theadvisability of using this medium at two levels of selectivity has beenrecognised.
Formula g/litre
Columbia Agar Base 41.0
Aesculin 1.0
Ferric ammonium citrate 0.5
Lithium chloride 15.0
Method for reconstitutionWeigh 57.5 grams of powder. Add to 1 litre of deionised water. Allowto soak for 10 minutes, swirl to mix then sterilise by autoclaving at121˚C for 15 minutes. Allow to cool to 47˚C, add 2 vials of selectivesupplement X122, mix well and pour plates.
Appearance: Pale yellow, clear.
pH: 7.2 ± 0.2
Minimum Q.C. organisms: L. monocytogenes NCIMB 50007E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation: Surface, streak out to single colonies. This medium ishighly selective, a heavy inoculum can be used.
Incubation: 30˚C aerobically for 24-48 hours.
Growth Characteristicscolony size shape &
organism (mm) surface colour other
L. monocytogenes 0.5-1.0 CV.E.G. Black Black/brownaround colonies
diffusion
Gram-ve Bacilli No growth
Enterococci p.p. 0.5 CV.E.G. Black Usually no growth
ReferencesGarayzabal, J.F.F., Rodriguez, L.D., Boland, J.A.V., Cancelo, J.L.B.,Fernandez, G.S. 1986. Listeria monocytogenes dans le lait pasteurise.Can. J. Microbiol. 32: 149-150.
Donnelly. C.W., Gregory J. Baigent 1986. Method for flowcytometric detection of Listeria monocytogenes in milk. Appl. &Environ. Microbiol.Oct. 689-695.
Bolton, C.F.J. Preston P.M.L. Personal communication. Lovett, J.Francis, D.W. Hunt. J.M. (1987). Listeria monocytogenes in rawmilk: Detection, Incidence and Pathogenicity. Journ. Food Protect.Vol. 50. No. 3: 188-192.
Van Netten, P., Van de Van, A., Perales, I., Mossel, P.A.A. 1988. Aselective and diagnostic medium for use in enumeration of Listeriaspp. in foods. International Journal of Food Microbiology 6:187-198.
58
M17 AgarLAB 92
DescriptionA medium for the enumeration of lactic streptococci (Streptococcuslactis, Streptococcus cremoris, Streptococcus diacetylactis) in dairyproducts. The medium can also be used to investigate thebacteriophage susceptibilities of these organisms. Anotherapplication is for the enumeration of Streptococcus thermophilus inyoghurts.
Formula g/litre
Balanced Peptone 5.0
Soy Peptone 5.0
Yeast Extract 2.5
Beef Extract 5.0
Lactose 5.0
Sodium glycerophosphate 19.0
Magnesium sulphate 0.25
Ascorbic acid 0.5
Agar No. 2 15.0
Method for reconstitutionWeigh 57.2 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then bring to boil todissolve agar before dispensing in 15ml aliquots. Sterilise byautoclaving at 115˚C for 20 minutes.
Appearance: Pale straw, translucent agar.
pH: 7.1 ± 0.2
Minimum Q.C. organisms: S. lactis
Storage of Prepared Medium: Capped containers – up to 1 monthat 15-20˚C in the dark.
Inoculation: Pour plate technique.
Incubation: 30˚C for 48-72 hours for mesophilic streptococci, 37˚Cfor 48 hours for Streptococcus thermophilus.
Interpretation: Count all colonies. Streptococci form colonies of 1-2mm in diameter.
ReferencesTerzaghi, B. E. Sandine, W. E.. 1975. Improved medium for lacticStreptococci and their Bacteriophages. Appl. Microbiol. 29 No. 6 pp807-813.
MacConkey Agar(With Salt)
LAB 30
DescriptionA selective medium for the isolation of bile tolerant organisms fromfaeces, urine, sewage and foodstuffs. Bile-tolerant Gram positiveorganisms as well as Gram negative organisms will grow on thismedium. This formula is recommended by W.H.O. and other bodiesfor the examination of water and milk. Some strains of Proteus spp.will spread on this medium making interpretation difficult, for thisreason LAB 2 MacConkey Agar (without salt) may be preferred as itis less prone to this phenomenon.
Formula g/litre
Peptone 20.0
Lactose 10.0
Bile Salts 5.0
Sodium chloride 5.0
Neutral red 0.05
Agar No. 2 12.0
Method for reconstitutionWeigh 52 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C and mix wellbefore pouring into petri dishes.
Appearance: Pink/red, clear
pH: 7.4 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in thedark.
Inoculation: Surface plating, streaking out to single colonies.
Incubation: 37˚C aerobically for 24 hours.
Growth Characteristicsapprox. size shape &
organism (mm) surface colour other
E. coli 2.0-3.0 CV.E.G. Red (ppt around colony)
K. aerogenes 2.0-4.0 CV.E.G. Pink/Red (mucoid)
Citrobacter spp. 2.0-4.0 CV.E.G. Pink/Red (ppt aroundcolony)
Proteus spp. 1.5-2.5 CV.E.G. Yellow(spreading)
Salmonella spp. 1.5-2.5 CV.E.G. Colourless
Shigella spp. 1.0-2.0 CV.E.G. Colourless(pink)
Transp.
S. aureus 0.5-2.0 CV.E.G. White/ (dependent onPink lactose
fermentationOrange and pigmentOpaque production)
Enterococcus spp. P.P.-0.5 CV.E.G. Pink/Deep
RedOpaque
P. aeruginosa 1.0-3.0 F.CR.D. Transp. (colonial-Pinkish variation)
ReferencesMinistry of Health. Public Health Laboratory Service WaterCommittee 1969. The Bacteriological Examination of WaterSupplies, 4th Edn. report No. 71, H.M.S.O., London.
59
World Health Organisation 1971. International Standards forDrinking Water. 3rd Edn. W.H.O., Geneva.
Taylor, E. W. 1958. The Examination of Water and Water Supplies.7th Edn. Churchill, London.
Cruikshank, R. 1973. A Guide to the Laboratory Diagnosis andControl of Infection. Medical Microbiology. 12th Edn. Churchill.
MacConkey Agar(without salt)
LAB 2
DescriptionA medium first introduced by MacConkey in 1905 for the isolationand differentiation of lactose and non lactose fermenting entericbacteria. The medium has since been modified to improve therecovery of staphylococci and enterococci, it is used for culturing awide range of clinical material and has applications in food, waterand dairy bacteriology.
Formula g/litre
Mixed Peptones 20.0
Lactose 10.0
Bile 5.0
Neutral red 0.05
Agar No. 2 13.5
Method for reconstitutionWeigh 48.5 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving for 15 minutes at 121˚C. Cool to 47˚C and mix wellbefore pouring plates. Prior to inoculation, dry the surface of the agarby partial exposure at 37˚C.
Appearance: Pink/red, clear.
pH: 7.4 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034S. aureus NCIMB 50080
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation method: Surface inoculation, streaking for singlecolonies.
Incubation: 37˚C aerobically for 24 hours.
Growth Characteristicscolony size shape &
organism (mm) surface colour other
E. coli 2.0-3.0 CV.E.G. Red (non lactosefermenting
yellow)
K. aerogenes 3.0-4.0 CV.E.G. Pink-Red (mucoid)
Proteus spp. 2.0-3.0 CV.E.G. Yellow fishy odour
Ps. aeruginosa 2.0-4.0 F.CR.D. Pink- characteristicYellow- odour if green(Green)
Shigella spp. 1.0-3.0 CV.E.G. Yellow
Salmonella spp. 2.0-3.0 CV.E.G. Yellow
S. aureus 0.5-1.0 CV.E.G. Pink- (lactose-negative)Orange
Enterococcus spp. 0.5 CV.E.G. Pink-Deep Red
ReferencesMacConkey, A. T. 1905 Lactose-fermenting bacteria in faeces. J.Hyg.(Camb), 5: 333-379.
MacConkey, A. T. 1908 Bile salt media and their advantages in somebacteriological examinations, J.Hyg. (Camb.), 8: 322-341.
Ministry of Health, Public Health Laboratory Service WaterCommittee 1969. The Bacteriological Examination of WaterSupplies, 4th Edn. report No. 71. H.M.S.O., London.
World Health Organisation 1971, International Standards forDrinking Water, 3rd Edn. W.H.O., Geneva. Taylor, E. W. 1958. TheExamination of Water Supplies, 7th Edn. Churchill, London.
MacConkey Agar No. 3LAB 45
DescriptionA modification recommended by the W.H.O. and the AmericanPublic Health Association for the isolation of Enterobacteriaceaefrom waters and sewage. The medium has been made more selectivethan MacConkey’s original formula by the use of crystal violet aswell as bile salts. Gram positive organisms will not grow on thismedium.
Formula g/litre
Peptone 20.0
Lactose 10.0
Bile Salts No. 3 1.5
Sodium chloride 5.0
Neutral red 0.03
Crystal violet 0.001
Agar No. 2 15.0
Method for reconstitutionWeigh 51.5 grams of powder and add to 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise for 15minutes at 121˚C. Cool to 47˚C and pour into petri dishes. Dry thesurface before inoculation.
Appearance: Pale red slight violet tinge.
pH: 7.1 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034Ent. faecalis (inhibition)NCIMB 50030
Storage of prepared medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation: Surface, streaking for single colonies.
Incubation: 37˚C aerobically for 18-24 hours.
Growth Characteristicscolony size shape &
organism (mm) surface colour other
E. coli 3.0-4.0 CV.E.G. Red (red ppt around(D) colony)
Kleb. aerogenes 4.0-5.0 CV.E.G. Pink-red (mucoid)
Proteus spp. 3.0-4.0 CV.E.G. Pale- (fishy odour)yellow
Ps. aeruginosa 1.0-1.5 F.CR.D. Yellow-green
Shigella spp. 2.0-3.0 CV.E.G. Pale- (pink)(D) yellow
Staph. aureus No growth
Other Staphylococcus spp. No growth
Enterococcus spp. No growth
60
ReferencesAmerican Public Health Association 1950. Diagnostic Proceduresand Reagents. 3rd edn. A.P.H.A., New York. American Public HealthAssociation 1946. Standard Methods for the examination of Waterand Sewage. 9th edn. A.P.H.A., New York.
MacConkey Broth (Purple)
LAB 5
DescriptionThis medium is used in the detection and enumeration of faecalcoliforms (37˚C) and E. coli (44˚C). The replacement of neutral redused in the original formulation by bromocresol purple makes thecolour change caused by acid producing organisms easier to read.
Formula g/litre
Peptone 20.0
Lactose 10.0
Bile Salts 5.0
Bromocresol purple 0.01
Method for reconstitutionWeigh 35 grams of powder, disperse in 1 litre of deionised water. Mixwell and dispense into tubes or bottles with inverted Durham tubes.Sterilise by autoclaving for 15 minutes at 121˚C. Prepare doublestrength broth (70g/l) if 50ml or 10ml amounts of inoculum are to beadded to equal volumes of broth. Prepare single strength broth (35g/l)if 1ml or 0.1ml amounts of inoculum are to be added to 10ml of broth.
Appearance: Purple, clear.
pH: 7.3 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034B. subtilis (inhibition)
Storage of Prepared Medium: Capped containers – up to 1 monthat 15-20˚C in the dark.
Incubation: 37˚C aerobically for coliforms, 44˚C aerobically for E. coli. Use Durham tubes to detect gas production for E. coli.
Growth Indicators: Turbidity, gas production. Lactose-fermentingorganisms cause a colour change from purple to yellow.
ReferencesMinistry of Health 1937. Bacteriological Tests for Graded Milk,Memo 139/Foods. H.M.S.O., London.
Minister of Health, Public Health Laboratory Service WaterCommittee 1969. The Bacteriological Examination of WaterSupplies, 4th Edn. report No. 71. H.M.S.O., London.
World Health Organisation 1971. International Standards forDrinking Water, 3rd Edn, W.H.O., Geneva.
Malt Extract AgarLAB 37
DescriptionAn acidic medium which will support the growth of most yeasts andmoulds whilst inhibiting most bacteria. It was first described byThom and Church in 1926 in a study of Aspergillus spp. claiming thehigh carbohydrate content ensured rapid growth. Selectivity can beincreased by further lowering the pH with the addition, aftersterilisation, of XO37 Lactic Acid. It should be noted that excessheating of this medium together with its low pH can easily result inhydrolysis of the agar gel producing soft plates.
Formula g/litre
Malt Extract 30.0
Mycological Peptone 5.0
Agar No. 2 15.0
Method for reconstitutionWeigh 50 grams of powder, disperse in 1 litre of deionised water,allow to soak for 10 minutes, swirl to mix then sterilise at 115˚C for10 minutes. If the addition of XO37 Lactic Acid is required thisshould be done after sterilisation. One 5ml vial of XO37 will lowerthe pH of 250ml of medium to 3.5-4.0. Cool to 47˚C before makingadditions and pouring plates.
Appearance: Pale brown/straw, clear.
pH: 5.4 ± 0.2 (if XO37 is added pH 3.5-4.0)
Minimum Q.C. organisms: Candida spp. NCIMB 50010
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark. Capped container – up to 1 month at 15-20˚C in the dark.
Inoculation: Pour plate technique or surface streaking for singlecolonies.
Incubation: 25˚C aerobically for 5 days.
Growth Characteristicscolony size shape &
organism (mm) surface colour other
Candida albicans 4 CV.E.D. White
Candida krusei 10 F.CR.D. White
Penicillium notatum 25 Green (white/yellow -
velvet strain dependent)
Aspergillus niger 25 White (yellow/blackborder, centre)blackcentre
ReferencesGalloway, L.D. and Burgess, R. 1952. Applied Mycology andBacteriology, Leonard Hill, London. Thom and Church, 1926. The Aspergilli.
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Malt Extract BrothLAB 159
DescriptionA liquid medium of low pH for the growth of yeasts and moulds,typically employed as part of sterility testing protocols for variousproducts. The high carbohydrate content of the medium ensures rapidgrowth of yeasts and moulds.
Formula g/litre
Malt Extract 17.0
Mycological Peptone 3.0
Method for reconstitutionWeigh 20g of powder and disperse in 1 litre of deionised water. Allowto soak for 10 minutes, swirl to dissolve and dispense into finalcontainers. Sterilise by autoclaving at 115˚C for 10 minutes.
Appearance: Pale brown/straw, clear broth.
pH 5.4 + 0.2
Inoculation:Inoculate samples direct into tubes of broth according to theparticular method being employed.
Incubation:25˚C (or 37˚C) for up to 7 days aerobically, depending upon protocolused.Subculture turbid tubes onto solid media for identification ofgrowth.
Minimum QC organism: Candida albicans NCIMB 50010Aspergillus niger NCIMB 50097
References:Galloway L D, and Burgess R (1952) Applied Mycology andBacteriology, 3rd ed, Leonard Hill, London pp 54 & 57.
Mannitol Salt AgarLAB 7
DescriptionA medium for staphylococci which is selective because the highsodium chloride level inhibits most other species with the exceptionof halophilic vibrios. The majority of Staphylococcus aureus fermentmannitol producing yellow colonies, occasional strains of coagulasenegative staphylococci may also ferment mannitol. It is necessary toconfirm the identity of presumptive S. aureus colonies by othermeans e.g. coagulase, protein A, DN’ase, thermonuclease or latexagglutination.
Formula g/litre
Beef Extract 1.0
Balanced Peptone No. 1 10.0
Sodium chloride 75.0
D-Mannitol 10.0
Agar No. 2 12.0
Phenol Red 0.025
Method for reconstitutionWeigh 108 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, mix by swirling. Sterilise byautoclaving for 15 minutes at 121˚C. Cool to 47˚C before pouringinto petri dishes.
Appearance: Red, clear gel.
pH: 7.4 ± 0.2
Minimum Q.C. organisms: S. aureusE. coli (inhibition)
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation Method: Surface plating, streak out for single colonies.
Incubation: 37˚C for 48 hours aerobically.
Growth Characteristicscolony size shape &
organism (mm) surface colour other
S. aureus 1.5-2.0 CV.E.G. BrightYellow
Other Staphylococci 1.0-1.5 CV.E.G. White or (some ferment
Yellow mannitol)
Enterobacteriaceae no growth
Vibrio spp. & other 1.5-4.0 various usually (yellow ifhalophiles Pink mannitol +ve)
ReferencesAmerican Public Health Association 1966. Recommended Methodsfor Microbiological Examination of Foods, 2nd Edn. (ed. J. M. Sharf)A.P.H. Washington.
Davis, J. G., 1959. Milk Testing 2nd edn, Dairy Industries, London.
Maximum Recovery Diluent(Peptone/Saline diluent)
LAB 103
DescriptionAn osmotically controlled solution which is an alternative to, and areplacement for, 1/4 strength Ringers LAB 100. The presence of alow level of peptone lessens the physiological shock normallyexperienced by bacterial cells when they are introduced to a diluentsuch as Ringers Solution. The level of peptone is such thatmultiplication of the organisms is not possible in the time in whichthe sample will be present in the diluent (1-2 hours). This formula isrecommended by ISO 6887: BS5763.
Formula g/litre
Peptone 1.0
Sodium chloride 8.5
Method for reconstitutionDissolve 9.5 grams of powder in 1 litre of deionised water, heatgently to dissolve then distribute into final containers. Sterilise byautoclaving at 121˚C for 15 minutes.
Appearance: Clear fluid.
Storage of Prepared Medium: Capped containers – up to 3 monthsat 15-20˚C in the dark.
ReferencesStraka, R. P. and Stokes, J. L. 1957. Rapid destruction of bacteria incommonly used diluents and its elimination. Appl. Microbiol. 5: 21-25. ISO 6887. 1983. Microbiology-General guidance for thepreparation of dilutions for microbiological examination. BS5763Part 6. Preparation of dilutions.
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Membrane Lauryl Sulphate BrothLAB 82
DescriptionThis medium superseded Membrane Enriched Teepol broth whenShell Chemicals withdrew Teepol 610 from sale. Sodium laurylsulphate was found to be an adequate reproducible substitute and thismedium is recommended for the enumeration of coliform andorganisms in water and sewage.
Formula g/litre
Peptone 39.0
Yeast Extract 6.0
Lactose 30.0
Phenol red 0.2
Sodium lauryl sulphate 1.0
Method for reconstitutionWeigh 76.2 grams of powder, disperse in 1 litre of deionised water.Distribute into screw cap containers and sterilise by autoclaving at115˚C for 10 minutes.
Appearance: Red, clear solution.
pH: 7.4 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034
Storage of Prepared Medium: Capped containers – up to 3 monthsat 15-20˚C in the dark.
Inoculation: E. coli and coliform counts should be made on separatesamples of water. The volumes should be chosen so as the number ofcolonies on the membrane lies between 10 and 100. With watersexpected to contain less than 1 coliform perml, a sample of 100mlshould be filtered. The membrane filter should be placed faceupwards on a pad soaked in Membrane Lauryl Sulphate Broth, afterfiltration. These membranes should be incubated in a container whichdoes not allow evaporation to occur. Water tight metal containersplaced in an accurate water bath are required for incubation ofmembranes at 44˚C.
Incubation: E. coli 4 hours at 30˚C 14 hours at 44˚C; Coliforms 4hours at 30˚C 14 hours at 35˚C.
Interpretation: No colonies:- assume a nil count. Small colonies ofan intermediate colour:- return to incubation for a full period.
E. coli: Yellow-coloured colonies from membranes incubated at 44˚Cshould be subcultured to Lactose Broth LAB 126 and TryptoneWater, LAB 129 to confirm gas and indole production respectively,after 24 hours incubation at 44˚C.
Coliform organisms: Yellow colonies from membranes incubated at35˚C or 37˚C should be subcultured into Lactose Broth LAB 126.After 48 hours incubation at 37˚C a result should be obtainedregarding the production of gas.
Full details of the methodology can be found in The BacteriologicalExamination of Water Supplies 71, 1969.
ReferencesBurnham, N.P. 1967. Proc. Soc. Wat. Treat, Exam. 16:40. Report 71.1994. The Bacteriological Examination of Water Supplies. 4th Ed.London, HMSO. Windle Taylor E. 1961 Glutamic acid medium, 40thAnn Rep. Div. Water Exam. Met. Water Board London pp 18-22.
Milk AgarLAB 19
DescriptionAn approved formulation for the enumeration of micro-organisms inmilk, rinse waters and dairy products. With the addition of a further5 g/l Agar No. 1 the medium is suitable for the preparation of Roll-Tubes using established mechanical equipment. Also see Milk PlateCount Agar LAB 115. Milk Agar can also be used with the P-INCsupplement (X019, X219) for accelerated shelf-life determination ofdairy products, (see page 103).
Formula g/litre
Yeast Extract 3.0
Peptone 5.0
Antibiotic Free Skim Milk Powder 1.0
Agar No. 1 15.0
Method for reconstitutionWeigh 24 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 15 minutes, swirl to mix then sterilise for 15minutes at 121˚C. Cool to 45˚C before mixing with sample dilutions.
Appearance: White, opalescent gel.
pH: 7.2 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034S. aureus NCIMB 50080
Storage of Prepared Medium: Capped containers – up to 3 monthsat 15-20˚C in the dark.
Inoculation: Pour plate technique.
Incubation: Aerobically at 30˚C for 72 hours.
ReferencesMinistry of Health 1937. Bacteriological Tests for Graded Milk.Memo 139/Foods H.M.S.O., London. British Standard 4285:Methods of Microbiological Examination for Diary Purposes.
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Milk Plate Count AgarLAB 115
DescriptionA medium recommended by the British Standards Institute and theInternational Organisation for Standardisation for the enumeration ofviable bacteria in milk and other dairy products.
Formula g/litre
Tryptone 5.0
Yeast Extract 2.5
Dextrose 1.0
Antibiotic Free Skim Milk Powder 1.0
Agar No. 1 10.0
Method for reconstitutionWeigh 19.5 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then bring to the boil todissolve the agar. Allow to cool to 47˚C and dispense into suitablecontainers. Sterilise by autoclaving at 121˚C for 15 minutes.
Appearance: Pale cream, opalescent gel.
pH: 6.9 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034S. aureus NCIMB 50080
Storage of Prepared Medium: Capped container – up to 3 monthsat 15-20˚C in the dark.
Inoculation: Pour plate technique.
Incubation: 30˚C aerobically for 72 hours.
Interpretation: Count all colonies. Calculate back to determineviable organisms per ml.
ReferencesBritish Standards Institute. 1984. BS 4285 Section 1.2. InternationalOrganisation for Standardisation Draft International Standard. 1982ISO/DIS 6610. D.I.N. 10192.
Minerals Modified Glutamate MediumLAB 80A & LAB 80B
DescriptionThis medium was developed for use with the Most Probable NumbersTechnique (M.P.N.) for the enumeration of coliforms in watersupplies. The medium is an improved version of the chemicallydefined glutamic acid medium described by Gray in 1964. Theproduct is supplied in two parts because it has been shown thatseparating the sodium glutamate from the base improves its stability.
Formula g/litre
LAB 80a (double strength)
Lactose 20.0
Sodium formate 0.5
L-Cystine 0.04
L(-) Aspartic acid 0.048
L(+) Arginine 0.04
Thiamine 0.002
Nicotinic acid 0.002
Pantothenic acid 0.002
Magnesium sulphate (MgSO4.7H20) 0.2
Ferric ammonium citrate 0.02
Calcium chloride (CaCl2.2H2O) 0.02
Dipotassium hydrogen phosphate 1.8
Bromocresol purple 0.02
LAB 80b
Glutamic acid (sodium salt) 12.7
Method for reconstitutionDouble strength: Dissolve 22.7 grams of base medium (LAB 80a)together with 12.7 grams of sodium glutamate (LAB 80b) in 1 litre ofdeionised water containing 5 grams of ammonium chloride eg BDHcat no. 27149. Dispense 10ml and 50ml volumes into tubes withinverted Durham tube.
Single strength: Dissolve in 11.35 grams of base medium (LAB 80a)together with 6.35 grams of sodium glutamate (LAB 80b) in 1 litre ofdistilled water containing 2.5 grams ammonium chloride. Dispense5ml volumes into tubes with inverted Durham tubes.
Sterilise by autoclaving for 10 minutes at 115˚C, alternatively heat to100˚C for 30 minutes on three successive days.
Appearance: Purple, clear solution.
pH: 6.7 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034
Storage of Prepared Medium: Capped containers – up to 3 monthsat 15-20˚C in the dark.
Inoculation: Use the Most Probable Number technique. With 10mland 50mls of sample add to equal volumes of double strengthmedium. With 1ml volumes of sample add to 5mls of single strengthmedium. Ensure the Durham tube is free of bubbles.
Incubation: 37˚C for 18-24 hours aerobically.
Interpretation Tubes showing the production of acid (medium turnsyellow) and gas in the Durham’s tube are considered presumptivepositive. Each presumptive positive tube should be subcultured toBrilliant Green Bile Broth LAB 51 with Durham tube and incubatedat 44˚C for 24 hours and examined for gas production. A tube of Tryptone Water LAB 129 should also be inoculated and incubatedat 44˚C for 24 hours for the production of indole. The production at 44˚C of gas from lactose and the formation of indole are evidenceof E. coli.
ReferencesGray, R. D. 1964. An improved formate lactose glutamate mediumfor the detection of Escherichia coli and other coliform organisms inwater. J. Hyg. Camb. 62: 495-508.
PHLS Water Sub-Committee. 1958. A comparison betweenMacConkey broth and Glutamic acid media for the detection ofcoliform organisms in water. J. Hyg. Camb. 56: 377-388.
PHLS Standing Committee on Bacteriological Examination of WaterSupplies. 1968. Comparison of MacConkey Broth, Teepol Broth andGlutamic Acid Media for the enumeration of Coliform organisms inwater. J. Hyg. Camb. 66: 67-87.
Report 71. 1969. The Bacteriological Examination of Water Supplies.4th Edn., London, HMSO.
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M.L.C.B. Agar(Mannitol Lysine Crystal Violet Brilliant Green Agar)
LAB 116
DescriptionA medium for the selective isolation of Salmonella spp. (with theexception of S. typhi and S. paratyphi A) from food and faeces.Salmonella colonies are recognised by distinctive colonialappearance and H2S production and like the Bismuth Sulphite Agarof Wilson & Blair, this medium will detect lactose and sucrosefermenting strains. Some problems may occur with H2S negativestrains, eg S. pullorum, S. senftenberg, S. sendai and S. berta. Thismedium should not be used to detect S. typhi and S. paratyphi A, asthese strains are more susceptible to the brilliant green dye.
Formula g/litre
Yeast Extract 5.0
Tryptone 5.0
Meat Peptones 7.0
Sodium chloride 4.0
Mannitol 3.0
L-Lysine HCL 5.0
Sodium thiosulphate 4.0
Ferric ammonium citrate 1.0
Brilliant green 0.012
Crystal violet 0.01
Agar No. 2 15.0
Method for reconstitutionWeigh 49 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then bring to the boil withfrequent agitation to completely dissolve the powder. Cool to 47˚Cand pour plates. DO NOT AUTOCLAVE OR OVERHEAT.
Appearance: Pale purple, translucent gel.
pH: 6.8 ± 0.2
Minimum Q.C. organisms: Salmonella spp. NCIMB 50076E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation: Surface plating, streaking for single colonies.Inoculation can be carried out directly, or from enrichment broths.Because of the low selectivity of this medium the inoculum shouldnot be heavy, and it is recommended that this medium should be usedin conjunction with other more selective media.
Incubation: 37˚C aerobically for 24 hours.
Growth Characteristicscolony size shape &
organism (mm) surface colour
Salmonella spp. 2.0-3.0 CV.E.G. Black
Salmonella spp. 2.0-3.0 CV.E.G. Pale(H2S negative)
Proteus spp. 1.0-2.0 CV.E.G. Grey brown
Shigella spp. mainlyinhibited
Citrobacter spp. 0.5-2.5 CV.E.G. Mainlyinhibitedpale may
have blackcentre
E. coli mainly inhibited
Klebsiella spp mainly inhibited
Gram positive organisms mainly inhibited
ReferencesInove et al. 66th meeting of the Japanese Vet. Medicine Society.
M.R.S. Agar(de Man, Rogosa and Sharpe Agar)
LAB 93
DescriptionThis medium was developed for the cultivation and enumeration ofLactobacillus spp. from various sources and is intended as asubstitute for Tomato Juice Agar. The medium is suitable for mostlactic acid bacteria. When acidified to pH 5.4 M.R.S. Agar can beused to enumerate Lactobacillus bulgaricus in yoghurts. Tween 80,sodium acetate, magnesium and manganese sulphates act as growthstimulants.
Formula g/litre
Mixed Peptones 10.0
Yeast Extract 5.0
Beef Extract 10.0
Glucose 20.0
Potassium phosphate 2.0
Sodium acetate 5.0
Ammonium citrate 2.0
Magnesium sulphate 0.2
Manganese sulphate 0.05
Tween 80 1.08
Agar No. 1 15.0
Method for reconstitutionWeigh 70 grams of powder and add 1 litre of deionised water. Allowto soak for 10 minutes, swirl to mix. Heat to dissolve and sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C and adjust pH ifthe acidified medium is required.
Appearance: Light amber, clear.
pH: 6.4 ± 0.2
Minimum Q.C. organisms: Lactobacillus acidophilus
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Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark. Capped containers – up to 1 month at 15-20˚C in the dark.
Inoculation: Surface, spread to cover surface, or use pour platetechnique.
Incubation: 25˚C microaerobically for 2-5 days.
Interpretation: Count all colonies exhibiting typical morphology.
Growth Characteristicscolony size shape &
organism (mm) surface colour
Lactobacillus 0.5-2.5 F.E.G. White Roughacidophilus
Lactobacillus sake 0.5-1.0 F.E.G. White
Streptococcus lactis 0.5-1.5 CV.E.G. White
Lactobacillus bulgaricus 1.0-1.3 CV.E. G. White
Referencesde Man, J. C., M. Rogosa and M. E. Sharpe, 1960. A medium for thecultivation of lactobacilli. J. Appl. Bacteriol. 23: 130-135.
M.R.S. Broth(de Man, Rogosa and Sharpe Broth)
LAB 94
DescriptionA medium for the cultivation and enumeration of Lactobacillus spp.this product has the same formulation as LAB 93 M.R.S. agar withthe omission of agar. The medium can be used for confirmatory testson organisms isolated on M.R.S. agar. The medium can also be usedfor enumeration by the Miles and Misra technique.
Formula g/litre
Mixed Peptones 10.0
Yeast Extract 5.0
Beef Extract 10.0
Glucose 20.0
Potassium phosphate 2.0
Sodium acetate 5.0
Magnesium sulphate 0.2
Manganese sulphate 0.05
Tween 80 1.08
Ammonium citrate 2.0
Method for reconstitutionWeigh 55 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then warm to completelydissolve solids. Dispense into suitable tubes or bottles then steriliseby autoclaving at 121˚C for 15 minutes.
Appearance: Light amber, clear.
pH: 6.4 ± 0.2
Minimum Q.C. organisms: Lactobacillus acidophilus
Storage of Prepared Medium: Capped containers – up to 3 monthsat 15-20˚C in the dark.
Inoculation: Either with suspect colonies from M.R.S. agar or withserial dilutions of test material.
Incubation: 25˚C microaerobically for 2-5 days.
Interpretation: For enumeration purposes count tubes showing signsof growth as positive.
Referencesde Man, J. C., Rogosa, M. and Sharpe, M. E. 1960. A medium for thecultivation of lactobacilli. J. Appl. Bacteriol. 23, 130-138.
MSRV(Semi-solid Rappaport Medium)
LAB 150
DescriptionMSRV was developed in 1986 by De Smedt, Bolderdijk and Rappoldas a rapid means of Salmonella detection. The medium, based uponRappaport Vassiliadis broth, is inoculated directly from the pre-enrichment medium, in the centre of the plate. Motile organismsspread from the centre in the semi-solid agar, but non-salmonellas areinhibited by the selective agents.
After overnight incubation the use of polyvalent salmonella antiseraor a latex kit can confirm the presence of a Salmonella. Alternatively,a paper disc wetted with polyvalent H antiserum can be placed 1/3 ofthe way from the edge of the dish, and will signal the presence of aSalmonella by inhibiting the mobility of the organism around thedisc.
Using this medium De Smedt and Bolderdijk have reported thepossibility of detecting Salmonella in 24hrs (1987)
Formula g/litre
Tryptone 2.3
Meat Peptone 2.3
Acid Hydrolysed Casein 4.7
Sodium chloride 7.3
Potassium dihydrogen phosphate 1.5
Magnesium chloride 10.9
Malachite green 0.037
Agar No. 1 2.5
Method for reconstitutionWeigh 31.5 grams of powder and disperse in 1 litre of deionisedwater. Soak for 10 minutes, swirl to mix and bring to the boil. Coolto 47˚C. and add 2 vials of X150 novobiocin supplement (10mg/vial).Mix well before dispensing.
Appearance: Turquoise/blue, clear, soft gel.
pH: 5.2 0.2
Minimum Q.C organisms: Salmonella typhimuriumNCIMB 50076E.coli (inhibition) NCIMB 50034
Storage of prepared medium: Plates – up to 7 days at 4˚C.
Inoculation: From pre-enrichment broth (6-24hrs) adding 0.1ml tothe centre of the plate.
Incubation: 37˚C. or 42 ±0.5˚C. for 18-24 hours. Keep lid uppermostat all times.
Interpretation: A spreading growth indicates a Salmonella may bepresent, substantiated if a disc with polyvalent H antiserum has beenadded and is inhibiting the zone. This should be confirmed bysubculturing from the edge of the mobility zone onto XLD andbrilliant green agar and performing biochemical and serological tests.Direct latex agglutination may be carried out from the edge of themobility zone.
ReferencesDe Smedt J.M. and Bolderdijk R.F. (1987): ‘One Day Detection ofSalmonella from Foods and Environmental Samples by MobilityEnrichment’. Fifth International Symposium on Rapid Methods andAutomation in Microbiology and Immunology, Florence 1987. BrixiaAcademic Press.
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De Smedt J.M. and Bolderdijk R.F., Rappold H. and LautenschlaegerD. Rapid Salmonella Detection in Foods in Mobility Enrichment ona Modified Semi-Solid Rappaport-Vassiliadis Medium. Journal ofFood Protection 49 510-514. 1986.
De Smedt J.M. and Bolderdijk R.F. Dynamics of SalmonellaIsolation with Modified Semi-Solid Rappaport-Vassiliadis Medium.Journal of Food Protection 50 658-661. 1987.
De Smedt J.M. and Bolderdijk R.F. Collaborative Study of theInternational Office of Cocoa. Chocolate and Sugar Confectionery onthe Use of Mobility Enrichment for Salmonella Detection in Cocoaand Chocolate. Journal of Food Protection 53 659-664. 1990.
Goossens H., Wauters G., De Boeck M., Janssens M., and Butzler J.P.Semi-solid selective mobility enrichment medium for isolation ofSalmonella from faecal specimens J. Clin. Microbiol 19 940-941.1984.
Mueller Hinton Agar IILAB 39
DescriptionA medium for antimicrobial sensitivity testing by the disc diffusionmethod. This medium, used in the technique of Bauer and Kirby, hasbeen adopted by the National Committee for Clinical LaboratoryStandards (NCCLS) in the USA as the definitive method forsusceptibility testing. The medium has a very low thymine andthymidine content, making it suitable for trimethoprim andsulphonamide testing, controlled to ensure correct zone sizes withaminoglycoside and tetracyline antibiotics. The medium wasoriginally formulated as a heat labile protein free medium for theisolation of pathogenic Neisseriaceae.
Formula g/litre
Beef Extract 2.0
Acid Hydrolysed Casein 17.5
Starch 1.5
Agar No. 1 17.0
Calcium ions 50-100mg/litre
Magnesium ions 20-35mg/litre
Method for reconstitutionWeigh 38 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise at 121˚C for15 minutes. Cool to 47˚C, mix well and pour plates.
Appearance: Straw coloured, clear gel.
pH: 7.3 ± 0.1
Minimum Q.C. organisms: E. coli ATCC 25922S. aureus (antibiotic sensitivityzones) ATCC 25923
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation: Surface, inoculum as described by N.C.C.L.S.
Incubation: As recommended by methodology for particularorganisms and antibiotics by NCCLS.
ReferencesMueller, J.H. and Hinton, J. 1941. Protein-free medium for primaryisolation of gonococcus and meningococcus. Proc. Soc. Exp. Biol.and Med., 48: 330-333.
Goodale, W.I., Gould G. and Schwab, L. 1943. LaboratoryIdentification of sulphonamide resistant gonococcic infection. J.Am.Med. Ass., 123: 547-549.
American Public Health Association. 1950. Diagnostic Proceduresand Reagents. 3rd edn., A.P.H.A., New York.
NCCLS. 1986. Performance standards for antimicrobialsusceptibility testing – second informational supplement.
Mueller Hinton Broth IILAB 114
DescriptionThis medium is the broth version of Mueller Hinton Agar. It is anantagonist free medium for use in the tube dilution technique for thedetermination of antibiotic M.I.C. values. The medium is carefullystandardised to meet N.C.C.L.S. standards for antimicrobialsusceptibility tests on bacteria which grow aerobically.
Formula g/litre
Beef Extract 2.0
Acid Hydrolysed Casein 17.5
Starch 1.5
Calcium ions 50 mg/litre
Magnesium ions 20 mg/litre
Method for reconstitutionWeigh 21 grams powder, disperse in 1 litre distilled water. Allow tosoak for 10 minutes, swirl to mix then heat gently to dissolve.Distribute into tubes or bottles, and sterilise at 121˚C for 10 minutes.
Appearance: Pale straw , clear.
pH: 7.4 ± 0.2
Minimum Q.C. organisms: S. aureus ATCC 25923E. coli ATCC 25922(M.I.C. values)
Storage of Prepared Medium: Capped containers – up to 3 monthsat 15-20˚C in the dark.
Inoculation: Standard inocula are required. As described by NCCLS.
Incubation: As recommended by methodology for particularorganisms and antibiotics by NCCLS.
ReferencesMacFaddin, J. 1985. Media for isolation cultivation, identificationmaintenance of medical bacteria. Williams & Williams, Baltimore.
N.C.C.L.S.-M7-A. 1985. Methods for dilution antimicrobiolsusceptibility tests for bacteria that grow aerobically. ApprovedStandard.
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Mueller Kauffman Tetrathionate BrothLAB 42
DescriptionA selective enrichment broth for salmonellae first described byMueller in 1923 then modified by Kauffman in 1935 with theaddition of Brilliant Green and Ox Bile increasing its selectivity.Organisms which can utilise tetrathionate, such as most Salmonella,flourish. However some salmonellas will be missed in this mediumeither because they are sensitive to Brilliant Green or cannot utilisetetrathionate (included in this group is S. typhi.) This medium is usedin the standard European Community Salmonella Isolation Procedureand in International Standards Organisation (ISO) Methods.
Formula g/litre
Tryptone 7.0
Soy Peptone 2.3
Sodium chloride 2.3
Calcium carbonate 25.0
Sodium thiosulphate anhydrous 40.7
Ox Bile 4.75
Method for reconstitutionWeigh 82 grams of powder, disperse in 1 litre of deionised water.Bring to the boil and cool to below 45˚C. Prior to use, add 30ml 1Niodine solution (or 19ml of a solution containing iodine 20g,potassium iodine 25g, distilled water 100ml) and 9.5ml of brilliantgreen 0.1% w/v solution. Mix well and dispense into sterilecontainers, keeping the chalk in suspension.
Appearance: Green, turbid solution which precipitates on standing.
pH: 7.8 ± 0.2
Minimum Q.C. organisms: Salmonella sp. NCIMB 50076E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Capped container – up to 7 days at2-8˚C in the dark.
Inoculation: 1 part sample to 9 parts medium or 1 part pre-enrichment medium to 10 parts medium.
Incubation: 43˚C aerobically for 24-48 hours.
Subculture: Subculture onto selective agars. e.g. B.G.A. LAB 34,XLD LAB 32, M.L.C.B. LAB 116.
ReferencesMueller. 1923. Un nouveau milieu d’enrichissment pour la recherchedu bacille typhique et des paratyphiques C.R. Soc. Biol (Paris) 89:434. 1975.
International Organisation for Standardisation. Meat and meatproducts. Detection of salmonellae (Reference method).
ISO 3565 (E). International Organisation for StandardisationMicrobiology-1981 General guidance on methods for the detection ofSalmonella. ISO 6579(E).
International Organisation for Standardisation. Milk and milkproducts- 1985. Detection of Salmonella. ISO 6785(E).
Edel, W. and E.H. Kampelmacher, 1968. Comparative studies onSalmonella isolations in eight European laboratories, Bull. Wld. Hlth.Org. 41: 297-306.
Kauffman, F., 1935. Weitere Erfahrungen mit dem kombiniertenAnreicherungsverfahren fur Salmonellabacillen. Z. Hyg. 117: 26-32.
van Leusden, F.M., M. van Schothorst and H.J. Beckers, 1982. Thestandard Salmonellae isolation method. In: Isolation andidentification methods for food poisoning organisms, edited by J.E.L.Corry, D. Roberts and F.A. Skinner. SAB Technical Series, No. 1735-49. Academic Press, London.
Nu Sens AgarLAB 74
DescriptionThis is a sensitivity test medium developed by LAB M in response torequests for a nutritionally rich sensitivity medium. This medium willgrow all organisms not having a specific need for blood, givingreproducible zone sizes. It is composed of specially selected peptoneswith a small amount of glucose, solidified with a very pure agar andis free from antagonists.
Formula g/litre
Acid Hydrolysed Casein 2.0
Peptone 7.5
Beef Extract 5.0
Sodium chloride 5.0
Glucose 2.0
Agar No. 1 15.0
Method for reconstitutionWeigh 36.5 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C and pour plates.
Appearance: Transparent, almost colourless.
pH: 7.4 ± 0.2
Minimum Q.C. organisms: S. aureus NCTC 6571E. coli NCTC 10418(antibiotic sensitivity zones)
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation method: Joan Stokes method, surface inoculum for semiconfluent growth, or breakpoint technique.
Incubation: 37˚C aerobically for 18-24 hours.
ReferencesEricsson, H. M., Sherris, J. C. 1971. Antibiotic sensitivity testing.Report of an international collaborative study. Acta. Pathol.Microbiol. Scand. Suppl. 217: 1-90.
Garrod, L. P. and Waterworth, P. M. 1969. Effect of mediumcomposition on the apparent sensitivity of Pseudomonas aeruginosato gentamicin. J. Clin. Pathol. 22: 534-538.
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Nutrient AgarLAB 8
DescriptionA general purpose medium for the cultivation of organisms that arenot demanding in their nutritional requirements e.g. organisms thatcan be isolated from air, water, dust etc. Nutrient Agar is suitable forteaching and demonstration purposes, it is isotonic and can beenriched with biological fluids such as sterile blood and egg yolk.
Formula g/litre
Peptone 5.0
Beef Extract 3.0
Sodium chloride 8.0
Agar No. 2 12.0
Method for reconstitutionWeigh 28 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving for 15 minutes at 121˚C. Cool to 47˚C, mix well thenpour plates.
Appearance: Buff, opalescent gel.
pH: 7.3 ± 0.2
Minimum Q.C. organisms: S. aureus NCIMB 50080E. coli NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark. Capped containers – up to 3 months at 15-20˚C in the dark.
Inoculation Method: Surface streaking for single colonies.
Incubation: Temperature and time to suit organisms. Usuallyaerobic.
Growth Characteristicscolony size shape &
organism (mm) surface colour other
S. aureus 1.0-2.0 CV.E.G. White-Yellow
other Staphylococcus 0.5-2.0 CV.E.G. White-spp. Yellow
Strep. pyogenes P.P.-0.5 CV.E.G. Transp.
E. coli 1.5-2.5 CV.E.G. Grey
Proteus spp. spreading - Grey fishy odour
Klebsiella spp. 2.0-4.0 CV.E.G. Grey mucoid
Bacillus spp. 2.0-6.0 various Grey may spread
Ps. aeruginosa 2.0-4.0 F.CR.D. Grey-Green odour ifpigmented
Nutrient Broth No. 2 B.P.LAB 14
DescriptionA general purpose broth which can be used for sterility testing foraerobic organisms as recommended by the British Pharmacopoeia.This broth can also be used as the suspending medium for cookedmeat granules for the cultivation of anaerobic organisms.
Formula g/litre
Beef Extract 10.0
Balanced Peptone No. 1 10.0
Sodium chloride 5.0
Method for reconstitutionWeigh 25 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then dispense into tubesor bottles, and sterilise for 15 minutes at 121˚C.
Appearance: Pale straw, clear.
pH: 7.3 ± 0.2
Minimum Q.C. organisms: S. aureus NCIMB 50080E. coli NCIMB 50034
Storage of Prepared Medium: Capped containers – up to 3 monthsat 15-20˚C in the dark.
Incubation: 37˚C for 18-24 hours aerobically.
ReferencesBritish Pharmacopoeia. 1973. H.M.S.O., London. Cruikshank, R.1972. Medical Microbiology. 11th edn. Livingstone, London.
Nutrient Broth “E”LAB 68
DescriptionAn inexpensive broth for the growth of nutritionally non-demandingorganisms. Ideal for teaching purposes.
Formula g/litre
Beef Extract 1.0
Yeast Extract 2.0
Peptone 5.0
Sodium chloride 5.0
Method for reconstitutionWeigh 13 grams of powder, add to1 litre of deionised water. Heat todissolve then dispense into bottles or tubes. Sterilise by autoclavingat 121˚C for 15 minutes.
Appearance: Straw coloured, clear.
pH: 7.4 ± 0.2
Minimum Q.C. organisms: S. aureus NCIMB 50080E. coli NCIMB 50034
Storage of Prepared Medium: Capped containers – up to 3 monthsat 15-20˚C in the dark.
Inoculation and incubation: To suit chosen organism.
Growth indicator: Turbidity.
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O157 Broth MTSB(Modified Tryptone Soy Broth)
LAB 165
DescriptionModified tryptone soy broth has emerged as the medium of choice forthe enrichment of E.coli O157:H7 in red meats1,2. As concernregarding this organism has grown due to the severity of the diseasesyndromes caused, and the increase in foodborne infection3, so too hasthe need to optimise methods for its efficient isolation. Symptomsstart with severe stomach cramps and watery, bloody diarrhoea, and apercentage of individuals infected will develop Haemolytic UraemicSyndrome (HUS) leading to acute renal failure4. In a comparison of 4different selective broth media, MTSB was the most productive andselective for the isolation of E.coli O157:H71. MTSB is madeselective for O157:H7 by including bile salts in the dehydratedmedium, and the addition of novobiocin supplement (X150).
Formulation g/litre
Tryptone 17.0
Sodium chloride 5.0
K2HPO4 4.0
Dextrose 2.5
Soy Peptone 3.0
Bile Salts No.3 1.5
pH: 7.2 ± 0.2Appearance: Clear straw broth
Method for reconstitutionWeigh 33 grams of powder and add to 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix and autoclave at 121˚C for15 minutes. Cool to 47˚C and add 2 vials of Novobiocin supplementX150. Mix well and distribute aseptically into sterile containers.
InoculationAdd 25g sample to 225ml of supplemented MTSB and homogenisefor 2 minutes.
Incubation42˚C aerobically for 24hrs. Subculture onto CT-SMAC (LAB161plus X161) and examine for non-sorbitol fermenting colonies. Someworkers1 recommend the use of an immunomagnetic separation stepafter 6hrs incubation.
InterpretationTurbidity in the broth indicates growth. All broths should besubcultured to selective media whether turbid or not.
Minimum QC organisms: E.coli O157:H7(non-toxigenic) NCTC 12900E.coli NCIMB 50034(inhibition)
References1) Bolton E.J., Crozier L., Williamson J.K. (1995) Optimisation ofmethods for the isolation of Escherichia coli O157 from beefburgers.PHLS Microbiology Digest 12 (2) 67-70.
2) Willshaw G.A., Smith H.R., Roberts D., Thirlwell J., Cheasty T.,Rowe B. (1993) Examination of raw beef products for the presenceof verocytotoxin producing Escherichia coli, particularly those ofserogroup O157. J.Appl.Bacteriol. 75 420-426.
3) Sharp J.C.M., Coia J.E., Curnow J., Reilly W.J. (1994) Escherichiacoli O157 infections in Scotland. J.Med.Microbiol 40 3-9.
4) Doyle M.P. (1991) Escherichia coli O157:H7 and itssignificancein foods. Int.J.Food Microbiol. 12 289-302.
Orange Serum AgarLAB 147
DescriptionA medium developed for the investigation of organisms involved inthe spoilage of citrus products including fruit juices and citrusconcentrates. The low pH of these products restricts the growth oforganisms to those capable of tolerating an acid environment such asyeasts and moulds and bacteria belonging to the genera Bacillus,Lactobacillus, Leuconostoc, Streptococcus and Clostridium. Byhaving a low pH and incorporating orange extract, Orange SerumAgar is the ideal isolation medium.
Formula g/litre
Tryptone 10.0
Yeast Extract 3.0
Orange Extract 5.0
Glucose 4.0
Di-potassium phosphate 3.0
Agar No.2 7.0
Method for reconstitutionWeigh 42 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes. Bring to the boil swirling frequently,cool to 47˚C. and dispense into containers. Sterilise by autoclaving at115˚C. for 15 minutes.
Appearance: Amber, slightly opalescent gel.
pH: 5.5 ± 0.2
Minimum Q.C. organisms L. acidophilusPen. roquefortii
Storage of Prepared Medium: Plates – up to 7 days at 4˚C. in thedark. Capped containers – up to 3 months at 15-20˚C. in the dark.
Inoculation: Pour plate technique.
Incubation: 3 days at 30˚C. for bacteria, 5 days at 30˚C for yeastsand moulds.
Interpretation: Count bacterial colonies and yeast/mouldsseparately. Calculate the colony forming units (C.F.U.’s) per ml of thesample allowing for dilution factors.
ReferencesHays G.L. (1951) The isolation, cultivation and identification oforganisms which have caused spoilage in frozen concentrated orangejuice. Proc. Florida State Hort. Soc.
Hays G.L. and Reister D.W. (1952) The control of ‘off-odour’spoilage in frozen concentrated orange juice. Food Tech 6 p386.
Murdock D.I., Folinazzo J.F., and Troy V.S. (1952) Evaluation ofplating media for citrus concentrates. Food Tech. 6 p181.
70
Oxytetracycline-Glucose-Yeast ExtractAgar Base(O.G.Y.E.)
LAB 89
DescriptionA selective medium for the enumeration of yeasts and moulds infood, introduced by Mossel in 1970. Unlike many selective media foryeasts O.G.Y.E. has a neutral pH and it has been shown to give betterrecovery rates than those media with a low pH. Oxytetracycline isused to inhibit bacteria, certain high protein foods may reduce theeffectiveness of this antibiotic as a selective agent. Rose BengalChloramphenicol LAB 36 is recommended in these instances.
Formula g/litre
Yeast Extract 5.0
Dextrose 20.0
Biotin 0.001
Agar No. 2 12.0
Method for reconstitutionWeigh 37 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 115˚C for 10 minutes. Cool to 47˚C and asepticallyadd two vials of X089 Oxytetracycline selective supplement. Mixthoroughly and pour into petri dishes.
Appearance: Pale yellow, clear.
pH: 7.0 ± 0.2
Minimum Q.C. organisms: Aspergillus sp. NCIMB 50097Saccharomyces cerevisiae
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in thedark. Capped containers – up to 1 month at 4˚C in the dark.
Inoculation: Surface spreading or pour plate.
Incubation: 25˚C aerobically for 5 days.
Growth Characteristicscolony size shape &
organism (mm) surface colour
Candida spp. 3.0-4.0 CV.E.D. Cream
Candida krusei 7.0-8.0 F.CR.D. White
S. cerevisiae 3.0-4.0 CV.E.D. White
Pen. notatum 1.5 Green/blue centre
Pen. flavescens 1.5-2.0 Yellow centre
Aspergillus niger 3.0-5.0 Yellow with black centre
Rhizopus spp. confluent Aerial hyphae withblack conidia
Tetracycline resistant bacteria may grow, colonial morphologydependent upon species.
ReferencesMossel, D. A. A. et al. 1970. O.G.Y.E. for Selective Enumeration ofMoulds and Yeast in Foods and Clinical Material. J. Appl. Bact. 35:454-457.
Banks, J. G., Board, R. G. 1987. Some factors influencing therecovery of yeasts and moulds from chilled foods. Int. J. FoodMicrobiol. 4: 197-206.
Palcam Agar Base(Polymixin, Acriflavine, Lithium chloride, Ceftazidine,Aesculin, Mannitol)
LAB 148DescriptionPalcam Agar was developed by Van Netten et al in 1989 as animproved selective differential medium for the isolation of Listeriamonocytogenes from food, clinical and environmental specimens.
Improved selectivity is achieved by the combination of antibioticsupplements and microaerobic incubation, whilst the double indicatorsystem of aesculin hydrolysis and mannitol fermentation aidsdifferentiation of Listeria spp from enterococci and staphylococciwhich can be confused with Listeria spp on other types of culturemedia.
Formula g/litre
Columbia Peptone Mix 23.0
Sodium chloride 5.0
Corn Starch 1.0
Yeast Extract 3.0
Glucose 0.5
Mannitol 10.0
Aesculin 0.8
Lithium chloride 15.0
Ferric ammonium citrate 0.5
Phenol red 0.08
Agar No. 2 12.0
Method for reconstitutionWeigh 71 grams of powder and disperse in 1 litre of deionised water.Soak for 10 minutes, swirl to mix then sterilise by autoclaving at121˚C. for 15 minutes. Cool to 47˚C and add 2 vials of P.A.C.Supplement – X144. Mix thoroughly before dispersing.
Appearance: Red, translucent
pH: 7.2 ± 0.2
Minimum Q.C. organisms L. monocytogenesNCIMB 50007E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 4˚C. in the dark.
Inoculation: 0.1ml of sample selectively enriched in Palcam Broth(or other enrichment medium) spread over surface of plate.
Incubation: 30˚C. aerobically or microaerobically for 24-48 hours.
Growth Characteristicscolony size shape &
organism (mm) Surface colour other
L. monocytogenes 1.5-2.0mm F.E.D. Grey/green Black halo,(draughtsman
colonies).
Other Listeria spp. 0.5-2.0mm F.E.D. Grey/green Black halo,
(draughtsmancolonies).
Enterococci Inhibited - - (Small yellowcolonies withyellow/green
halo).
Staphylococci Inhibited - - (Small white/yellow colonies
with yellow/green halo)
Bacillus spp. Inhibited - - -
71
ReferencesVan Netten P., Perales I., Curtis G.D.W., Mossel D.A.A. (1989)Liquid and solid selective differential media for the enumeration of L. Monocytogenes Int. J. Food Micro. 8 (4) 299-316.
Palcam Broth(Liquid, Polymixin, Acriflavine, Lithium chloride,Ceftazidime, Aesculin, Mannitol)
LAB 144
DescriptionDeveloped by Van Netten et al (1989) L-Palcam is a selectivedifferential medium for the enrichment of Listeria spp. in food,environmental and clinical samples. It is unique amongst Listeriaenrichment media in that it contains an indicator system (aesculin)which will signal the presence of a possible Listeria by abrowning/blackening of the broth; the result being the indication of apotential problem up to 48 hours before growth on plating media canbe observed.
Formula g/litre
Columbia Peptone Mix 23.0
Yeast Extract 5.0
Peptonised Milk 5.0
Sodium chloride 5.0
Mannitol 5.0
Aesculin 0.8
Ferric ammonium citrate 0.5
Phenol red 0.08
Lithium chloride 10.0
Method for ReconstitutionWeigh 54.4 grams of powder and disperse in 1 litre of deionisedwater. Soak for 10 minutes, swirl to mix and autoclave at 121˚C for15 minutes. Cool to 47˚C and add two vials of X144. Mix well anddispense into sterile tubes or bottles.
Appearance: Clear red broth
pH: 7.2 ± 0.2
Minimum Q.C. organisms: L. Monocytogenes NCIMB 50007E. coli (inhibition) NCIMB 50034
Storage of prepared medium: Capped containers – up to 7 days at 4˚C.
Inoculation: Sample or pre-enriched sample added to the broth in theratio 1 : 10.
Incubation: 30˚C for 24 hours and 48 hours.
Subculture: Onto Palcam Agar – LAB 148. If low numbers ofListeria are present the medium may not produce the brown blackcolour. All tubes should be subcultured onto selective agar before asample is scored as negative.
ReferencesVan Netten P., Perales I., Curtis G.D.W., Mossel D.A.A. (1989)Liquid and solid selective differential media for the enumeration of L. Monocytogenes Int. J. Food Micro. 8 (4) 299-316.
Peptone WaterLAB 104
DescriptionA general purpose growth medium that can be used as a base forcarbohydrate fermentation studies. The medium has a high level oftryptone making it suitable for use in the indole test. Adjustment ofthe pH to 8.4 makes the medium an enrichment broth for Vibrio spp.
Formula g/litre
Peptone 5.0
Tryptone 5.0
Sodium chloride 5.0
Method for reconstitutionWeigh 15 grams of powder, and disperse in 1 litre of deionised water.Allow to dissolve then distribute into final containers. Sterilise byautoclaving at 121˚C for 15 minutes. If sterile additions are to bemade to this medium e.g.: carbohydrates, the volume of water forreconstitution must be reduced accordingly. A pH indicator may beadded to detect acid production from carbohydrate utilisation.
Appearance: Clear, colourless.
pH: 7.2 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034
Storage of Prepared Medium: Capped containers – up to 3 monthsat 15-20˚C in the dark.
Inoculation: A light inoculum from a pure culture.
Incubation: According to organism.
ReferencesBergey’s Manual of Systematic Bacteriology, Vol. 1, 1984. Williamsand Wilkins, Baltimore/London.
MacFadden, J. F. 1983. Biochemical Tests for the Identification ofMedical Bacteria, 2nd edn. Williams and Wilkins, Baltimore/London.
Perfringens Agar (O.P.S.P.)
LAB 109
DescriptionThis medium was developed by Handford in 1974 to overcome someof the problems associated with enumerating Clostridium perfringensin foods. The medium is buffered and utilises sodium metabisulphiteand liver extract as sources of H2S with ferric ammonium citrate asthe indicator. The medium is made selective with the addition ofX109 Sulphadiazine and X110 Oleandomycin/Polymixinsupplements.
Formula g/litre
Tryptone 15.0
Yeast Extract 5.0
Soy Peptone 5.0
Liver Extract 7.0
Ferric ammonium citrate 1.0
Sodium metabisulphite 1.0
Tris buffer 1.5
Agar No. 2 10.0
72
Method for reconstitutionWeigh 45.5 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes then sterilise by autoclaving at 121˚Cfor 15 minutes. Allow to cool to 47˚C before adding 2 vials each ofselective supplements X109 and X110.
Appearance: Pale straw, clear.
pH: 7.3 ± 0.2
Minimum Q.C. organisms: C. perfringensE. coli (inhibition)
Storage of Prepared Medium: Use on day of preparation.
Inoculation: Pour plates, 1ml sample plus 9ml medium. When setoverlay with sterile medium.
Incubation: 37˚C anaerobically for 24 hours.
Interpretation: Count large black colonies, presumptively identifiedas C. perfringens.
ReferencesHandford, P. M. 1974. A new medium for the detection andenumeration of C. perfringens in foods. J. Appl. Bact. 37: 559-570.
Shahidi, S. A. and Ferguson, A. R. 1971. A new quantitative andconfirmatory medium for C. perfringens in food. Appl. Microbiol.21: 500-506.
Marshall, R. S., Steenberger, J. F. and McClung, L. S. 1965. Appl.Microbiol. 13: 559. A rapid technique for the enumeration of C.perfringens.
Pharmacopoeia of culture media for food microbiology. 1987. Int. J.Food Microbiol. 5: 3: 240-241.
Plate Count AgarLAB 149
DescriptionA medium designed for use with the spiral plating system and othersurface inoculation techniques. The formula is equivalent to A.P.H.A.Plate Count Agar and is suitable for the determination of total viablecounts in food products by surface count and pour plate methods.
Formula g/litre
Tryptone 5.0
Yeast Extract 2.5
Glucose 1.0
Agar No. 2 12.0
Method for reconstitutionWeigh 20.5 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C then pour intopetri dishes.
Appearance: Pale straw colour, clear.
pH: 7.0 ± 0.2
Minimum Q.C. organisms: S. epidermidis NCIMB 50082E. coli NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in thedark.
Inoculation: Surface, or pour plate.
Incubation: 30˚C aerobically for 48 hours for aerobic mesotrophcount. 6˚C aerobically for 10 days for aerobic psychrotroph count.55˚C aerobically for 48 hours for aerobic thermotroph count.
Interpretation: Count all colonies or use spiral plating colony countequipment.
ReferencesReasoner D.J., Geldreich E.E. (1985) A New Medium for theEnumeration and Subculture of Bacteria from potable water. App &Env. Microbiol. Jan. 1985 p.1-7.
American Public Health Association (1985) Standard Methods for theEnumeration of Water and Wastewater. 16th Edition. AmericanPublic Health Association Inc. Washington D.C.
Report on Public Health and Medical Subjects No. 71. (1994)Methods for the Examination of Waters and Associated Materials.HMSO.
Plate Count Agar (A.P.H.A.)(Standard Methods Agar, Tryptone Glucose Yeast Agar)
LAB 10
DescriptionFormulated to A.P.H.A. specifications, this medium is used forestablishing total viable counts for aerobes in food, dairy and waterbacteriology. The product uses agar of very high gel strength in orderthat it can be used in pour-plate as well as surface inoculationtechniques. The product can be remelted prior to use although itshould not be held for a prolonged period in the molten stage.
Formula g/litre
Tryptone 5.0
Yeast Extract 2.5
Glucose 1.0
Agar No. 1 15.0
Method for reconstitutionWeigh 23.5 grams of powder, disperse in 1 litre of deionised water.Bring to the boil with frequent stirring to dissolve. Dispense intotubes and sterilise by autoclaving at 121˚C for 15 minutes. Cool to44-46˚C for not more than 3 hours prior to use.
ROLL-TUBES. Add an additional 10g/litre Agar No. 1 prior toreconstitution of the medium.
Appearance: Pale straw coloured, clear gel.
pH: 7.0 ± 0.2
Minimum Q.C. organisms: S. epidermidis NCIMB 50082E. coli NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark. Capped containers – up to 3 months at 15-20˚C in the dark.
Inoculation method: Pour plate technique or surface inoculation.
Incubation: 30˚C aerobically for 48 hours for aerobic mesotrophcount. 6˚C aerobically for 10 days for aerobic psychrotroph count.55˚C aerobically for 48 hours for aerobic thermotroph count.
Interpretation: Count all colonies and calculate the number oforganisms (or ‘colony forming units’ c.f.u.) per ml of sampleallowing for dilution factors.
ReferencesAmerican Public Health Association 1972. Standard Methods for theExamination of Dairy Products. 13th edn. (ed. W. J. Hausler)A.P.H.A., Washington.
American Public Health Association 1966. Recommended Methodsfor the Microbiological Examination of Foods, 2nd edn. (ed. J. M.Sharf) A.P.H.A., Washington.
American Public Health Association 1976. Standard Methods for theExamination of Water and Waste Water, 14th edn. (ed. M. A. Franson)A.P.H.A., Washington.
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Potato Dextrose AgarLAB 98
DescriptionPotato Dextrose Agar is recommended by the American PublicHealth Association for the enumeration of yeasts and moulds inexamination of dairy products, soft drinks, dried and frozen foods andother types of product. Depending on whether the medium is to beused as a selective or non-selective agar it can be used with orwithout acidification.
Formula g/litre
Potato Extract 4.0
Dextrose 20.0
Agar No. 1 15.0
Method for reconstitutionWeigh 39 grams of powder, disperse in 1 litre of deionised water, thensterilise at 121˚C for 15 minutes. Mix well before pouring into sterilePetri dishes. In certain cases it may be desirable to lower the pH ofthe medium to 3.5 in order to suppress bacterial growth. This can bedone by adding 10ml of sterile 10% Lactic Acid X037, to one litre ofPotato Dextrose Agar LAB 98. This addition must be afterautoclaving and cooling to 47˚C. Once the pH has been lowered themedium may not be heated again without resultant loss of gelstrength caused by agar hydrolysis.
Appearance: Translucent white agar.
pH: 5.6 ± 0.2 (3.5-4.0 if X037 is added)
Minimum Q.C. organisms: Aspergillus sp. NCIMB 50097Saccharomyces cerevisiae
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark. Capped containers – up to 1 month at 15-20˚C in the dark.
Inoculum: Pour plate technique.
Incubation: 21˚C aerobically for 5 days.
Growth Characteristicscolony size shape &
organism (mm) surface colour
Candida spp. 2.0 C.V.E.D. White
Candida kruseii 2.0 F.Rz.D. Grey/White
Tryc. mentagrophytes 4.0 Fluffy YellowWhite obverse
Tryc. verrucusum 5.0 Fluffy YellowWhite obverse
Toro. glabrata 3.0 C.V.E.G. White
Asp. niger 4.0 Black spores Yellowcentre obverse
White surround
Pen. notatum 4.0 Green spores Greencentre
White surround obverse
ReferencesAssociation of Official Analytical Chemists (AOAC). BacteriologicalAnalytical Manual, 5th ed. 1978. Washington D.C. Hausler, W. J. (ed.).
Standard Methods for the Examination of Dairy Prod. 14th edn.,Washington D.C.: American Public Health Association, 1976.
Pseudomonas Agar Base (C.F.C./C.N. Agar)
LAB 108
DescriptionThe base medium is a modification of King’s medium A which usesmagnesium and potassium salts to enhance production of thepigments pyocyanin (green) and fluorescein (detected by U.V./bluelight). The medium is made selective for Pseudomonas aeruginosaby the addition of X107 C.N. supplement. Alternatively the mediumcan be made selective for Pseudomonas species generally by theaddition of X108 C.F.C. supplement.
Formula g/litre
Acid Hydrolysed Casein 10.0
Gelatin Peptone 16.0
Potassium sulphate 10.0
Magnesium chloride 1.4
Agar No. 2 11.0
Method for reconstitutionWeigh 48.4 grams of powder and disperse in 1 litre of deionisedwater. Add 10mls of glycerol. Sterilise by autoclaving at 121˚C for 15minutes. Allow the medium to cool to 47˚C then add the contents of2 vials of either X107 C.N. supplement or X108 C.F.C. supplement.Mix well and pour into petri dishes.
Appearance: Pale straw, opaque.
pH: 7.1 ± 0.2
Minimum Q.C. organisms: Ps. aeruginosa NCIMB 50067E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation: Surface, spread 0.1 to 0.5mls of sample over entiresurface.
Incubation: 25-30˚C aerobically for 48 hours.
Interpretation: Count all colonies as Pseudomonas species.Colonies that exhibit the pyocyanin and fluorescein pigments countas Ps. aeruginosa.
Growth Characteristicscolony size shape &
organism (mm) surface colour fluorescence
Ps. aeruginosa 2.0-3.0 CV.Cr.D. Green/Blue yes
Ps. fluorescens 2.0-3.0 CV.Cr.D. Yellow yes
Ps. fragi 1.0-3.0 CV.Cr.D. Grey no
Ps. maltophila 2.0-3.0 CV.Cr.G. Grey no
Ps. putida 2.0-3.0 F.Cr.D. Grey no
Ps. cepacia 2.0-3.0 F.Cr.D. Grey no
ReferencesBurton, M. O., Campbell, J. J. R. and Eagles, B. A. 1948. The mineralrequirement for pyocyanin production. Can. J. res. Sect. C. Bot. Sci.26:15.
King, E. O., Ward, M. K. and Raney, D. E. 1954. Two simple mediafor the demonstration of pyocyanin and fluorescein. J. Lab. Clin.Med. 44: 301.
Goto, S. and Enomoto, S. 1970. Jap. J. Microbiol. 14: 65-72.
Mead, G. C. and Adams, B. W. 1977. Br. Poult. Sci. 18: 661-667.
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R2A MediumLAB 163
DescriptionR2A medium was developed to determine the bacterial count inpotable waters during treatment and distribution, and has been shownto give significantly higher counts than plate count agar (PCA) orsimilar high-nutrient media. The standard plate count (SPC) methodusing PCA provides an enumeration of bacteria which grow best at,or near, body temperature and this estimation at best may correlate tothe coliforms present in the sample. However, there will be apopulation of heterotrophic bacteria which cannot grow at all underthe conditions of the SPC method or may grow so slowly that thecolonies fail to reach a size detectable to the eye in the 48-hincubation period. In order to enumerate this section of the bacterialpopulation in water, a medium of low nutritional content (R2A) andextended incubation times are required. R2A medium isrecommended in Report 71, Methods for the Examination of Watersand Associated Materials, and Standard Methods for the Enumerationof Water and Wastewater.
Formulation g/litre
Yeast Extract 0.5
Meat Peptone 0.5
Casamino acids 0.5
Glucose 0.5
Starch 0.5
Dipotassium hydrogen Phosphate 0.3
Magnesium sulphate 0.05
Sodium pyruvate 0.3
Agar No.2 15.0
pH: 7.2 + 0.2
Appearance: Clear opalescent gel
Method for reconstitutionWeigh 18 grams of powder and disperse in 1 litre of deionised water.Swirl to mix and sterilise at 121˚C for 15 minutes. (If required, bringto the boil to dissolve the agar, and pour into smaller volumes beforesterilising). Cool to 44-46˚C for not more than 3 hours before use.
Inoculation: Pour 15ml into a petri dish containing 1ml of sample,mix well and allow to set. Pour a further 10ml as an overlay and againallow to set. Alternatively it may be used as a spread plate,inoculating 0.1ml onto the plate and spreading over the entire surfaceof the medium. It can also be used with membrane filters if required.
Incubation: When plates have set, incubate at 22˚C for 5-7 days or30˚C for 3 days. Other incubation temperatures between 20˚C and28˚C may be used.
Interpretation: Count all colonies and report the number of bacteriain the original sample as the heterotrophic plate count
Minimum QC organisms: Pseudomonas fluorescens,Aeromonas hydrophila
ReferencesReasoner D.J., Geldreich E.E. (1985) A New Medium for theEnumeration and Subculture of Bacteria from potable water. App &Env. Microbiol. Jan. 1985 p. 1-7.
American Public Health Association (1985) Standard Methods for theEnumeration of Water and Wastewater. 16th Edition. AmericanPublic Health Association Inc. Washington DC.
Report on Public Health and Medical Subjects No. 71. (1994)Methods for the Examination of Waters and Associated Materials.HMSO.
Rappaport Vassiliadis (R.V.) SingleComponent Enrichment Broth
LAB 86
IntroductionRappaport Vassiliades Broth (R10 modification) was born out of along series of experiments carried out to determine the correct levelsof malachite green and magnesium chloride that would allowSalmonella to multiply freely yet still inhibit the other entericorganisms.
This formulation has been shown to be superior to MuellerKauffmann and Selenite Broth for the isolation of Salmonella frommeat products.
The development work carried out on the formulation shows that it isextremely efficient in detecting small numbers of Salmonella inheavily contaminated products. This formulation is very hygroscopicand will produce a slight exothermic reaction when mixed with water.
Formula g/litre
Soy Peptone 4.5
Sodium chloride 7.2
Potassium dihydrogen phosphate 1.26
Dipotassium hydrogen phosphate 0.18
Magnesium chloride anhydrous 13.58
Malachite green 0.033
Method for reconstitutionWeigh 26.8 grams powder, disperse in 1 litre of deionised water, swirlto mix, when dissolved dispense in 10ml. volumes in screw cappedbottles and sterilise by autoclaving at 115˚C for 15 minutes.
Appearance: Clear, green fluid.
pH: 5.2 ± 0.2
Minimum Q.C. organisms: E. coli (inhibited) NCIMB 50034S. enterididis NCIMB 50073
Storage of Prepared Medium: Capped container – 6 months at 2-8˚C
Inoculation: From pre-enrichment broth in the proportions of 1-partinoculum to 99 parts R.V. Broth. Sub-culture onto either XLD Agar,M.L.C.B. Agar or other salmonella selective agars.
Incubation: 41.5 ± 0.5˚C for 24 hours (incubator) or 42 ± 0.1˚C for24hrs (water bath).
ReferencesVassiliades P., 1983 The Rappaport Vassiliades (R.V.) EnrichmentMedium for the Isolation of salmonellas: An overview J. Appl.Bacteriol. 56 69-76.
Vassiliades P., Mavromatti CH. Efstratiou, M. and Chronas G. 1985.A note on the stability of Rappaport-Vassiliades Enrichment MediumJ. Appl. Bacteriol. 59 143-145.
Bolton F.G. Preston P.H.L. Personal communication.
Int. J. Food Micro. Pharmacopoeia of culture media for FoodMicrobiology.
Peterz M., Wiberg C., and Norberg P. 1989. The effect of incubationtemperature and magnesium chloride concentration on growth ofSalmonella in home-made and in commercially available dehydratedRappaport-Vassiliadis broths.
75
Reinforced Clostridial AgarLAB 23
DescriptionThis is a solidified version of R.C.M. (LAB 22) and can be used forthe enumeration of anaerobes by pour plate, shake tube or membranefiltration methods. When solidified in tubes or bottles with minimalhead space it can be used for anaerobic culture without the need foranaerobic atmosphere.
Formula g/litre
Yeast Extract 3.0
Beef Extract 10.0
Peptone 10.0
Glucose 5.0
Soluble Starch 1.0
Sodium chloride 5.0
Sodium acetate 3.0
L-Cysteine hydrochloride 0.5
Agar No. 2 12.0
Method for reconstitutionWeigh 49.5 grams of powder, disperse in 1 litre of deionised water,allow to soak for 10 minutes, swirl to mix then sterilise for 15minutes at 121˚C. Cool to 47˚C. and distribute into sterile dishes ortubes containing decimal dilutions of the sample under test.
Appearance: Pale straw, translucent gel.
pH: 6.8 ± 0.2
Minimum Q.C. organisms: C. perfringens NCIMB 50027
Storage of Prepared Medium: Capped container – up to 3 monthsat 15-20˚C in the dark.
Inoculation: Pour plate technique or tube culture.
Incubation: 30˚C for up to 72 hours. Anaerobic conditions for pourplate. Count as early as possible as prolonged incubation may resultin the medium being disrupted due to gas production.
Interpretation: Count all colonies as presumptive clostridia.
ReferencesMiller, N. J., Garrett, O. W. and Prickett, P. S. 1939. Anaerobictechnique – a modified deep agar shake. Food Res. 4: 447-451.Ingram, M. and Barnes, E. M. 1956. A simple modification of thedeep shake tube for counting anaerobic bacteria. Lab. Pract. 5: 145.
Reinforced Clostridial Medium (R.C.M.)
LAB 22
DescriptionThis medium was formulated by Hirsch and Grinstead to recoversmall numbers of Clostridium spp. from a variety of sources. Variousworkers have reported its efficiency with many products andspecimens, R.C.M. is rich, non-selective and uses cysteinehydrochloride and glucose as reducing agents. The small amount ofagar reduces diffusion of oxygen through the fluid.
Formula g/litre
Yeast Extract 3.0
Beef Extract 10.0
Peptone 10.0
Soluble Starch 1.0
Glucose 5.0
L-Cysteine hydrochloride 0.5
Sodium chloride 5.0
Sodium acetate 3.0
Agar No. 1 0.75
Method for reconstitutionWeigh 38 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then bring to the boil todissolve. Distribute 25ml into 1oz Universal containers. Sterilise for15 minutes at 121˚C.
Appearance: Straw coloured, clear.
pH: 6.8 ± 0.2
Minimum Q.C. organisms: C. perfringens NCIMB 50027
Storage of Prepared Medium: Capped containers – up to 3 monthsat 15-20˚C in the dark.
Inoculation: Homogenised food sample to a ratio of 1:10 with R.C.M.
Incubation: 30˚C for up to 72 hours.
Growth IndicatorsTurbidity, colonies in medium, gas production.
ReferencesHirsch, A. and Grinstead, E. 1954. Methods for the growth andenumeration of anaerobic spore-formers from cheese. J. Dairy Res.21: 101-110.
Barnes, E. M. and Goldberg, H.S. 1962. The isolation of anaerobicGram-negative bacteria from poultry. J. Appl. Bact. 25: 94-106.
76
Ringers Solution (1/4 strength)LAB 100 LAB 100Z (TABLETS)
DescriptionAn osmotically controlled solution for the preparation of suspensionsof food samples and for use as a diluent in dilution techniques forbacterial enumeration. The solution can also be used in the samplingof food production apparatus by the rinse and swab method.
Formula g/litre
Sodium chloride 2.25
Potassium chloride 0.105
Calcium chloride 0.12
Sodium bicarbonate 0.05
Method for reconstitutionLAB 100 – Dissolve 2.5 grams of powder in 1 litre of deionisedwater, when completely dissolved dispense into containers asrequired and sterilise by autoclaving at 121˚C for 15 minutes.
LAB 100Z – Dissolve 1 tablet in 500mls of deionised water. Proceed as for LAB 100.
Ringers Solution (Calgon)LAB 101
DescriptionFor use with calcium alginate swabs. The sodium hexametaphosphatein the formula will enable the alginate swabs to completely dissolve.This ensures total release of all the organisms taken up by the swabto increase the accuracy of a quantitative bacterial count.
Formula g/litre
Sodium chloride 2.25
Potassium chloride 0.105
Calcium chloride 0.12
Sodium bicarbonate 0.05
‘Calgon’ (Sodium hexametaphosphate) 10.0
Method for reconstitutionDissolve 12.5 grams of powder in 1 litre of deionised water. Dispense10ml amounts into screw cap containers and sterilise by autoclavingat 121˚C for 15 minutes.
Ringers Solution (Thiosulphate)LAB 102
DescriptionAn isotonic rinse for use in hygiene studies on plant and equipmentthat have been treated with chlorine disinfectants. 100ml of LAB MThiosulphate Ringer Solution will neutralise 7mg of chlorine.
Formula g/litre
Sodium chloride 2.15
Potassium chloride 0.075
Calcium chloride 0.12
Sodium thiosulphate 0.5
Method for reconstitutionDissolve 2.8 grams of powder in 1 litre of deionised water. Dispenseinto containers as required then sterilise by autoclaving at 121˚C for15 minutes.
ReferencesMin. of Health, 1937. Bacteriological Tests for Graded Milk. Memo139/Foods. H.M.S.O. London. Davis, J. G. 1956. Laboratory Controlof Dairy Plant. Dairy Industries, London. Higgins, M. 1950.Comparison of recovery rate of organisms from cotton wool andcalcium alginate wool swabs. Mon. Bull. Min. Health, P.H.L.S., 9:50-51.
Rose Bengal Chloramphenicol Agar Base
LAB 36
DescriptionA selective medium for the enumeration of moulds and yeasts infoods. The original formulation of Jarvis (1973) usedchlortetracycline, this has been substituted by chloramphenicolbecause of superior selectivity. The Rose Bengal dye is taken up bythe growing colonies making them easier to see and inhibiting theirspreading. Rose Bengal becomes increasingly toxic on exposure tolight so it is important to store plates in the dark.
Formula g/litre
Mycological Peptone 5.0
Dextrose 10.0
Dipotassium phosphate 1.0
Magnesium sulphate 0.5
Rose bengal 0.05
Agar No. 2 12.0
Method for reconstitutionWeigh 28.5 grams of powder, disperse in 1 litre of deionised water,allow to soak for 10 minutes, swirl to mix then sterilise for 15minutes at 121˚C. Allow to cool to 47˚C then add 2 vials of X009.Chloramphenicol (or 2 vials X089 oxytetracycline). Mix well, thenpour into petri dishes. This medium should be protected from light.Chloramphenicol may be added before autoclaving.
Appearance: Deep purple/red, clear.
pH: 7.2 ± 0.2
Minimum Q.C. organisms: Aspergillus sp. NCIMB 50097Sacchromyces cerevisiae E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark. Capped Container – up to 1 month at 2-8˚C in the dark.
Inoculation: Surface spreading.
Incubation: 25˚C aerobically for 24 hours to 5 days.
Growth Characteristicscolony size shape &
organism (mm) surface colour other
Rhizopus spp. 13.5 Fluffy White
Mucor spp. 12.5 Fluffy White
Fusarium spp. 10 Fluffy White
Aspergillus flavus 8 Flat & Yellow/ hyphaehyphae Green
Candida spp. 1.5 CV.E.G. White
Sacchromyces spp. 1.0 CV.E.G. White
E. coli no growth
Ent. faecalis no growth
B. subtilis no growth
Staphylococcus spp. no growth
77
ReferencesJarvis, B, 1973. Comparison of an improvised Rose-BengalChlortetracycline Agar with other media for the selective isolationand enumeration of moulds and yeasts in food. J. Appl. Bact. 36: 723-727.
Overcast, W.W. and Weakley, D.L. 1969. An aureomycin rose-bengalagar for the enumeration of yeast and mould in cottage cheese. J.Milkand Fd.Tech. 32: 442-445.
Banks, J. G. Board, R. G. 1987. Some factors influencing therecovery of yeasts and moulds from chilled foods. Int. J. FoodMicrobiol. 4: 197-206.
Sabouraud Dextrose AgarLAB 9
DescriptionIntroduced by Sabouraud in 1910 as a selective medium for fungi andyeasts. The acidic pH (5.6) of this medium inhibits many species ofbacteria. The medium can be made more selective by the addition ofchloramphenicol supplement (X009). Diagnostic features, such assporing structures and pigmentation are well developed on thismedium. Because of its low pH this medium is very sensitive tooverheating which will soften the agar and caramelise thecarbohydrate.
Formula g/litre
Balanced Peptone No. 1 10.0
Dextrose 40.0
Agar No. 2 12.0
Method for reconstitutionWeigh 62 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving for 15 minutes at 121˚C. NOTE: The gel strength of themedium may diminish if recommended sterilising time ortemperature is exceeded.
Cool to 47˚C, mix well then pour plates.
Appearance: Buff opalescent gel.
pH: 5.6 ± 0.2
Minimum Q.C. organisms: Candida sp. NCIMB 50010E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark. Capped container – up to 3 months at 15-20˚C in the dark.
Inoculation method: Surface streaking for single colonies or stabmethod.
Incubation: Aerobically, yeasts 37˚C for 48 hours; fungi 25-30˚C forup to 3 weeks.
Growth Characteristicscolony size shape &
organism (mm) surface colour other
C. albicans 0.5-2.0 CV.E.D. White Yeasty smell
C. krusei 1.0-3.0 F.CR.D. Grey- Yeasty smellWhite
T. rubrum 25 White- Reverse-fluffy shades of
red
T. mentagraphytes 25 White- Reverse-fluffy yellow-
orange
M. canis 25 White- reverse-centre- yellow-yellowradial
E. floccosum 25 White- Reverse-fluffy tan
ReferencesSabouraud, R. 1910. Les Teignes Paris. Pagano. J., Levin, J. D. andTrejo, W. 1957-8. Diagnostic medium for the differentiation ofspecies of Candida. Antibiotics Annual, 137-143.
Sabouraud Liquid Medium U.S.P.(Fluid Sabouraud Medium)
LAB 33
DescriptionA liquid sterility test medium for the detection of yeasts, moulds andacidophilic bacteria in pharmaceutical products. This mediumconforms with the United States Pharmacopeia. It can also be used asa growth medium for the determination of fungistatic activity inpharmaceutical products.
Formula g/litre
Pancreatic digest of casein 5.0
Peptic digest of fresh meat 5.0
Glucose 20.0
Method for reconstitutionWeigh 30 grams of powder, disperse in 1 litre of deionised water,allow to soak for 10 minutes, swirl to mix. Heat to dissolve, thendispense into final containers before sterilising at 121˚C for 15minutes.
Appearance: Pale straw clear
pH: 5.7 ± 0.2
Minimum Q.C. organisms: Candida sp. NCIMB 50010E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Capped containers – up to 3 monthsat 15-20˚C in the dark.
Inoculation: As recommended in the U.S.P.
Incubation: 22-25˚C aerobically for 10 days.
ReferencesThe Pharmacopeia of the United States of America. XVII 1965.
Sabouraud Maltose AgarLAB 111
DescriptionThis is a modification of Sabouraud Dextrose Agar, substitutingmaltose for dextrose, recommended by the American Public HealthAssociation.
Formula g/litre
Balanced Peptone 10.0
Maltose 40.0
Agar No. 2 12.0
Method for reconstitutionWeigh 62 grams of powder, disperse in 1 litre of deionised water,allow to soak for 10 minutes, swirl to mix then autoclave at 121˚C for15 minutes. Do not overheat or the agar gel will be softened and thecarbohydrate will be caramelised. This medium may be madeselective by the addition of 2 ampoules X009 Chloramphenicolselective supplement which may be added either before or afterautoclaving. Cool to 47˚C and mix well before pouring plates.
Appearance: Cream/yellow, translucent.
pH: 5.6 ± 0.2
Minimum Q.C. organisms: Candida sp. NCIMB 5001078
E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark. Capped containers – up to 3 months at 15-20˚C in the dark.
Inoculation: Surface streaking or stab inoculum.
Incubation: Aerobic; yeasts 37˚C for 48 hours; other fungi 25˚C forup to 3 weeks.
Growth Characteristicscolony size shape &
organism (mm) surface colour other
C. albicans 0.5-2.0 CV.E.D. White Yeasty smell
C. krusii 1.0-3.0 F.CR.D. Grey- Yeasty smellWhite
T. rubrum 25mm White- Reverse-fluffy shades of
Red
T.mentagraphytes 25mm White- Reverse-fluffy yellow-
orange
M. canis 25mm White- Reverse-centre yellow-yellow orangeradial
E. floccosum 25mm White- Reverse-fluffy tan
ReferencesSabouraud R. 1910. Les Teignes. Paris. Pagano. J., Levin. J. D. andTrejo, W. 1957-8 Diagnostic medium for the differentiation of speciesof Candida. Antibiotics Annual. 137-143.
Salt Meat Broth TabletsLAB 113Z
DescriptionAn enrichment medium for the isolation of staphylococci fromheavily contaminated samples. It can also be used for the isolation ofhalophilic micrococci which contaminate hides and raw salt.
Formula g/litre
Beef Extract 10.0
Meat Peptone 5.0
Tryptone 5.0
Cooked Meat Granules 30.0
Sodium Chloride 100.0
Method for reconstitutionAdd 2 tablets to 10ml of deionised water in a narrow container. Allowto soak for 15 minutes then sterilise by autoclaving at 121˚C for 15minutes.
Appearance: Straw broth over meat particles
pH: 7.6 ± 0.2
Minimum Q.C. organisms: S. aureus NCIMB 50080E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Capped containers – up to 3 monthsat 15-20˚C in the dark.
Inoculation: Up to 1g of sample to 10ml of medium.
Incubation: 37˚C aerobically. Subculture at 24 and 48 hours.
Subculture: On to Baird Parker Medium LAB 85, Mannitol SaltAgar LAB 7 or 4S agar LAB 84.
Selenite BrothLAB 44A & LAB 44B
DescriptionA medium for the selective enrichment of salmonellae from faeces,food and sewage. First described by Leifson in 1936 the medium is apeptone lactose broth, moderately buffered, which utilises sodiumbiselenite as a selective agent. This medium can be incubated atvarious temperatures from 35-43˚C to vary the selectivity.Subcultures should be performed after no more than 24 hoursincubation as there is an increasing loss of selectivity if incubation isprolonged.
Formula g/litre
Selenite Broth Base LAB 44A:
Peptone 5.0
Lactose 4.0
Sodium phosphate 10.0
Sodium biselenite LAB 44B:
Sodium hydrogen selenite 4.0
Method for reconstitutionDissolve 4 grams of sodium biselenite in 1 litre of cold deionisedwaer. Add 19 grams of Selenite Broth Base and warm to dissolve.Distribute into tubes or bottles and sterilise for 5-10 minutes in aboiling water bath, or by free steaming. DO NOT AUTOCLAVE.
Appearance: Pale orange/red with slight precipitate (overheatingwill cause excessive precipitate and loss of selectivity).
pH: 7.1 ± 0.2 (complete medium)
Minimum Q.C. organisms: Salmonella sp. NCIMB 50076E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Capped containers – up to 3 monthsat 15-20˚C in the dark.
Inoculation: Approximately 0.5-1 gram of sample per 10ml tube.
Incubation: Up to 24 hours aerobically at 35-43˚C.
Subculture: Onto two or more selective agars.
ReferencesLeifson, E. 1936. New selenite enrichment media for isolation oftyphoid and paratyphoid (Salmonella) bacilli. Amer. J.Hyg. 24: 423-432.
MacFaddin, J.F. 1985. Media for the isolation, cultivation,identification of Medical Bacteria Vol 1. Williams and Wilkins,Baltimore.
79
WARNING: SODIUM BISELENITE
Toxic by inhalation and ingestion.Danger of cumulative effects. Causessevere burns. After contact with skinwash with water immediately. If youfeel unwell seek medical advice.
Selenite Cystine BrothLAB 55A & 44B
DescriptionThis formulation is a result of the investigation of North and Bartramin 1953. They examined the effect of varying concentrations ofcystine and phosphate on the recovery of salmonellae in egg productsusing selenite broth. It was found that the addition of 10micrograms/ml of cystine to Leifson’s selenite broth enhancedrecovery of salmonellae.
Formula g/litre
Selenite Cystine Broth Base LAB 55A
Balanced Peptone No. 1 5.0
Lactose 4.0
Sodium phosphate 10.0
L-Cystine 0.01
Sodium biselenite LAB 44B
Sodium hydrogen selenite 4.0
Method for reconstitutionDissolve 4 grams of Sodium biselenite (LAB 44B) in 1 litre ofdeionised water. Add 19 grams of Selenite Cystine Broth Base andheat to dissolve. Distribute into tubes or bottles, and sterilise for 10minutes in a boiling water bath, or steamer. DO NOT AUTOCLAVETHIS MEDIUM.
Appearance: Pale straw colour, clear with slight precipitate. (A brickred precipitate indicates overheating).
pH: 7.0 ± 0.2
Minimum Q.C. organisms: Salmonella sp. NCIMB 50076E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Capped containers – up to 3 monthsat 15-20˚C in the dark.
Inoculation: Add sample to broth in the ratio of 1:10. Use a pre-enrichment broth if damaged organisms are to be recovered.
Incubation: 37˚C for 24-48 hours aerobically. Subculture ontoSalmonella selective media.
ReferencesInternational Organisation for Standardization. Microbiology 1981.General guidance on methods for the detection of Salmonella. ISO, 6579-1981.
International Organization for Standardization. Milk and milkproducts 1985. Detection of Salmonella. ISO 6785-2985 (E). ICMSF,1978. Micro-organisms in foods. 1. Their significance and methodsof enumeration, 2nd edn., University of Toronto Press, Toronto, Ont.
Leifson, E., 1936. New selenite enrichment media for the isolation oftyphoid and paratyphoid (Salmonella) bacilli. Am, J. Hyg. 24, 423-432.
North, W. R. and M. T. Bartram, 1953. The efficiency of selenitebroth of different compositions in the isolation of Salmonella. Appl.Microbiol. 1, 130, 134.
Speck, M. L. 1984. Compendium of methods for the microbiologicalexamination of foods, 2nd edn., American Public Health Association.
Sensitivity Test Agar (S.T.A.)
LAB 12
DescriptionA medium formulated for antibiotic susceptibility testing by the JoanStokes technique. S.T.A. is inhibitor-free, very rich and includesvarious nucleotides to enable fastidious organisms to be tested. It isnecessary to add lysed or ‘chocolated’ blood for some organisms.
Formula g/litre
Peptone-Infusion Solids 21.5
Starch 0.6
Sodium chloride 5.0
Disodium citrate 1.0
Adenine sulphate 0.01
Guanine hydrochloride 0.01
Uracil 0.01
Xanthine 0.01
Aneurine hydrochloride 0.01
Uridine 0.1
Agar No. 2 12.0
Method for reconstitutionWeigh 40 grams of powder, disperse in 1 litre of deionised water,allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving for 15 minutes at 121˚C. To prepare blood agar cool to45˚C and add 7% lysed blood or 6% defibrinated blood according topreference. Mix well then pour plates.
Appearance: Dependent upon the blood additive.
pH: 7.4 ± 0.2
Minimum Q.C. organisms: S. aureus NCTC 6571E. coli NCTC 10418(antibiotic sensitivity zones)
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation method: Surface, according to technique.
Incubation: 37˚C, atmosphere to suit organisms metabollicrequirements.
Interpretation: There are no defined zone sizes as in MuellerHinton, but all antibiotics should give adequate zone sizes whencompared to controls using standard organisms, e.g. S. aureus NCTC6571, E. coli NCTC 10418, Ps. aeruginosa NCTC 10662.
ReferencesStokes, E. J. (1968). Clinical Bacteriology 3rd edn. Arnold, London.Committee of the A.C.P. 1965. Report on the Antibiotic Sensitivitytest trial organised by the bacteriology committee of the Associationof Clinical Pathologists. J.Clin. Pathol., 18: 1-5.
Hanus, F. J. Sands, J. G. and Bennett, E. O. 1967. Antibiotic activityin the presence of agar. Appl. Microbiol., 15: 31-34.
Bechtle, R. M. and Scherr. G. H. 1958. A new agar for in vitroantimicrobial sensitivity testing. Antibiot. Chemother., 8: 599-606.
80
WARNING: SODIUM BISELENITE
Toxic by inhalation and ingestion.Danger of cumulative effects. Causessevere burns. After contact with skinwash with water immediately. If youfeel unwell seek medical advice.
Simmons Citrate AgarLAB 69
DescriptionA medium devised by Simmons in 1926 to help in the differentiationof enteric bacteria and in the isolation of fungi. CertainEnterobacteriacae have the ability to utilize citrate as the sole sourceof carbon and utilize inorganic ammonium salts as the sole source ofnitrogen resulting in an increase in alkalinity. Bromothymol Blue isused as a pH indicator.
Formula g/litre
Magnesium sulphate 0.2
Ammonium dihydrogen phosphate 1.0
Dipotassium phosphate 1.0
Sodium citrate 2.0
Sodium chloride 5.0
Bromothymol blue 0.08
Agar No. 2 15.0
Method for reconstitutionWeigh 24 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then heat to dissolve theagar and solids. Dispense into tubes or bottles then sterilise byautoclaving at 121˚C for 15 minutes. Allow to set as slopes.
Appearance: Green, opalescent.
pH: 6.9 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034C. freundii
Storage of Prepared Medium: Capped containers – up to 3 monthsat 15-20˚C in the dark.
Inoculation: Streak on surface and stab into the butt.
Incubation: 37˚C aerobically for 24-48 hours, with loose caps toallow gaseous exchange.
Interpretation: Utilisation of citrate and ammonium salts results ingrowth and a change in colour of the medium from green to blue.
Growth Characteristicsorganism growth colour of medium
(most) Salmonella spp yes blue
S. typhi no green
S. cholerasuis no green
S. gallinarum no green
S. paratyphi A no green
Serratia spp. yes blue
Shigella spp. no green
Yersinia spp. no green
Klebsiella spp. yes blue
Proteus mirabilis yes blue
P. vulgaris yes blue
Providencia rettgeri no green
P. providenciae no green
E. coli no green
C. freundii yes blue
ReferencesSimmons, J. S. 1926. A Culture medium for differentiating organismsof typhoid – colon aerogenes groups and for isolation of certain fungi.J.Inf. Dis. 39: 209-215.
Koser, S. A. 1923. Utilisation of the salts of organic Acids by theColon-aerogenes group. J. Bact. 8: 493-520. MacFaddin, J. F. 1983.Biochemical Tests for Identification of Medical Bacteria. Williamsand Wilkins.
Single Step Staphyloccocus SelectiveAgar (4-S medium)
LAB 84
DescriptionThis staphylococcal isolation and identification medium is based onthe ability of S. aureus to grow and produce lecithinase in thepresence of a high sodium chloride concentration and potassiumtellurite at 42˚C. The high temperature inhibits the production oflipase (which causes clearing of egg protein) allowing the effect oflecithinase (which causes opacity of egg protein) to be more easilydetected. For the recovery of heat stressed staphylococci a 3 hourpreincubation in Brain Heart Infusion Broth LAB 49 at 37˚C isrecommended.
Formula g/litre
Tryptone 4.0
Yeast Extract 3.0
Dextrose 10.0
Sodium chloride 50.0
Agar No. 2 13.0
Method for reconstitutionWeigh 80 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Cool to 48˚C and addaseptically 30mls X073 egg yolk emulsion and 1ml X027 3.5%potassium tellurite solution. Mix well then pour plates.
Appearance: Pale cream, opaque.
pH: 6.7 ± 0.2
Minimum Q.C. organisms: S. aureus NCIMB 50080S. epidermidis NCIMB 50082
Storage of Prepared Medium: Capped container – up to 3 monthsat 15-20˚C in the dark.
Inoculation: Surface spreading or streak out to single colonies.
Incubation: 42˚C aerobically for 48 hours.
Growth Characteristicscolony size shape &
organism (mm) surface colour other
S. aureus 0.5-1.0 CV.E.G. Grey (Opaque zone ofppt around colony)
S. epidermidis 0.5 CV.E.G. White/ (No ppt zone)grey
S. faecalis P.P. CV.E.G. Grey (Usually nogrowth)
Most other organisms are inhibited by this medium at 42˚C.
ReferencesMintzer-Morgenstern, L. and Katzenelson, E. 1982. A SimpleMedium for Isolation of Coagulase-Positive Staphylococci in aSingle Step. J. Food Protect. 45:3:218-22.
81
Slanetz and Bartley Medium(Membrane Enterococcus Agar)
LAB166
DescriptionThis medium was originally described by Slanetz and Bartley for theenumeration of enterococci from water samples using a membranefiltration technique, but it may also be used as a spread plate for theexamination of other sample types. Enterococci reduce tetrazoliumchloride to the insoluble red dye formazan, producing colonies whichare dark red or maroon on the surface of the membrane or agar. Thisreaction is not exclusive to enterococci, and the count at this stageshould be considered presumptive. Colonies may be confirmed asenterococci by demonstrating aesculin hydrolysis using KanamycinAesculin Azide Agar LAB106
Formula g/litre
Tryptone 20.0
Yeast Extract 5.0
Glucose 2.0
Dipotassium hydrogen phosphate 4.0
Sodium azide 0.4
2,3,4 Tetrazolium chloride 0.1
Agar 12.0
Method for reconstitutionWeigh 43.5 grams of powder and mix with 1 litre of deionised water.Bring to the boil with frequent stirring to dissolve completely. Coolto 47˚C and pour into sterile petri dishes. DO NOT AUTOCLAVE,OVERHEAT, OR LEAVE FOR GREATER THAN 4hr AT 47˚C.
Appearance: Rose coloured gel.
pH: 7.2 ± 0.2
Mininmum QC organisms: Enterococcus faecalisNCIMB 50030 Escherichia coli NCIMB
50034.
Storage: Plates – upto 7 days at 2-8˚C. Storage in bottles is notrecommended as re-melting the medium will cause damage.
InoculationWater: Filter 100ml of the water through a suitable membrane, andplace this on the surface of a properly dried Slanetz and Bartley plate.
Other samples: Dilute as necessary and spread 0.5ml over the surfaceof the plate using a spreader, and allow to soak into the agar.
IncubationWater: at 37˚C for 48hr if testing potable waters or processed foods.At 37˚C for 4hr then 44˚C for 44hr if testing untreated waters or rawmaterials.
InterpretationCount all red and maroon colonies as presumptive enterococci.Confirmation of isolates can be achieved by demonstration of apositive aesculin reaction on KAAA LAB106.
Reference:Slanetz L.W., and Bartley C.H. (1957) J.Bact. 74 591-595
Report on Public health and Medical Subjects No.71 Methods for theExamination of Waters and Associated Materials. The Microbiologyof Water 1994, Part 1 – Drinking Water. HMSO 1SBN 011 753010 7.
Sorbitol MacConkey Agar(SMAC, CT-SMAC)
LAB 161DescriptionThis is a selective differential medium for the isolation ofEscherichia coli 0157:H7, the primary serovar associated withhaemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS).Pathogenicity of the organism is linked to the production ofverocytotoxins (VT1 and VT2), but it should be noted that not allstrains of 0157:H7 produce verocytotoxins, and that strains fromother serovars can be toxin producers (e.g. 026, 0111, 0113, 0145).
0157:H7 has been associated epidemiologically with food poisoningoutbreaks involving beefburgers and cold cooked meats 3, 4, 5, 6.The medium is a modification of MacConkey Agar No. 3 with thesubstitution of the fermentable carbohydrate from lactose to sorbitol.0157:H7 is sorbitol negative and produces translucent colonieswhereas most other E.coli strains are sorbitol positive and so producepink/red colonies. Selectivity of the medium can be increased byadding Cefixime-Tellurite (C.T.) supplement X161.
Formula g/litre
Peptone 20.0
Sorbitol 10.0
Bile salts no.3 1.5
Sodium chloride 5.0
Neutral red 0.03
Crystal violet 0.001
Agar 12.0
Method for reconstitutionWeigh 48.5 grams of powder and add to 1 litre of de-ionised water.Allow to soak for 10 minutes, swirl to mix and sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C, add 2 vials of CTsupplement X161, and pour plates. Dry the surface prior toinoculation.
Appearance: Pale red, slight violet tinge.
pH: 7.1 + 0.2
Minimum QC organisms: E.coli 0157:H7 (non-toxigenic)NCTC 12900 (translucent)E.coli NCIMB 50034 (Pink/red) Ent. faecalis NCIMB 50030(inhibition)
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark
Inoculation: Surface streak for single colonies
Incubation: 37˚C aerobically for 18-24 hr.
Growth Characteristics:Organism Size (mm) Shape Colour
E.coli 0157:H7 2.5 – 4.0 CV.E.G Translucent
Other E.coli 2.5 – 4.0 CV.E.G Pink/red
Sorbitol +ve organisms 2.5 – 5.0 Any Pink/red
References
Law D., Ganguli L.A., Donohue-Rolfe A., Acheson D.W.K. (1992)J.Med.Micro. 36 198-202.
Hitchins A.D., Hartman P.A., and Todd E.C.D. (1992) in“Compendium of methods for the microbiological examination offoods” Ch.24. Published by American Public Health Association.
Varnam A.H., Evans M.G., (1991) Foodborne Pathogens anIllustrated Text. Published by Wolfe Publishing Ltd.
Riley L.W. (1991) Ann.Rev.Micro. 41 383-408
Riley L.W. et al (1983) New Eng.J.Med 308 681-685
Salmon R.L., Farrel I.D., Hutchinson J.G.P. (1989) Epid.Inf. 103249-254
82
S.S. Agar(Salmonella Shigella Agar)
LAB 52
DescriptionThis medium is a modification of Leifson’s DCA Medium firstdescribed in 1941 by Mayfield and Goeber shortly before Hynesdescribed his modification of DCA. The selectivity of the mediumwas increased by the addition of extra bile salts, sodium citrate andthe addition of brilliant green dye. There is also the extra thiosulphategiving good H2S production which reduces the ferrous ammoniumsulphide giving black centred colonies with H2S positive organisms.
The selectivity of this medium can be such that it was suggested byTaylor et al in 1965 to be unsuitable for the isolation of Shigellaspecies. Greater understanding of the selection mechanisms involvedenable us to adjust the reaction and allow the more delicate Shigellato grow without unduly impairing the medium’s selective properties.
Formula g/litre
Beef Extract 5.0
Balanced Peptone No. 1 5.0
Lactose 10.0
Bile Salts No. 3 8.5
Sodium citrate 8.5
Sodium thiosulphate 8.5
Ferric citrate 1.0
Brilliant Green 0.00033
Neutral Red 0.025
Agar No. 2 13.5
Method for reconstitutionWeigh 60 grams of powder, disperse in 1 litre of deionised water, andallow to soak for 10 minutes. Swirl to mix, then bring to the boil, andallow to cool to 47˚C. Mix well then pour plates. Dry the surfacebefore incubation. DO NOT AUTOCLAVE THIS MEDIUM.
Appearance: Pale Pink, clear.
pH: 7.0 ± 0.2
Minimum Q.C. organisms: Salmonella sp. NCIMB 50076E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 4˚C in the dark.
Inoculation method: Surface plating, streaking for single colonies.
Incubation: 37˚C aerobically for 18-24 hours.
Growth Characteristicscolony size shape &
organism (mm) surface colour other
E. coli 0.1-2.0 CV.E.D. Red Red ppt aroundcolonies
(No growth)
K. aerogenes 0.1-3.0 CV.E.G. Pink-Red (No growth)
Proteus spp. 1.0-2.0 CV.E.G. Yellow (grey centre)(fishy odour)
Ps. aeruginosa 0.1-1.0 CV.CR.D. Pink (Green pigment)Yellow
Salmonella spp. 2.0-3.0 CV.E.G. Yellow (black centre)
Shigella spp. 0.5-2.0 CV.E.G. Pink-Yellow
Gram positive organisms – no growth.
ReferencesIsenberg, H. D., Kominos, S., and Siegel, M. 1969. Isolation ofsalmonellae and shigellae from an artificial mixture of fecal bacteria.Appl. Microbiol., 18: 4, 656-659.
Leifson, E. 1935. New culture media based on sodium desoxycholatefor the isolation of intestinal pathogens and for the enumeration ofcolon bacilli in milk and water. J. Pathol. Bacteriol., 40: 581-589.
Taylor, W. I., Harris, B. 1965 Isolation of shigellae. II. Comparison ofplating media and enrichment broths. Am. J. Clin. Pathol. 44: 4, 476-479.
Sugar Free AgarLAB 87
DescriptionA formula described by the International Dairy Federation for theenumeration of psychrotrophic and mesophilic Gram negative rods inbutter and other dairy products. The Gram negative rods are able todeaminate proteins as a carbon source, whilst some enterococci areinhibited by this formula. The medium conforms to the formulationof the International Dairy Federation (I.D.F.).
Formula g/litre
Gelatin Peptone 7.5
Tryptone 7.5
Sodium chloride 5.0
Agar No. 1 14.0
Method for reconstitutionWeigh 34 grams of powder and disperse in 1 litre of deionised water.Allow to soak for 10 minutes, boil to dissolve and disperse into tubesor flasks. Sterilise by autoclaving at 121˚C for 15 minutes.
Appearance: Light straw, clear.
pH: 7.6 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034
Storage of Prepared Medium: Capped container – up to 3 monthsat 15-20˚C in the dark.
Inoculation: 0.2ml of butter fat in a pour plate technique.
Incubation: 30˚C for 2 days then 20˚C for a further two days –aerobically.
Interpretation: Count colonies.
ReferencesInternational Dairy Federation (1964). International standard count of contaminating organisms in butter. International Standard FIL-IDF30.
Ritter, P. and Eschmann, K. H. 1966. Alimenta 5(2), 43-45. Thomas,S. B. 1969. J. Appl. Bact. 32, 269-296.
Mossel, D. A. A., Krol, B. and Moerman, P.C. 1972. Alimenta 11(2),51-60.
83
T.C.B.S. Cholera Medium(Thiosulphate Citrate Bile Salts Sucrose Agar)
LAB 96
DescriptionT.C.B.S. is designed for the selective isolation of Vibrio species,particularly V. cholerae. The formulation was developed byKobayashi, Enomoto, Sakazaki and Kuwahara and inhibits most ofthe Enterobacteriaceae for at least 24 hours. Therefore heavyinoculation of the medium is possible.
Formula g/litre
Yeast Extract 5.5
Peptone Mix 10.0
Sodium thiosulphate 10.0
Sodium citrate 10.0
Bile salts 9.0
Sucrose 17.0
Sodium chloride 10.0
Ferric citrate 1.0
Bromothymol blue 0.04
Thymol blue 0.04
Agar No. 1 15.0
Method of reconstitutionWeigh 88 grams of powder and add to 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then bring to the boil. Coolto 47˚C and pour into petri dishes. DO NOT AUTOCLAVE OROVERHEAT THIS MEDIUM.
Appearance: Dark green clear agar.
pH: 8.6 ± 0.2
Minimum Q.C. organisms: V. cholerae (type F)E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 4˚C in thedark. Capped containers – up to 1 month at 15-20˚C in the dark.
Inoculation: Surface plating with a heavy inoculum, streak out tosingle colonies.
Incubation: 37˚C aerobically for 18-24 hours.
Growth Characteristicscolony size shape &
organism (mm) surface colour other
Vibrio. cholerae 2.0-3.0 CV.E.G. Yellow may revert to green at R.T.
V. parahaemolyticus 3.0-5.0 CV.E.G. Blue orGreen
V. alginolyticus 3.0-5.0 CV.E.G. Yellow
V. metschnikovii 2.0-4.0 CV.E.G. Yellow
V. fluvialis 2.0-3.0 CV.E.G. Yellow
V. vulnificus 2.0-3.0 CV.E.G. Yellow
V. mimicus 2.0-3.0 CV.E.G. Green
Enterococci 1.0 CV.E.G. Yellow
Proteus spp. 1.0 F.CR.G. Green/Yellow
Ples. shigelloides P.P. Green
ReferencesKobayashi, T., Enomoto, S., Sakazaki, R. and Kuwahara, S. 1963.Jap. Bacteriol 18: 10-11, 387-391.
Furniss, A. L., Lee J. V. and Donovan, T. J. 1978. The Vibrios, PHLSMonograph No. 11.
Tetrathionate Broth Base A.P.H.A.LAB 97
DescriptionA selective enrichment broth for the growth of Salmonella typhi andother Salmonella spp. from faeces, foods etc. It conforms to theformulation recommended by the American Public HealthAssociation for use in the examination of dairy products and foodsfor salmonellae. Organisms which reduce tetrathionate, such assalmonellae, proliferate in the medium, whilst most enteric organismsare inhibited. Certain members of the Proteus group will also reducetetrathionate thereby impairing the performance of the medium insome cases. To overcome this, Novobiocin may be added to themedium at a level of 40 microgram/ml before addition of the iodine.Gram-positive organisms are inhibited by the inclusion of bile salts.
Formula g/litre
Balanced Peptone No. 1 5.0
Bile Salts 1.0
Calcium carbonate 10.0
Sodium thiosulphate 30.0
Method for reconstitutionWeigh 46 grams of powder and add to 1 litre of deionised water.Bring to the boil with frequent swirling to fully dissolve the medium.Cool to 45˚C and add 20ml of iodine solution prepared as indicatedbelow. Mix well before dispensing into bottles and continue swirlingwhilst dispensing to avoid the calcium carbonate sedimenting. Forthe best results the medium should be used the same day as prepared.
Iodine solution: Dissolve 5g of potassium iodide and 6g of iodinecrystals in 20mls of distilled water.
Appearance: Turbid white.
pH: 8.4 ± 0.2
Minimum Q.C. organisms: Salmonella sp. NCIMB 50076E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Capped containers – up to 7 days at4˚C in the dark (without iodine solution).
Inoculation: Add 1 part of sample suspension or inoculated pre-enrichment medium to 9 parts of Tetrathionate Broth.
Incubation: 12-24 hours at 37˚C.
Subculture: Onto LAB 34 Brilliant Green Agar and either LAB 32XLD or LAB 110 Hektoen Enteric or other Salmonella selectivemedia.
ReferencesStandard methods for the Examination of Dairy products, 10thEdition. APHA, 1953.
84
Thioglycollate Medium (Brewer)LAB 64
DescriptionThis is the original formula introduced by Brewer in 1940 as a clearmedium for the cultivation of anaerobes. It has found applications asa sterility test medium and as a blood culture medium although it hasbeen superseded by Fluid Thioglycollate LAB 25 and FastidiousAnaerobe Broth LAB 71 for these purposes.
The agar makes the medium viscous slowing down the permeation ofoxygen and any convection currents. Sodium thioglycollate acts as areducing agent and also neutralises the bacteriostatic properties ofmercurial compounds. Methylene blue is a redox indicator which iscolourless at low Eh but turns blue on exposure to oxygen.
Formula g/litre
Beef Extract 1.0
Yeast Extract 2.0
Balanced Peptone No. 1 5.0
Dextrose 5.0
Sodium chloride 5.0
Sodium thioglycollate 1.1
Methylene blue 0.002
Agar No. 1 1.0
Method for reconstitutionWeigh 20 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes then bring to the boil with gentleagitation to dissolve the solids. Distribute into screw top containersleaving minimal headspace. Sterilise by autoclaving at 121˚C for 15minutes. Tighten caps as soon as possible after autoclaving.
Appearance: Straw coloured, translucent, viscous liquid which mayhave a blue surface due to contact with oxygen. If the medium has adiffuse blue tinge it should not be used until the oxygen has beendriven off by holding in a boiling water bath for 5 minutes. Do notreheat more than once.
pH: 7.2 ± 0.2
Minimum Q.C. organisms: C. perfringens NCIMB 50027
Storage of Prepared Medium: Capped container – up to 3 monthsat 15-20˚C in the dark.
Inoculation: Ensure adequate dispersal of the inoculum in the broth.
Incubation: 37˚C for 24-72 hours.
Growth Indicators: A diffuse turbidity or individual colonies.
ReferencesBrewer, J. H. 1940. Clear liquid mediums for the culture ofanaerobes. J. Amer. Med. Ass. 115: 598-600.
Todd Hewitt BrothLAB075
DescriptionA nutritious broth medium formulated by Todd and Hewitt for theproduction of antigenic streptococcai haemolysin. Todd Hewitt Brothis also used to cultivate streptococci prior to serological grouping.The use of a fermentable sugar in the formulation leads to theproduction of acid which would normally inactivate the haemolysin.This is prevented by the inclusion of buffers to maintain the pH of themedium thus preserving the haemolysin, as well as promoting thegrowth of pneumococci.
Formula g/litre
Infusion from fat-free minced meat 10.0
Tryptone 20.0
Dextrose 2.0
Sodium bicarbonate 2.0
Sodium chloride 2.0
Disodium phosphate anhydrous 0.4
Method for ReconstitutionWeight 36.4grams of powder and disperse in 1 litre of deionisedwater. Allow to soak for 10 minutes, swirl to mix and warm todissolve. Dispense into 10ml volumes in screw capped containers andsterilise by autoclaving at 121˚C for 15 minutes.
Appearance: Pale straw, clear broth
pH: 7.8 ± 0.2
Inoculation: Pick a well isolated colony for subculture into ToddHewitt Broth.
Incubation: 37˚C for 18-48hrs, aerobically.
Storage: Capped containers - up to 3 months at 15-20oC in the dark.
Minimum Q.C. Organisms Streptococcus pyogenesATCC 19615.
Reference:Todd E.W., and Hewitt L.F., (1932) A New Culture Medium for theProduction of Antigenic Streptococcal Haemolysin. J. Path. Bact. 35(1) 973-974.
Updyke E., L., and Nickle M.I. (1954) A Dehydrated Medium for thePreparation of Type Specific Extracts of Group A Streptococci. Appl.Microbiol. 2 117-118.
85
Triple Sugar Iron AgarLAB 53
DescriptionThis is a modification of the Krumwiede and Kohn medium of 1917which differentiates some of the Enterobacteriaceae on the basis offour reactions; fermentation of lactose, glucose and sucrose and H2Sproduction. This medium should be used in conjunction with a ureasetest to eliminate Proteus spp. when screening for Salmonella spp.
Formula g/litre
Beef Extract 3.0
Yeast Extract 3.0
Balanced Peptone No. 1 20.0
Sodium chloride 5.0
Lactose 10.0
Sucrose 10.0
Glucose 1.0
Ferric citrate 0.3
Sodium thiosulphate 0.3
Phenol red 0.025
Agar No. 2 12.0
Method for reconstitutionWeigh 65 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then bring to the boil withfrequent swirling to dissolve the solids. Distribute into tubes andsterilise at 121˚C for 15 minutes. Allow to set as a slope ensuring thatthe slant is over a butt approximately 3cm deep.
Appearance: Reddish-orange gel.
pH: 7.4 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034Ps. aeruginosa NCIMB 50067
Storage of Prepared Medium: Capped containers – up to 3 monthsat 15-20˚C in the dark.
Inoculation: A heavy inoculum is streaked over the surface of theslope and stabbed into the butt.
Incubation: 37˚C aerobically for 24 hours.
InterpretationSlant/butt Colour Utilisation
Alkaline/acid Red/yellow Glucose only fermented Peptones utilised
Acid/acid Yellow/yellow Glucose fermented Lactose + or sucrose fermented
Alkaline/alkaline Red/Red Neither glucose, lactose, nor sucrose fermented
Peptones utilised
Organism Butt Slant Sulphideproduction
Shigella dysenteriae NC
S. sonnei Acid or -
S. flexneri Alk.
Salmonella typhi Acid NC +
S. paratyphi Acid NC -S. cholerasuis Gas
S. typhimurium AcidS. enteritidis Gas NC +S. pullorum
S. gallinarum Acid NC +
E. coliEnterobacter Acid Acid -aerogenes GasE. cloacae
Proteus mirabilis Acid Acid +Gas
Providencia rettgeri Acid NC -
NC = No Change
ReferencesAmerican Public Health Association 1963. Diagnostic Proceduresand Reagents, 4th edn., A.P.H.A., New York.
American Public Health Association 1966. Recommended Methodsfor the Microbiological Examination of Foods. 2nd edn., A.P.H.A.,New York.
Edwards, P. R. and Ewing, W. H. 1962. Identification ofEnterobacteriaceae. Burgess Publishing Co., Minneapolis.
86
Tryptone Bile AgarLAB 72
DescriptionFirst introduced by Delaney, McCarthy and Grasso in 1962 as amethod for detecting faecal coliforms in water supplies based on theproduction of indole on a bile medium at 44˚C. The idea was appliedto foodstuffs by Anderson and Baird-Parker in 1975. The inoculum isplaced onto the membrane on a resuscitation agar and incubated at37˚C for 4 hours. The membrane is then transferred to a TryptoneBile Agar plate and incubated at 44˚C: after incubation the membraneis flooded with indole reagent. Indole positive colonies produce a redcolour on the membrane and are easily counted.
Formula g/litre
Tryptone 20.0
Bile Salts No. 3 1.5
Agar No. 2 15.0
Method for reconstitutionWeigh 36.5 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. Cool to 47˚C and pour into petridishes.
Appearance: Straw coloured, clear gel.
pH: 7.2 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034
Storage of Prepared Medium: Plates – up to 4 days at 2-8˚C in thedark. Capped container – up to 1 month at 2-8˚C in the dark.
Inoculation: 1ml of a 1:10 dilution of homogenised sample onto amembrane. The recommended membranes are 85mm in diameterwith a 0.45 micron pore size manufactured from cellulose esters.
Incubation: 4 hours at 37˚C on Nutrient Agar LAB 8 or MineralsModified Glutamate Medium LAB 80A and 80B plus agar – then 18-24 hours on Tryptone Bile Agar at 44˚C.
Indole reagent: 5% p-dimethylaminobenzaldehyde in N-HC1(Vracko & Sherris 1963).
Indole reaction: Pipette 1-2ml of reagent into petri dish lid, removemembrane with forceps and place on reagent. Allow to stand for 5minutes for reaction to develop, then dry in sunlight to ‘fix’ thecolour. Count all pink colonies as E. coli.
Growth characteristicscolony size shape & indole reaction on
organism (mm) surface membrane
E. coli 1.0-3.0 CV.E. positive – pink colour
otherEnterobacteriaceae 0.5-2.0 negative – no colour
Klebsiella spp. no growth
Pseudomonas spp. no growth
Staphylococcus spp. no growth
Bacillus spp. no growth
ReferencesAnderson, J. M., Baird-Parker, A. C. 1975. A rapid and direct methodfor enumerating Eschericia coli biotype I in food. J. Appl. Bact. 39:111-117.
Delaney, J. E., McCarthy, J. A. & Grasso, R. J. 1962. Measurementof E. coli type I by the membrane filter technique Wat. Sewage Wks.109, 289.
Baird, R. M., Corry, J. E. L., Curtis, G. D. U. 1988. Pharmacopoeiaof culture media for food microbiology. Int. J. Food Microbiol. 276-277.
Tryptone Bile Glucuronide AgarLAB 162
DescriptionA medium developed for the simple enumeration of E.coli without theneed for membranes, or pre-incubation on Minerals ModifiedGlutamate Medium. Based upon the formulation of Tryptone BileAgar, LAB 072, the medium has been modified by the addition of achromogenic substrate to detect the B-glucuronidase enzyme, whichis highly specific for E.coli*, and is detected by the MUG reagent inother formulations. The advantage of the chromogenic substrate is thatit requires no UV lamp to visualise the reaction, and it is concentratedwithin the colony, facilitating easier enumeration in the presence ofother organisms, or when large numbers are present on the plate.
Formula g/litre
Tryptone 20.0
Bile Salts No.3 1.5
X-glucuronide 0.075
Agar 15.0
Method for reconstitutionWeigh 36.5 grams of powder, disperse in 1 litre of deionised waterand allow to soak for 10 minutes. Swirl to mix and sterilise at 121˚Cfor 15 minutes. Cool to 47˚C and pour into petri dishes. Dry thesurface prior to inoculation.
Appearance: Straw, clear gel.
pH: 7.2 + 0.2
Minimum QC organisms: E. coli (blue). NCIMB 50034Enterobacter sp (cream).NCIMB 50029
Incubation: 30˚C for 4 hours, followed by 18 hours at 44˚C.
Interpretation: Count all blue colonies as presumptive E.coli,calculate the cfu/g in the original material. A simple indole test maybe performed by placing 1 drop of Kovac’s reagent onto a colony. Ifindole positive a red halo will appear in the medium around thecolony. If negative then the halo will be white.
Storage: Plates – up to 7 days at 2-8˚C in the dark. Capped container – up to 3 months at 15-20˚C in the dark
ReferencesDibb W.L. and Bottolfsen K.L. (1984) Evaluation of RoscoDiagnostic B-glucuronidase Tablets in the Identification of UrinaryIsolates of Escherichia coli. Acta Path. Microbiol. Immunol. Scand.Sect. B.92 261-264.
Hansen W. and Yourassowsky E. (1984) Detection of B-glucuronidase in Lactose Fermenting Members of the FamilyEnterobacteriaceae and its Presence in Bacterial Urine Cultures. J.Clin. Micro. 20 (6) 1177-1179.
Robinson B.J. (1984) Evaluation of a Fluorogenic Assay forDetection of E.coli. App. & Env. Microbiol. 48 (2) 285-288
Perez J.L., Berrocal C.I., and Berrocal L. (1986) Evaluation of aCommercial B-glucuronidase Test for the Rapid and EconomicalIdentification of Escherichia coli. J. App. Bacteriol. 61 541-545.
Raghubeer E. and Matches J.R. (1990) Temperature Range forGrowth of Escherichia coli Serotype 0157:H7 and SelectedColiforms in E.coli Medium. J. Clin. Micro. 28 (4) 803-805.
Bolton F.J. (1995) Personal Communication.
*96-97% of E.coli strains positive. A notable exception is E.coli 0157 H7.
87
Tryptone Glucose Extract AgarLAB 63
DescriptionA plate count agar suggested by the American Public HealthAssociation (A.P.H.A.) for estimation of total viable counts in foodand dairy products. This medium is also recommended by theAssociation of Official Analytical Chemists (A.O.A.C.).
Formula g/litre
Beef Extract 3.0
Tryptone 5.0
Glucose 1.0
Agar No. 1 15.0
Method for reconstitutionWeigh 24 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then boil to dissolvebefore distributing into tubes or bottles. Sterilise at 121˚C for 15minutes.
Appearance: Pale straw colour, clear.
pH: 7.0 ± 0.2
Minimum Q.C. organisms: S. epidermidis NCIMB 50082E. coli NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in thedark. Capped containers – up to 3 months at 15-20˚C in the dark.
Inoculation: Pour plate technique.
Incubation: 30˚C aerobically for 48 hours for aerobic mesophilecount. 6˚C aerobically for 10 days for aerobic psychrotroph count.55˚C aerobically for 48 hours for aerobic thermophile count.
ReferencesAssociation of Official Analytical Chemists (AOAC). 1978.Bacteriological Analytical Manual, 5th ed.: Association of OfficialAnalytical Chemists.
Hausler, W. J. (Ed.) 1976. Standard Methods for the Examination ofDairy Products, 14th edn.: American Public Health Association.
Speck, M. J. (Ed.) 1976. Compendium of Methods for theMicrobiological Examination of Foods.: American Public HealthAssociation.
Tryptone Soy Agar (U.S.P.)LAB 11
DescriptionA general purpose agar which will support the growth of a wide rangeof micro organisms. The formula conforms with that laid down by theUnited States Pharmacopeia for sterility testing. The medium can beused for phage typing, colicine typing and for testing the X and Vfactor requirements of Haemophilus spp.
Formula g/litre
Tryptone 15.0
Soy Peptone 5.0
Sodium chloride 5.0
Agar No. 2 12.0
Method for reconstitutionWeigh 37 grams of powder, disperse in 1 litre of deionised water,allow to soak for 10 minutes, swirl to mix then sterilise for 15minutes at 121˚C. Cool to 47˚C, mix well and then pour plates.
Appearance: Pale straw coloured, clear gel.
pH: 7.3 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034S. epidermidis NCIMB 50082
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in thedark. Capped containers – up to 3 months at 15-20˚C in the dark.
Inoculation: Surface plating.
Incubation: Time and temperature to suit organisms, usuallyaerobic.
Growth Characteristicscolony size shape &
organism (mm) surface colour other
S. aureus 1.0-1.5 CV.E.G. White-Yellow
Other staphylococci 1.0-1.5 CV.E.G. White- strain-dependent
Yellow
Strep. pyogenes PP-0.5 CV.E.G. Transp.
S. pneumoniae PP CV.E.G. Transp. (CO2)
Ent. faecalis 0.5-1.0 CV.E.G. Grey-White
Klebsiella spp. 2.0-3.0 CV.E.G. White mucoid
Ps. aeruginosa 0.5-3.0 F.CR.D. Grey- (marked strainGreen variation)
ReferencesUnited States Pharmacopeia 21st edition. 1985.
Blair, J. E. and Carr, M. 1953. The bacteriophage typing ofstaphylococci. J. Infect. Dis. 93: 1-13.
Examination of Dairy Products. A.P.H.A., New York.
88
Tryptone Soy Broth U.S.P.(Soybean Casein Digest Medium U.S.P.)
LAB 4
DescriptionA general purpose nutritious broth capable of growing a wide rangeof bacteria and fungi. The medium is recommended by the UnitedStates Pharmacopeia for the sterility testing of a wide range ofpharmaceutical products. The medium is also widely used for bloodcultures although the high carbohydrate level may cause rapid growthand subsequent death of acid-producing organisms.
Formula g/litre
Tryptone (Casein Digest USP) 17.0
Soy Peptone 3.0
Sodium chloride 5.0
Dipotassium phosphate 2.5
Dextrose 2.5
Method for reconstitutionWeigh 30 grams of powder, disperse in 1 litre of deionised water.Swirl to mix and warm if necessary to dissolve. Dispense into tubesor flasks and sterilise at 121˚C for 15 minutes. Do not exceedtemperature.
Appearance: Straw coloured, clear.
pH: 7.3 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034S. aureus NCIMB 50080
Storage of Prepared Medium: Capped containers – up to 3 monthsat 15-20˚C in the dark.
Incubation: 20-25˚C aerobically for 14 days, for sterility tests. 37˚Caerobically for 14 days for blood cultures.
Growth indicators: Turbidity or precipitate.
ReferencesUnited States Pharmacopeia 21st edition 1985.
Tryptone WaterLAB 129
DescriptionA substrate for the testing of an organism’s ability to produce indolefrom tryptophane. The indole test is frequently used in theclassification of coliform organisms. This product is preferable topeptone water LAB 104 because it has a higher content of tryptophane.
Formula g/litre
Tryptone 10.0
Sodium chloride 5.0
Method for reconstitutionWeigh 15 grams of powder, disperse in 1 litre of deionised water.Heat to dissolve then distribute into screw cap bottles. Sterilise byautoclaving at 121˚C for 15 minutes.
Appearance: Colourless, clear.
pH: 7.5 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034
Storage of Prepared Medium: Capped containers – up to 3 monthsat 15-20˚C in the dark.
Inoculation: From pure culture.
Incubation: 37˚C for 24-48 hours.
Interpretation: Indole positive organisms will give a distinct colourchange when either Kovac’s or Ehrlich’s indole reagent is added.
ReferencesAmerican Public Health Association. 1955. American Water WorksAssociation 10th edn. 391-392.
MacFaddin, J. 1983. Biochemical tests for the identification ofmedical bacteria. 2nd edn. Williams & Wilkins, Baltimore.
Tryptose Phosphate BrothLAB 62
DescriptionThis is a versatile nutritionally rich buffered glucose broth. Themedium is a general purpose broth that has been used as a bloodculture medium, a supplement for animal cell culture, and with theaddition of sodium azide 0.025% as a selective medium forstreptococci in dairy products.
Formula g/litre
Tryptose 20.0
Dextrose 2.0
Sodium chloride 5.0
Disodium phosphate 2.5
Method for reconstitutionWeigh 29.5 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, heat to dissolve solids then distributeinto final containers. Sterilise by autoclaving at 121˚C for 15 minutes.
Appearance: Very pale straw, clear.
pH: 7.3 ± 0.2
89
Minimum Q.C. organisms: E. coli NCIMB 50034S. aureus NCIMB 50080
Storage of Prepared Medium: Capped containers – up to 3 monthsat 15-20˚C in the dark.
Inoculation: For blood culture work dilute sample at least 1:10 in broth.
Incubation: Dependent on application.
ReferencesAmerican Public Health Association 1948. Standard Method for theExamination of Dairy Products, 10th edn. A.P.H.A., New York.American Public Health Association, 1950. Diagnostic Proceduresand Reagents, 3rd edn., A.P.H.A., New York.
T.Y.C. Medium(Tryptone Yeast Cystine)
LAB 35
DescriptionA medium designed by J. D. de Stoppelaar in 1967 to differentiateStreptococcus sanguis (frequently found in dental plaque) fromStreptococcus mutans (implicated in dental caries). The medium usesa high sucrose content to promote the formation of specific glucansby S. mutans thus forming distinctive colonies. It can be madeselective by the addition of 0.2 units per ml of Bacitracin.
Formula g/litre
Tryptone 15.0
Yeast Extract 5.0
L-Cystine 0.2
Sodium sulphite 0.1
Sodium chloride 1.0
Disodium phosphate anhydrous 0.8
Sodium bicarbonate 2.0
Sodium acetate anhydrous 12.0
Sucrose 50.0
Agar No. 2 12.0
Method for reconstitutionWeigh 98 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix, then sterilise for 15minutes at 121˚C. Cool to 47˚C, mix and pour plates.
Appearance: White, translucent gel.
pH: 7.3 ± 0.2
Minimum Q.C. organisms: S. mutans ATCC 25175
Storage of Prepared Medium: Plates – up to 7 days at 4˚C in the dark.
Inoculation: Surface, streaking out for single colonies.
Incubation: 37˚C for 4-5 days in an atmosphere of 90% H2, 10%CO2.
Growth Characteristicscolony size shape &
organism (mm) surface colour other
Strep. mutans 1.0-3.0 ‘heaped’ Yellow Crumbles whenType A colony touched with wire
granularsurface
irregularedge
Type B 1.0-3.0 As Type A Grey white soft consistency,
white precip in agar(glistening drop)
S. sanguis 1.0-3.0 Convex White very rubberyglossy (glistening drop)
crenated
Referencesde Stoppelaar, J. D. van de Houte, J. and de Moor, C. E. 1967. Thepresence of dextran forming bacteria resembling Streptococcus bovisand Streptococcus sanguis in dental plaque. Arch. Oral Biol. 12:1199-1201. de Stoppelaar, J. D. 1971. Streptococcus mutans,Streptococcus sanguis and dental caries. Thesis, Rijksuniversiteit,Utrecht.
Emilson, C. G., Bratthall, D. 1976. Growth of Streptococcus mutanson various selective media. Journal of Clinical Microbiology 4: 95-98.
Gold, O. G., Jordon, H. V., Van Houte, J. 1973. A selective mediumfor Streptococcus mutans. Archives of Oral Biology 19: 1357-1364.
Ikeda, T., Sandham, H. J. 1972a. A high-sucrose medium for theidentification of Streptococcus mutans. Archives of Oral Biol. 4: 781-783.
Wade, W. G., Alldred, M. J., Walker, D. M. (1986. J. Med. Microbiol.22: 319-323. An improved medium for isolation of Streptococcusmutans.
Urea Agar Base(Christensen)
LAB 130
DescriptionThis is a modification of Christensen’s urea base for the detection ofrapid urease production by Proteus spp. Other enterobacteria willsplit the urea, but this will be delayed. This delay is achieved by theincorporation of glucose and the introduction of a buffering systeminto the medium. The indicator for ammonia production is phenolred.
Formula g/litre
Peptone 1.0
Glucose 1.0
Sodium chloride 5.0
Disodium phosphate 1.2
Potassium dihydrogen phosphate 0.8
Phenol red 0.012
Agar No. 1 12.0
Method for reconstitutionWeigh 2.1 grams of powder, disperse in 95ml of deionised water.Allow to soak for 10 minutes, swirl to mix, then sterilise at 121˚C for15 minutes. Allow to cool to 47˚C, add aseptically 5ml sterile ureasolution X130. Distribute into sterile bottles and slopes, allow to setin the sloped position.
Appearance: Yellow/pale pink, translucent.
pH: 6.8 ± 0.2
90
Minimum Q.C. organisms: Proteus spp.E. coli NCIMB 50034
Storage of Prepared Medium: Capped container – up to 1 month at2-8˚C in the dark.
Inoculation: Pure culture using straight wire for stab/streaktechnique.
Incubation: 37˚C for 4-6 hours or overnight, aerobically.
Interpretation: Production of red colour in under 6 hours is positivefor rapid urease production.
Organism Growth CharacteristicsProteus spp. Red colour 4-6 hours
Citrobacter spp. Red colour 18-24 hours
Klebsiella spp. Red colour 18-24 hours
Staphylococcus spp. Red colour 24-48 Hours
Helicobacter pylori Red colour 30 minutes
ReferencesChristensen, W. B. 1946. Urea decomposition as a means ofdifferentiating Proteus and paracolon cultures from each other andfrom Salmonella and Shigella types. J. Bacteriol. 52: 461-466.
Urea Broth Base(Christensen)
LAB 131
DescriptionThis is a liquid version of Christensen’s medium (LAB 130)introduced by Maslen in 1952. This modification allows inoculationby Pasteur pipette, and it is easier to detect contamination in a fluidrather than in a slope. Maslen also claimed that it is easier to detectpositive results.
Formula g/litre
Peptone 1.0
Glucose 1.0
Disodium phosphate 1.2
Potassium dihydrogen phosphate 0.8
Sodium chloride 5.0
Phenol red 0.004
Method for reconstitutionWeigh 0.9 grams of powder, add to 95ml of deionised water. Swirl tomix then sterilise by autoclaving at 121˚C for 15 minutes. Allow tocool to 47˚C then add aseptically 5ml of X130 sterile urea solution.Distribute into sterile screw cap bijou bottles.
Appearance: Yellow, clear.
pH: 6.8 ± 0.2
Minimum Q.C. organisms: Proteus sp.E. coli NCIMB 50034
Storage of Prepared Medium: Capped container – up to 1 month at2-8˚C in the dark.
Inoculation: Fluid culture by pasteur pipette or straight wire frompure growth.
Incubation: 37˚C for 4-6 hours – preferably in a water bath for mostrapid growth, aerobically.
Interpretation: The production of a red colour in under six hours isa positive result for rapid urease.
Organism Growth CharacteristicsProteus spp. Red colour 4-6 hours
Corynebacterium hoffmani Red colour 18-24 hours
C. ulcerans Red colour 18-24 hours
Some strains Citrobacter Red colour 18-24 hours
Klebsiella ,, ,,
Escherichia ,, ,,
Yersinia ,, ,,
Staphylococcus ,, ,,
Pasteurella multocida Red colour 18-24 hours
ReferencesMaslen L.G.C. 1952. Routine use of liquid urea medium for identifying Salmonella and Shigella organisms. J. Brit. Med. 2:545-546.
UVM BaseLAB 155
DescriptionUVM (University of Vermont Medium) Base is a two stage selectiveenrichment broth for the isolation of Listeria from meat products andenvironmental swabs, and forms the basis of the USDA method. Theoriginal method has been modified to replace the second stage broth(UVM II) with Fraser broth LAB164 (McClain & Lee 1989)
Formula g/litre
Tryptone 5.0
Meat Peptone 5.0
Beef Extract 5.0
Yeast Extract 5.0
Sodium chloride 20.0
Disodium hydrogen phosphate 9.6
Potassium dihydrogen phosphate 1.35
Aesculin 1.0
pH: 7.4 ± 0.2
Appearance: Straw opalescent broth
Method for reconstitutionWeigh 52 grams of powder and add to 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix and sterilise at 121˚C for15 minutes. Cool to 47˚C and add 2 vials of UVM I supplement(X155) or UVM II supplement (X156) as required. Mix well anddistribute into sterile tubes or bottles.
Inoculation: UVM I – Add 25g sample to 225ml of UVM I andhomogenise. UVM II – Subculture 0.1ml of enriched UVM I into10ml UVM II
Incubation: UVM I – 30˚C aerobically for 24 hrs. Subculture ontoselective agars and into UVM II. UVM II – 30˚C for 24 and 48 hrs.Subculture onto selective agars at 24 and 48 hrs. Incubate selectiveagars for 24 and 48 hrs.
Minimum QC organism: Listeria sp. NCIMB 50007E.coli (inhibition) NCIMB 50034
ReferencesMcClain D., and Lee W.H. (1989) FSIS method for isolation ofL.monocytogenes from processed meat and poultry products.Lab.Comm.No.57, Revised May 24, 1989. US Dept of Agric.FSIS,Microbiol. Div.
Warburton D.W. et al (1991) A Canadian comparative study ofmodified versions of the FDA and USDA methods for the detectionof L.monocytogenes. J.Food Protection 54 (9) 669-676
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Violet Red Bile Agar (V.R.B.A.)LAB 31
DescriptionA medium for the enumeration of coliform organisms in food anddairy products. The selectivity of the medium is due to the presenceof bile salts and crystal violet. Lactose fermenters produce red/purplecolonies often surrounded by a halo of the same colour. Non lactosefermenters produce pale colonies. Selectivity can be increased byincubation at 42-44˚C.
Formula g/litre
Yeast Extract 3.0
Balanced Peptone No. 1 7.0
Sodium chloride 5.0
Bile Salts No. 3 1.5
Lactose 10.0
Neutral red 0.03
Crystal violet 0.002
Agar No. 2 12.0
Method for reconstitutionWeigh 38.5 grams of powder, disperse in 1 litre of deionised water.Dissolve by bringing to the boil with frequent swirling of the flask toprevent overheating. Autoclaving is not necessary. Cool to 45˚C anddistribute into bottles or tubes. If held molten in a water bath, usewithin 3 hr.
Appearance: Light purple-violet, clear gel.
pH: 7.4 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034S. epidermidis (inhibition)NCIMB 50082
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark. Capped containers – up to 1 month at 15-20˚C in the dark.
Inoculation: Pour plate (with or without overlay) or surface spread.
Incubation: 37˚C for 18-24 hours for ‘coliforms’; 4˚C for 10 daysfor psychrotrophs; 32˚C for 24-48 hours for mesotrophs; 42˚C for 18hours for thermotrophs.
Interpretation: Count all red/purple colonies > 0.5mm in diameter.Calculate the number of coliforms in original sample.
ReferencesAmerican Public Health Association 1972, Standard Methods for theExamination of Dairy Products. 13th edn. (ed. W. H. Hausler),A.P.H.A., Washington.
American Public Health Association 1966. Recommended Methodsfor the Micro biological Examination of Foods. 2nd end. (ed. J. M.Sharf) A.P.H.A., Washington.
Davis, J. G. 1951. Milk Testing Dairy Industries, London.
Mossel, D. A. A., Eelderink I. and Sutherland, J. P. 1977.Development and use of single, ‘polytropic’ diagnostic tubes for theapproximate taxonomic grouping of bacteria, isolated from foods,water and medicinal preparations. Zbl. Bakt. Hyg. I., Orig., A 278,66-79.
Mossel, D. A. A., Eelderink, I. Koopmans M. and van Rossem, F.1979. Influence of carbon source, bile salts and incubationtemperature on the recovery of Enterobacteriaceae from foods usingMacConkey type agars. J. Food Protec. 42, 470-475.
Mossel, D. A. A., van der Zee, H. Hardon, A. P. and van Netten, P.1986. The enumeration of thermotropic types amongst theEnterobacteriaceae colonizing perishable foods. J. Appl. Bacteriol.60, 289-295.
Violet Red Bile Glucose Agar(V.R.B.G.A.)
LAB 88
DescriptionA modification of Violet Red Bile Agar LAB 31 introduced byMossel in 1978. V.R.B.A. LAB 31 contains lactose which isfermented by members of the coli/aerogenes group, this mediumgives a ‘coliform’ count. V.R.B.G.A. LAB 88 has substituted lactosewith glucose. Glucose is fermented by all members of theEnterobacteriaceae thus V.R.B.G.A. gives a presumptiveEnterobacteriaceae count. Bile salts and crystal violet are used toinhibit Gram positive and non-enteric organisms. The overlayprocedure ensures anaerobic conditions and suppresses the growth ofnon-fermentative Gram negative bacteria.
Formula g/litre
Yeast Extract 3.0
Balanced Peptone No. 1 7.0
Sodium chloride 5.0
Bile Salts No. 3 1.5
Glucose 10.0
Neutral red 0.03
Crystal violet 0.002
Agar No. 2 12.0
Method for reconstitutionWeigh 38.5 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes then swirl to mix. Bring to the boil withfrequent swirling to prevent overheating. Further sterilisation is notrequired. Cool to 45˚C, mix well and dispense into tubes or bottles. If held molten in a water bath, use within 3 hr.
Appearance: Light purple-violet, clear.
pH: 7.4 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034S. epidermidis (inhibition)NCIMB 50082
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in thedark. Capped containers – up to 1 month at 15-20˚C in the dark.
Inoculation: Pour plate method with overlay.
Incubation: 37˚C aerobically for 18-24 hours.
Interpretation: Count all red/purple colonies > 0.5mm in diameter.Calculate the number of Enterobacteriaceae in original sample.
ReferencesPharmacopoeia of Culture Medium for Food Microbiology 1987. Int.J. Food Microbiol. 5: 3: 280-81.
Mossel, D. A. A., Mengerink, W. H. J. and Scholts, H. H. 1962. Useof a modified MacConkey agar medium for the selective growth andenumeration of Enterobacteriacaea. J. Bacteriol. 84: 381.
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W. L. Nutrient Agar(Wallerstein Laboratory)
LAB 79
DescriptionThis medium was developed by Green and Gray in 1950 for theisolation and enumeration of yeasts, moulds and bacteria in thebrewing process. The medium has a pH of 5.5 which is optimum forBrewers yeast and will allow the growth of a wide range of organismsincluding Enterobacteriaceae, Flavobacterium, Lactobacillus andPediococcus spp. as well as yeasts and moulds. If a process involvingbakers or distillers yeast is under examination the pH should beadjusted to 6.5. The medium may be adapted to detect bacteria onlyby the addition of 0.004 g/litre of Actidione to suppress the yeasts.
Formula g/litre
Yeast Extract 4.0
Tryptone 5.0
Dextrose 50.0
Potassium dihydrogen phosphate 0.55
Potassium chloride 0.425
Calcium chloride 0.125
Magnesium sulphate 0.125
Ferric chloride 0.0025
Manganese sulphate 0.0025
Bromocresol green 0.022
Agar No. 2 15.0
Method for reconstitutionWeigh 75 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then sterilise byautoclaving at 121˚C for 15 minutes. If adjustment of pH to 6.5 isrequired used 1% sodium bicarbonate.
Appearance: Green, clear.
pH: 5.5 ± 0.2
Minimum Q.C. organisms: S. cerevisiae.
Storage of Prepared Medium: Plates – up to 7 days at 4˚C in thedark. Capped container – up to 1 month at 4˚C in the dark.
Inoculation: Surface plating or pour plate.
Incubation: 30˚C aerobically for 48 hours (bacteria); 20˚Caerobically for 48 hours (yeasts).
Interpretation: Count all colonies. Calculate organisms per ml inoriginal sample.
ReferencesGreen, S. R. and Greay, P. P. 1950. Differential Procedure Applicableto Investigation in Brewing. Wallerstein Lab. Comm. 13,357.
Hall, Jean F. 1971. Detection of Wild Yeasts in the Brewery. J. Inst.Brewing, 77: 513-516.
Wort AgarLAB 38
DescriptionA medium for the enumeration of yeasts and moulds in butter,developed by Parfitt in 1933. The medium can be modified to enableit to isolate osmophilic yeasts from soft drinks and sugar products bythe addition of high concentrations of sucrose and glucose.
Formula g/litre
Malt Extract 15.0
Peptone 0.78
Maltose 12.75
Dextrin 2.75
Dipotassium phosphate 1.0
Ammonium chloride 1.0
Agar No. 2 15.0
Method for reconstitutionWeigh 48.3 grams of powder and disperse in 1 litre of deionisedwater. Add 2.35mls of glycerol. Allow to soak for 10 minutes, swirlto mix then sterilise for 15 minutes at 121 C. Use 60 grams per litreif required for inoculation by plate streaking with a wire loop. Do notexceed time or temperature of sterilisation. If osmophilicmodification is required add 48.3 grams of powder to 1 litre of asolution containing 35% w/v sucrose and 10% w/v glucose thensterilise at 108˚C (5 p.s.i.) for 20 minutes.
Appearance: Light Brown, translucent.
pH: 5.0 ± 0.2
Minimum Q.C. organisms: S. cerevisiae.
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in thedark. Capped container – up to 1 month at 15-20˚C in the dark.
Inoculation: Pour plate or surface spread.
Incubation: 25˚C aerobically for 5 days.
Growth Characteristicscolony size shape &
organism (mm) surface colour other
Candida spp. 4.0 CV.E.G White
Fungi Varies withspecies
S. cerevisiae 2.0-3.0 Varies with Creamstrain
ReferencesParfitt, E. H. 1933. The influence of media upon the yeast and mouldcount of butter. J. Dairy Sci. 16: 141-147.
Scarr, M. P. 1959. Selective media used in the microbiologicalexamination of sugar products. J. Sci. Fd. Agric. 10: 678-681.
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Wort BrothLAB 99
DescriptionA broth version of the medium LAB 38 Wort Agar developed byParfitt for the enumeration of yeasts and moulds, in butter. Themedium can be modified for the isolation of osmophilic yeasts fromsoft drinks and sugar products by the addition of high concentrationsof sucrose and glucose.
Formula g/litre
Malt Extract 15.0
Peptone 0.78
Maltose 12.75
Dextrin 2.75
Dipotassium phosphate 1.0
Ammonium chloride 1.0
Method for reconstitutionWeigh 33.3 grams of powder and disperse in 1 litre of deionisedwater, add 2.35mls. of glycerol. Allow to soak for 10 minutes, swirlto mix then sterilise by autoclaving at 121˚C for 15 minutes. Ifosmophilic version is required disperse 33.3 grams of powder in 1litre of a solution of 35% w/v sucrose and 10% w/v glucose thensterilise at 108˚C (5 p.s.i.) for 20 minutes.
Appearance: Light Brown, translucent.
pH: 4.8 ± 0.2
Minimum Q.C. organisms: S. cerevisiae.
Storage of Prepared Medium: Capped container – up to 1 month at15-20˚C in the dark.
Incubation: 25˚C aerobically for 5 days.
X.L.D. Agar(Xylose Lysine Decarboxylase Agar)
LAB 32
DescriptionThis medium was introduced by Taylor in 1965 to improve therecovery and recognition of Shigella spp, and has proved to be anexcellent medium for Salmonella spp. The medium is low in nutrientsand relies on a small amount of sodium desoxycholate for selectivity.The indicator system is novel and complex. Most enteric organismsexcept Shigella, will ferment xylose to produce acid. However thesalmonellae will also decarboxylate the lysine to keep the pH neutral.At near neutral pH Salmononella can produce H2S from the reductionof thiosulphate producing black or black centred colonies.Citrobacter spp. can also decarboxylate lysine, however, the acidproduced by fermentation of both lactose and sucrose will keep thepH too acid for H2S to be produced.
Formula g/litre
Xylose 3.75
L-Lysine 5.0
Lactose 7.5
Sucrose 7.5
Sodium chloride 5.0
Yeast Extract 3.0
Phenol red 0.08
Agar No. 2 13.0
Sodium desoxycholate 1.0
Sodium thiosulphate 6.8
Ferric ammonium citrate 0.8
Method for reconstitutionWeigh 53.5 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix. Bring rapidly to the boilwith frequent stirring, and transfer immediately to a 47˚C water bath.Pour into plates as soon as the medium has cooled. Protracted boilingor prolonged holding at elevated temperature induces precipitation.
Appearance: Light rose, clear gel.
pH: 7.4 ± 0.2
Minimum Q.C. organisms: Salmonella sp. NCIMB 50076E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation: Surface, streaking out for single colonies.
Incubation: 37˚C for 18-24 hours aerobically.
Growth Characteristicscolony size shape &
organism (mm) surface colour other
Salmonella spp. 1.0-2.5 CV.E.G. Trans. (clearing of acidblack ppt of coliforms)centre
Shigella sonnei 1.5-2.5 CV.E.G. Pink
S. flexneri 1.0-2.0 CV.E.G. Pink
S. dysenteriae 0.5-1.5 CV.E.G. Pink
E. coli 0.5-1.5 CV.E.G.(D) Yellow inhibited (pptaround colony)
Citrobacter spp. 1.0-1.5 CV.E.G.(D) Yellow (black centre)
Proteus spp 1.0-2.5 CV.E.G. Trans. Pink fishy odour(blackcentre)
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ReferencesTaylor, W. I. 1965. Isolation of shigellae. I. Xylose Lysine Agars:New media for the isolation of enteric pathogens. Am. J. Clin.Pathol., 44: 471-475.
Taylor, W. I., and Harris, B. 1965. Isolation of shigellae. II.Comparison of plating media and enrichment broths. Am. J. Clin.Pathol., 44(4), 476-479.
Taylor, W. I., and Harris, B. 1967. Isolation of shigellae. III.Comparison of new and traditional media with stool specimens. Am.J. Clin. Pathol., 48: 350-355. Taylor, W. I., and Schelhart, D. 1967.Isolation of shigellae. IV. Comparison of plating media with stools.Am. J. Clin. Pathol., 48: 356-362.
Yeast Extract Agar(Yeastrel Milk Agar)
LAB 18
DescriptionA nutrient agar corresponding to the Standard Formulation for theplate count of micro-organisms in water and dairy products. Thismedium is also useful for teaching and demonstration purposes usingnon-fastidious organisms.
Formula g/litre
Yeast Extract 3.0
Balanced Peptone No. 1 5.0
Agar No. 1 15.0
Method for reconstitutionWeigh 23 grams of powder, disperse in 1 litre of deionised water.Free steam or boil to dissolve. Mix well, and dispense into containers.Sterilise for 15 minutes at 121˚C.
To prepare Yeastrel Milk Agar add 10mls of fresh milk beforeautoclaving.
Appearance: Pale straw, clear gel.
pH: 7.2 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034S. epidermidis NCIMB 50082
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark. Capped container – up to 3 months at 15-20˚C in the dark.
Inoculation: Pour plate technique or surface spreading.
Incubation: 30˚C aerobically for 48 hours for aerobic mesophilcount. 6˚C aerobically for 10 days for aerobic psychrotroph count.55˚C aerobically for 48 hours for aerobic thermophil count.
ReferencesMinistry of Health, Public Health Laboratory Service WaterCommittee 1969. The Bacteriological Examination of WaterSupplies, 4th Edn. report No. 71. H.M.S.O., London.
British Standard 4285: Methods of Microbiological Examination forDairy Purposes.
Yeast Extract DextroseChloramphenicol Agar
LAB 119
DescriptionA selective medium for the enumeration of yeasts and moulds in milkand other dairy products. The formulation meets the requirements ofthe International Milk Union (1980), the International Organisationfor Standardisation (I.S.O.) and the British Standards Institute(B.S.I.). The medium is said to have superior storage properties toO.G.Y.E. and also has the advantage of incorporating an autoclavablesupplement.
Formula g/litre
Yeast Extract 5.0
Dextrose 20.0
Agar No. 1 15.0
Method for reconstitutionWeigh 40 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, swirl to mix then bring to boil. Add 2 vials of X009 which have been dissolved in ethanol andautoclave at 121˚C for 10 minutes. Allow to cool to 45˚C before usingwith poured plate technique. THIS MEDIUM MUST NOT BE RE-AUTOCLAVED.
Appearance: Pale yellow, clear.
pH: 6.6 ± 0.2
Minimum Q.C. organisms: Aspergillus sp. NCIMB 50097S. cerevisiaeE. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Incubation: 25˚C for 5 days, aerobically.
Inoculation: Pour plate technique.
Interpretation: Count all colonies.
ReferencesEngel, G. 1982. Verglich verschieden Nährböden zum quantitativenNachweis von Hefen und Schimmelpilzen in Milch undMilchprodukten. Milchwiss. 37: 727-730.
International Organisation for Standardization (ISO): Milk and milkproducts – enumeration of yeasts and moulds – colony counttechnique at 25˚C – standard method ISO/DIS 6611.
International Milchwirtschaftsverband: Milch und Milchprodukten –Zählung von Hefen und Schimmelpilzen (Kolonieählung bel 25 C). –International IMV Standard 94: 1980 in Milchwiss. 36: 220-222.
Normenausschufl Lebensmittel und landwirtshaft. Produkte in DINDeutsches Institut für Normung e.V. MikrobiologischeMilchuntersuchung. Bestimmung der Anzahl von Hefen undSchimmelpilzen. Reference method DIN 10186.
British Standards Institute. B.S. 4285. Section 3.6: 1986.
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Yersinia Selective Agar(Schiemann’s C.I.N. Agar)
LAB 120
DescriptionThis medium is based on the work of Schiemann. It is used for theisolation and enumeration of Yersinia spp. from clinical samples andfrom food. The selective components are sodium desoxycholate,crystal violet, cefsulodin, irgasan and novobiocin. Yersiniae fermentmannitol with an intense, localised, acid production in the centre ofthe colony which produces a red ‘bull’s eye’ appearance. The ratio oftransparent border to red centre varies with serotype andenvironmental strains may appear rough with an irregular edge. Mostother enteric bacteria, if they grow, produce a larger colony with adiffuse pinkish centre and opaque outer zone.
Formula g/litre
Peptone Mixture 22.5
Mannitol 20.0
Sodium chloride 1.0
Magnesium sulphate 0.01
Sodium pyruvate 2.0
Sodium desoxycholate 0.5
Neutral red 0.03
Crystal violet 0.001
Agar No. 2 12.0
Method for reconstitutionWeigh 58 grams of powder, disperse in 1 litre of deionised water.Allow to soak for 10 minutes, then bring to the boil for 1 minute only.DO NOT AUTOCLAVE. Allow to cool to 47˚C add 2 ampoulesC.I.N. supplement X120. Mix well, pour plates.
Appearance: Red, clear.
pH: 7.4 ± 0.2
Minimum Q.C. organisms: Y. enterocoliticaE. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the dark.
Inoculation: Surface, streaking out for single colonies.
Incubation: 30˚C aerobically for 24 hours.
Growth Characteristicscolony size shape &
organism (mm) surface colour other
Y. enterocolitica 1.0-2.5 CV.E.G. Red centre Colony varieswith strain,
may berough &irregular
Citrobacterspp. 2.5-3.0 CV.E.G. Pale pink (may
not grow)colony
Gram +veorganisms no growth
ReferencesSchiemann, D. A. 1979. Synthesis of a selective agar medium forYersinia enterocolitica. Can. J. Microbiol. 25: 1298-1304.
Schiemann, D. A. 1982. Development of a two step enrichmentprocedure for recovery of Yersinia enterocolitica from food. Appl.Eniviron. Microbiol. 43: 14-27.
Mossel, D. A. A. 1987. Cefsulodin Irgasan Novobiocin (C.I.N.) agar.Int. J. Food. Microbiol. 5: 208, 209.
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Lyophilised Selective Agents
Presentation and Shelf LifeLAB M lyophilised supplements are presented in packs of 10 vials,and for the majority of the supplements each vial is sufficient for500mls of medium. Larger and smaller volumes are indicated forrelevant products.
The shelf life of freeze-dried supplements is 2-3 years provided theyare stored in a refrigerator at 2-8˚C. Once rehydrated the stability ofantibiotics varies greatly and will determine the shelf life of theprepared agars and broths. For this reason any unused, rehydrated,supplement should be discarded, as even deep-freezing may notprevent the rapid degradation of the antibiotics. To ensure the correctlevel of selective supplements the entire vial contents must be addedto the stated volume of cooled, molten medium.
RehydrationVials should be rehydrated aseptically using a sterile needle andsyringe charged with 5mls of the specified diluent for the particularsupplement being added. The supplement should be rehydrated,withdrawn and added to the medium in a single process, followed byimmediate disposal of the syringe into an approved container. Underno circumstances attempt to re-sheath an exposed needle.
If sterile needles and syringes are not readily available, the rubberstopper may be completely removed and, using careful aseptictechnique, rehydrate the supplement using a sterile pipette.
AdditionMost antibiotics are heat labile, and so to prevent a reduction ofpotency the medium should be cooled to 47˚C by holding in a waterbath set at this temperature.
Once the supplement has been added the medium must be gently butthoroughly mixed to ensure that the selective agents are evenlydistributed. Failure to do this will result in a range of concentrationsin the plates/bottles and consequent inconsistency in results. Theshelf life of supplemented media is governed by the stability of theadded components, and is generally shorter than unsupplementedagars and broths. For information on the shelf life of prepared mediaconsult the individual product listings in the previous section of themanual.
AnaerobesX090, X290
NALIDIXIC ACID, VANCOMYCIN for the isolation of Gramnegative anaerobes from clinical material.
Suitable for use with LAB 90 Fastidious Anaerobe Agar. When usedwith other blood agar bases, e.g. LAB 1 Columbia Agar, furtherenrichment of the medium with haemin and menadione is beneficial.
Final Concentration mg/litre
Nalidixic acid 10
Vancomycin 2.5
Add 1 vial X090 to 500mls medium
Add 1 vial X290 to 1 litre medium
Rehydrate contents of vial with 5mls sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C together with otheradditives, mix gently and pour.
Reference:Wren, M. W. D., 1980. J. Clin. Path. 33: 61-65. Multiple SelectiveMedia for the isolation of anaerobic bacteria.
X091, X291NALIDIXIC ACID for the isolation of non-sporing anaerobesfrom clinical material.
Suitable for use with LAB 90 Fastidious Anaerobe Agar. When usedwith other blood agar bases, e.g. LAB 1 Columbia Agar, furtherenrichment of the medium with haemin, menadione and sodiumpyruvate is beneficial. The addition of Tween 80, which, enhances thegrowth of anaerobic cocci, to the medium is required for N.A.T.medium. The Tween 80 may be added before sterilisation at aconcentration of 0.1%.
Final Concentration mg/litre
Nalidixic acid 10
Add 1 vial X091 to 500mls medium
Add 1 vial X291 to 1 litre medium
Rehydrate contents of each vial with 5mls sterile deionised water.Add aseptically to sterilised medium cooled to 47˚C together withother additives, mix gently and pour.
Reference:Wren, M. W. D., 1980. J. Clin. Path. 33: 61-65. Multiple SelectiveMedia for the isolation of anaerobic bacteria.
X092METRONIDAZOLE, NALIDIXIC ACID for the isolation ofActinomyces spp. from clinical material.
Suitable for use with LAB 90 Fastidious Anaerobe Agar. Themetronidazole will suppress the growth of most other anaerobes.
Final Concentration mg/litre
Metronidazole 10
Nalidixic acid 30
Add 1 vial X092 to 500mls. medium.
Rehydrate contents of each vial with 5mls sterile deionised water.Add aseptically to sterilised medium cooled to 47˚C together withother additives, mix gently and pour.
Reference:Stannard, A. E., National Hospital for Nervous Diseases, London.Personal Communication.
X015, X215NEOMYCIN 75 for the isolation of Clostridium spp. and otheranaerobes.
When added to blood agar the resulting medium will allow thegrowth of clostridia, most Bacteriodes fragilis strains and someanaerobic cocci.
Final Concentration mg/litre
Neomycin 75
Add 1 vial X015 to 500mls medium
Add 1 vial X215 to 1 litre medium
Reconstitute each vial by the addition of 5mls of sterile deionisedwater. Add aseptically to sterilised medium cooled to 47˚C, mixgently and pour.
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X016NEOMYCIN 100 for the selective isolation of Clostridium spp.
When added to egg yolk medium this supplement will allow thegrowth of clostridia whilst inhibiting other lecithinase producingorganisms.
Final Concentration mg/litre
Neomycin 100
Add 1 vial X016 to 500mls medium
Reconstitution as X015.
X018KANAMYCIN 75 for the selective isolation of Clostridium spp.and other anaerobes.
An alternative to X015. Kanamycin is more inhibitory to anaerobiccocci.
Final Concentration mg/litre
Kanamycin 75
Add 1 vial X018 to 500mls medium
Reconstitution as X015, X016.
References:Lowbury, C. J. L., Lilly, H. A. 1955. A selective plate medium for Cl.welchi. J. Path. & Bact. 70: 105.
Collee, J. G., Watt, B. 1971. Changing approaches to the sporinganaerobes in medical microbiology. Spore Research ed. A. N.Barkeer.
Sutter, V. L., Citron, D. M., Edelstein, M. A. C., Finegold, S. M.1985. Wadsworth Anaerobic Bacteriology Manual 4 ed. Starpublishers, Belmont, California.
Wren, M. W. D. 1980. Multiple selective media for the isolation ofanaerobic bacteria from clinical specimens. J. Clin. Path. 33: 61-65.
Bacillus cereusX073
EGG YOLK EMULSION.
A concentrated sterile emulsion of top quality egg yolks which maybe incorporated into several media formulations for the detection oflecithinase production by micro organisms.
The most common use is in Bacillus Cereus Medium LAB073, but itcan also be used in media for staphylococci and with Fildes extractand serum to prepare Nagler plates for clostridia.
Presented in 100ml bottles, add 100ml to 900ml of Bacillus cereusmedium, or 50ml to Blood Agar Base LAB028 containing Fildesextract and serum.
X074POLYMYXIN for the isolation of B. cereus from foods.
Suitable for the preparation of LAB 73 Bacillus cereus Medium(P.R.E.P.). The addition of X073 sterile egg yolk emulsion is alsorequired.
Final Concentration
Polymyxin B 8mg/litre = 64,000i.u/litre
Add 1 vial X074 to 500mls medium
Rehydrate contents of vial with 5mls of sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C together with eggyolk emulsion, mix gently and pour.
Reference:Micro-organisms in Food. Ed. Thatcher, F. S., Clarke, D. F. publishedby Univ. of Toronto Press.
Campylobacter speciesX112, X212
CEFOPERAZONE, AMPHOTERACIN for the isolation ofCampylobacter spp. from clinical, environmental and foodsamples.
Suitable for use with LAB 112 Campylobacter Selective Medium(blood free) or with blood agar media. Incubation at 37˚C gives betterresults than at 42˚C and is generally more convenient.
Final Concentration mg/litre
Cefoperazone 32
Amphotericin 10
Add 1 vial X112 to 500mls medium
Add 1 vial X212 to 1 litre medium
Rehydrate contents of vial with 5mls of sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C, mix gently and pour.
Reference:Bolton, F. J., Hutchinson, D. N., Parker, G. 1988. Reassessment ofSelective Agars and Filtration Techniques for Isolation ofCampylobacter Species from Faeces. Eur. J. Clin. Microbiol. Infect.Dis. 7: 155-160.
X214VANCOMYCIN, POLYMYXIN, TRIMETHOPRIM, to makeSkirrow’s medium for the isolation of Campylobacter spp.
Suitable for use with LAB001 Columbia Agar or other blood agarbases, supplemented with lysed horse blood.
Final Concentration mg/litre
Vancomycin 10
Polymyxin 2500 iu/litre
Trimethoprim 5
Add 1 vial of X214 to 1 litre of medium
Rehydrate contents of vial with 5ml sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C, along with otheradditives, mix well and pour.
Reference:Skirrow, M.B. (1977) British Medical Journal 2 11-9.
X131C.V.T.C. For the isolation of Campylobacter spp. from food andenvironmental samples by the enrichment broth technique.
Developed for use with LAB 135 Campylobacter Enrichment Broth.Gives higher isolation rates than Preston broth and does not requiremodified atmosphere incubation.
Final Concentration mg/litre
Cefoperazone 20
Vancomycin 20
Trimethoprim 20
Cycloheximide 50
Add 1 vial X131 to 500mls medium
Rehydrate contents of vial with 5mls of sterile 50% alcohol. Addaseptically to sterilised medium cooled to 47˚C, mix gently anddispense into sterile containers.
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Reference:Bolton, F. J., Preston., P. H. L. S. Personal communication, 1989.
Clostridium difficileX093
CYCLOSERINE, CEFOXITIN for the isolation of Clostridiumdifficile from clinical materials.
Suitable for use with LAB 90 Fastidious Anaerobe Agar.
Final Concentration mg/litre
D-Cycloserine 250
Cefoxitin 8
Add 1 vial X093 to 500mls medium
Rehydrate contents of vial with 5mls of water. Add aseptically tosterilised medium cooled to 47˚C together with other additives, mixgently and pour.
Reference:George, W. L., Sutter, V. L., Citron, D., Finegold, S. M. 1976.Selective and differential medium for isolation of Clostridiumdifficile.
Clostridium perfringensX109, X110
SULPHADIAZINE (X109). OLEANDOMYCIN PHOSPHATE,POLYMIXIN (X110).
For use with LAB 109 Perfringens agar to prepare O.P.S.P. for theselective isolation of Clostridium perfringens from foodstuffs.
Final Concentration mg/litre
Sulphadiazine 100
Oleandomycin 0.5
Polymyxin 10,000 i.u./litre
Add 1 vial X109 and 1 vial X110 to 500mls medium
Rehydrate contents of vials with 5mls of sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C, mix gently and pour.
Reference:Handford, P. M. 1974. J. Appl. Bact. 37, 559-570.
Escherichia coliX150
NOVOBIOCIN for the enrichment of E.coli O157:H7 from food,environmental and clinical samples.
For the addition to LAB165 O157 Broth MTSB
Final concentration mg/litre
Novobiocin 20
Rehydrate contents of vial with 5ml sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C, mix well and pour.
Add 1 vial of X150 to 500ml of O157 Broth MTSB.
X161CEFIXIME TELLURITE supplement for the isolation of E.coliO157:H7 from food, environmental and clinical samples.
For the addition to LAB161 Sorbitol MacConkey Agar (SMAC)
Final concentration mg/litre
Cefixime 0.05
Potassium tellurite 2.5
Rehydrate contents of vial with 5ml sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C, mix well and pour.
Add 1 vial of X161 to 500ml of Sorbitol MacConkey Agar (SMAC).
X546V.C.C. Supplement for the selective enrichment of E.coli 0157:H7from food and other samples.
For use with Buffered Peptone Water LAB046
Final Concentration mg/litre
Vancomycin 8.0
Cefixime 0.05
Cefsulodin 10.0
Add 1 vial of X546 to 2.25 litres of LAB046
Rehydrate the contents of one vial with 20mL of sterile deionisedwater. Add aseptically to sterilised medium cooled to 47˚C. Mix welland dispense into 225mL aliquots.
Gardnerella vaginalisX011
COLISTIN, NALIDIXIC ACID for the isolation of G. vaginalisfrom clinical material.
Suitable for addition to LAB 1 Columbia Agar or LAB 15 BloodAgar Base No. 2 to produce a selective isolation medium.
Final Concentration mg/litre
Colistin 10
Nalidixic acid 15
Add 1 vial X011 to 500mls medium
Rehydrate contents of vial with 5mls sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C, together with anyother additives, mix gently and pour.
Reference:Goldberg, R. L., Washington, J. A. II 1976. “Comparison of Isolationof Haemophilus vaginalis (Corynebacterium vaginalae) fromPeptone-Starch-Dextrose Agar and Columbia, Colistin, NalidixicAcid Agar. J. Clin. Microbiol. 4(3): 245.
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Gram Positive CocciX012
COLISTIN, NALIDIXIC ACID for the preparation of ColumbiaC.N.A. medium.
A medium selective for Gram positive cocci is obtained when thisantibiotic mixture is added to LAB 1 Columbia Agar.
Final Concentration mg/litre
Colistin 10
Nalidixic acid 10
Add 1 vial X012 to 500mls medium
Rehydrate contents of vial with 5mls sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C, together with anyother additives, mix gently and pour.
Reference:Ellner, P. D., Stossel, C. I. Drakeford, E. Vasi, F. 1966. “A new culturemedium for medical bacteriology.” Amer. J. Clin. Path. 45: 502.
Haemophilus influenzaeX260
BACITRACIN for the isolation of Haemophilus influenzae.
Suitable for use with Columbia blood agar base and other blood agarssupplemented with heated (“chocolated”) blood.
Final Concentration mg/litre
Bacitracin 75
Add 1 vial of X260 to 1 litre of medium.
Rehydrate contents of vial with 5ml sterile deionised water. Addaseptically to sterilised medium with heated blood cooled to 47˚C,mix well and pour.
Helicobacter pyloriX040
VANCOMYCIN, CEFSULODIN, AMPHOTERICIN, for theisolation of Helicobacter pylori.
For addition to Helicobacter pylori medium LAB140
Final Concentration mg/litre
Cefsulodin 10
Vancomycin 10
Amphotericin 20
Add 1 vial to 500ml medium
Rehydrate contents of vial with 5ml sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C, along with otheradditives, mix well and pour.
Impedance MicrobiologyX137
T.M.A.O. Selenite for inclusion in Easter and Gibson SalmonellaDetection Medium LAB 137.
The growth of Salmonella in the medium reduces T.M.A.O. toT.M.A. and in so doing, significantly increases the conductivity of themedium. The incorporation of sodium biselenite makes the mediumselective for salmonellae.
Final Concentration g/litre
T.M.A.O. (Trimethylamine-N-oxide) 5.0
Sodium biselenite 4.0
Add 1 vial X137 to 100mls medium
Reconstitute contents with 5mls of sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C. Swirl to mix thendispense into sterile containers.
References:Easter, M. C., Gibson, D. M. 1985. Rapid and automated detection ofSalmonella by electrical measurements, J. Hyg. 94: 245-262.
Gibson, D. M. 1987. Some modifications to the media for rapidautomated detection of salmonellas by conductance. H. Appl.Bacteriol. 63: 299-304.
Odgen, I. D., Cann, D. C. 1987. A modified conductance medium forthe detection of Salmonella spp. J. Appl. Bacteriol. 63: 359-464.
Listeria monocytogenesX122
C.C.C.A.F. Cefotetan, Cycloheximide, Colistin, Acriflavine,Fosfomycin, for the isolation of Listeria monocytogenes fromenvironmental, clinical and food samples.
For addition to LAB 122 Listeria Isolation Medium.
Final Concentration mg/litre
Cefotetan 2
Cycloheximide 400
Colistin 20
Fosfomycin 10
Acriflavine 5
Add 1 vial X122 to 500mls medium
Reconstitute contents of vial by the addition of sterile 50% ethanol inwater. Add aseptically to sterilised medium cooled to 47˚C, mixgently then pour.
Reference:Curtis, et al. 1989. A selective differential medium for the isolation ofListeria monocytogenes. Lett. in Appl. Microbiol. 8: 95-98.
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X138N.A.C. Nalidixic Acid, Acriflavine, Cycloheximide for theselective enrichment broth culture of Listeria monocytogenes.
For addition to LAB 138 Listeria Enrichment Broth recommended bythe F.D.A. for Listeria isolation from food and environmentalsamples.
Final Concentration mg/litre
Nalidixic acid 40
Cycloheximide 50
Acriflavine 15
Add 1 vial of X138 to 500mls medium
Reconstitute contents of vial by the addition of sterile 50% ethanol inwater. Add aseptically to sterilised medium cooled to 47˚C, mixgently then pour.
Reference:Lovett et al. 1987. Listeria monocytogenes in raw milk: detectionincidence and pathogenicity. J. Food Protect. 50: 188-192.
X144P.A.C. supplement for the enrichment and isolation of Listeriaspp from food and environmental samples.
For the addition to LAB144 Palcam Broth and Lab148 Palcam Agar
Final concentration
Polymixin 10mg/litre
Acriflavine 5mg/litre
Ceftazidime 20mg/litre
Rehydrate contents of vial with 5ml sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C, along with otheradditives, mix well and pour.
Add 1 vial of X144 to 500ml of Palcam Broth or Palcam Agar.
X164, X5641/2 FRASER supplement for the primary enrichment of Listeriaspp from food and environmental samples.
For addition to LAB164 Fraser Broth Base
Final Concentration mg/litre
Ferric ammonium citrate 500
Acriflavine 12.5
Nalidixic acid 10
Rehydrate contents of vial with 2ml 50% methanol (5ml for X564).Add aseptically to sterilised medium cooled to 47˚C, mix well and pour.
Add 1 vial of X164 to 450ml of Fraser Broth Base
Add 1 vial of X564 to 2.25 litres of Fraser Broth Base
X165FRASER supplement for the secondary enrichment of Listeriaspp from food and environmental samples.
For addition to LAB164 Fraser Broth Base
Final Concentration mg/litre
Ferric ammonium citrate 500
Acriflavine 25
Nalidixic acid 20
Rehydrate contents of vial with 2ml 50% methanol. Add asepticallyto sterilised medium cooled to 47˚C, mix well and pour.
Add 1 vial of X165 to 500ml of Fraser Broth Base
X155, X555 UVMI. Supplement for the primary enrichment of Listeria sppfrom food and environmental samples.
For addition to LAB155 UVM Broth Base
Final Concentration mg/litre
Nalidixic acid 20
Acriflavine 12
Rehydrate contents of vial with 5ml sterile deionised water (10ml forX555). Add aseptically to sterilised medium cooled to 47˚C, mix welland pour.
Add 1 vial of X155 to 500ml of UVM Broth Base
Add 1 vial of X555 to 2.25 litres of UVM Broth Base
X156UVMII. Supplement for the secondary enrichment of Listeria sppfrom food and environmental samples
For the addition to LAB155 UVM Broth Base
Final Concentration mg/litre
Nalidixic acid 20
Acriflavine 25
Rehydrate contents of vial with 5ml sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C, mix well and pour.
Add 1 vial of X156 to 500ml of UVM Broth Base
Neisseria gonorrhoeaeX070, X270
L.C.A.T. Lincomycin, Colistin, Amphotericin, Trimethoprim for theisolation of Neisseria spp. from clinical material.
L.C.A.T. is often preferred to X068 V.C.N.T. for the isolation of N. gonorrhoeae because of the emergence of vancomycin sensitivestrains. The antifungal agent amphotericin is more readily soluble andtherefore a more active antifungal than nystatin. L.C.A.T. is quoted asthe selective agent for New York City G.C. agar but can readily besubstituted for V.C.N. or V.C.N.T. in Thayer Martin G.C. agar.
Final Concentration mg/litre
Lincomycin 1
Colistin 6
Amphotericin 1
Trimethoprim 6.5
Add 1 vial X070 to 500mls medium
Add 1 vial X270 to 1 litre medium
Rehydrate contents of vial with 5mls sterile 25% alcohol in water.Add aseptically to sterilised medium cooled to 47˚C together withother additives, mix gently and pour.
Reference:Young, H. 1978. Cultural Diagnosis of Gonorrhoea with modifiedN.Y.C. Medium. Brit. Journ. Ven. Dis. 54: 36-40.
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X069, X269L.C.T. Lincomycin, Colistin, Trimethoprim. A variant ofL.C.A.T. with the amphotericin omitted to permit the growth ofyeasts.
Concentrations and rehydration as L.C.A.T.
Add 1 vial X069 to 500mls medium
Add 1 vial X269 to 1 litre medium
X068, X268V.C.N.T. Vancomycin, Colistin, Nystatin, Trimethoprim forThayer Martin Medium.
The addition of trimethoprim in V.C.N.T. inhibits the swarming ofProteus spp. which occasionally make interpretation difficult.
Final Concentration mg/litre
Vancomycin 3
Colistin 7.5
Nystatin 12.5
Trimethoprim 5
Add 1 vial X068 to 500mls medium
Add 1 vial X268 to 1 litre medium
Rehydration as for X067.
Reference:Thayer, J. D. and Martin, J. E. 1966. Improved medium selective forthe cultivation of N. gonorrhoeae and N. meningitidis. Public Healthrep. 81: 559-562.
X271GROWTH SUPPLEMENT, to improve the isolation of Neisseriaspp from selective media.
For addition to GC agar base LAB067.
Final Concentration mg/litre
L-cystine 11
L-cysteine 259
Thiamine HCl 0.03
Ferric nitrate 0.2
Co-Carboxylase 1
NAD 1.0
Guanine HCl 0.3
Adenine 10
L-glutamine 100
PABA 0.13
Vitamin B12 0.1
Add 1 vial to 1 litre of medium
Rehydrate contents of vial with 5ml sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C, along with otheradditives, mix well and pour.
Pseudomonas speciesX108
MODIFIED C.F.C. – Cephalothin, Fucidin, Cetrimide for theselective isolation of Pseudomonas spp.
When added to LAB 108 Pseudomonas Agar, to prepare C.F.C.medium this supplement can be used to select pseudomonads fromfood and environmental samples.
Final Concentration mg/litre
Cephalothin 50
Fucidin 10
Cetrimide 10
Add 1 vial X108 to 500mls medium
Rehydrate contents of vial with 5mls of sterile 50% alcohol. Addaseptically to sterilised medium cooled to 47˚C, mix gently and pour.
Reference:Mead, G. C. and Adams, B. W. 1977. Br. Poult. Sci. 18: 661-667.
X107C.N. Cetrimide, Nalidixic acid for the isolation of Pseudomonasaeruginosa.
Suitable for use with LAB 108 Pseudomonas Agar to make themedium selective for Ps. aeruginosa.
Final Concentration mg/litre
Cetrimide 200
Nalidixic acid 15
Add 1 vial X107 to 500mls medium
Rehydrate contents of vial with 5mls of sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C, mix gently and pour.
Reference:Goto, S., Enomoto, S. 1970. Jap. J. Microbiol. 14: 65-72.
X140TICARCILLIN, POLYMYXIN, for the isolation of Burkholderia(Pseudomonas) cepacia
Suitable for use with LAB108 pseudomonas selective agar, orspecific selective bases such as that described by Gilligan et al.
Final Concentration mg/litre
Ticarcillin 100
Polymyxin 300,000 iu/litre
Add 1 vial to 500ml of medium
Rehydrate contents of vial with 5ml sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C, mix well and pour.
Reference:Gilligan P.H., Gage P.A., Bradshaw L.M., Schidlow D.V., DeCiccoB.T. (1985) Isolation medium for the recovery of Pseudomonascepacia from respiratory secretions of patients with cystic fibrosis.J.Clin.Microbiol. 22 (1) 5-8.
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Pre-Incubation Test (P-INC)X019, X219
PENICILLIN, NISIN, CRYSTAL VIOLET, for accelerated shelflife determination of dairy products.
The Pre-incubation test uses a selective mixture to inhibit Grampositive organisms whilst allowing the growth of Gram negativebacteria, the main cause of post-pasteurisation contamination and amajor factor in determining the shelf life of the product.The technique is also useful for monitoring plant hygiene.
Final Concentration mg/litre
Penicillin 20,000iu/litre
Nisin 40,000iu/litre
Crystal violet 2.0
Add 1 vial of X019 to 200ml of Milk Agar LAB019
Add 1 vial of X219 to 1 litre of Milk Agar LAB019
Rehydrate contents of 1 vial with 5mls sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C, mix thoroughly andpour plates.
Method APre-incubate test material at 21˚C for 24hr. Prepare suitable dilutionseries, and inoculate Milk Agar plates containing P-INC supplement.Incubate at 21˚C for 24hr, and count all colonies (some may be small,use of a hand lens is recommended). Calculate the CFU/ml and usingthe tables of Griffith’s et al the shelf life can be determined.
Method BRehydrate X219 with 1ml of deionised water only, add 0.1ml to thetest material and incubate at 20˚C for 24hr. Prepare suitable dilutionseries, and inoculate Milk Agar plates. Proceed as for Method Aabove.
References:Griffiths M.W., and Phillips J.D. (1985) J.Appl.Bact. 57, 107.
Griffiths M.W., and Phillips J.D., and Muir D.D. (1980) J. Soc. DairyTechnol. 33, 8.
Griffiths M.W., and Phillips J.D., and Muir D.D. (1981) J. Soc. DairyTechnol. 34, 142.
Griffiths M.W., and Phillips J.D., and Muir D.D. (1984) J. Soc. DairyTechnol. 37, 22.
Griffiths M.W., and Phillips J.D., and Muir D.D. (1984) Rapiddetection of post-pasteurised contamination. Hannah Research Inst.Bulletin No.10.
Griffiths M.W., and Phillips J.D., and Muir D.D. (1984) Dairy Ind.Int. 50 (3) 25
Griffiths M.W., and Phillips J.D., and Muir D.D. (1984) Post-pasteurisation contamination - the major cause of failure of freshdairy products. Hannah Research Inst.
Griffiths M.W., and Phillips J.D., and Muir D.D. (1986) Aust. J.Dairy Technol. 41, 77-79.
SalmonellaX150
NOVOBIOCIN, for the isolation of Salmonella using semi-solidtechnology
For addition to LAB150 MSRV and LAB537 Diassalm
Final Concentration mg/litre
Novobiocin 20 (MSRV)
Novobiocin 10 (Diassalm)
Add 1 vial to 500ml (MSRV)
Add 1 vial to 1 litre (Diassalm)
Rehydrate contents of vial with 5ml sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C, mix well and pour.
References:De Smedt J.M., and Bolderdijk R.F., (1986) Dynamics of salmonellaisolation with modified semi-solid Rappaport Vassiliadis medium.J.Food Protection 50 658-661
Van Netten P, Van Der Zee H., and Van Der Moosdijk A., (1991) Theuse of diagnostic selective semi-solid medium for the isolation ofSalsmonella enteritidis from poultry. Proceedings of the 10thsymposium on the quality of poultry meat. Spelderholt Beckbergen56-67.
StaphylococciX085
EGG YOLK TELLURITE
A sterile emulsion of egg yolk and potassium tellurite for use as aselective and differential agent in Baird Parker Medium BaseLAB085. The complete medium is selective for S.aureus, and theaddition of egg yolk tellurite aids differentiation of this organismfrom others capable of growing on the agar.
Presented in 100ml bottles with a tellurite concentration of 0.2% togive a final concentration in the complete medium of 0.01% (w/v).Add 50ml to 1 litre of Baird Parker Medium Base.
X207METHICILLIN, for the isolation of Methicillin ResistantS.aureus (MRSA)
Suitable for use with LAB007 Mannitol salt agar.
Final Concentration mg/litre
Methicillin 4
Add 1 vial of X207 to 1 litre of medium
Rehydrate contents of vial with 5ml sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C, mix well and pour.
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StreptococciX013
COLISTIN, OXOLINIC ACID for the selective isolation ofstreptococci from clinical material.
When added to LAB 1 Columbia agar or LAB 15 Blood Agar BaseNo. 2, X013 renders the medium selective for streptococci. Alterationin haemolysis patterns may occur when azide or crystal violet areemployed as selective agents but this does not occur with X013.
Final Concentration mg/litre
Colistin 10
Oxolinic acid 5
Add 1 vial X013 to 500mls medium
Rehydrate contents of vial with 5mls of sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C together with otheradditives, mix gently and pour.
Reference:Petts, D. 1984. Colistin - Oxolinic Acid - Blood Agar: a new selectivemedium for streptococci. J. Clin. Microbiol. 19: 4-7.
Yeasts and MouldsX009, X209
CHLORAMPHENICOL for the selective isolation of yeasts andmoulds from food, environmental and clinical specimens.
Chloramphenicol’s broad antibiotic spectrum suppresses mostcontaminating bacteria allowing the yeasts and moulds to grow. It canbe added to such media as LAB 95 Sabouraud Dextrose Agar, LAB36 Rose Bengal Chloramphenicol Agar, LAB 37 Malt Extract Agarand LAB 117 Dermatophyte Test Medium to increase their selectivitywhilst not lowering the pH. Reduction of pH will increase theselectivity of a yeast and mould medium but will also inhibit someyeasts as well as having a deleterious effect on the agar gel.
Final Concentration mg/litre
Chloramphenicol 100
Add 1 vial X009 to 500mls medium
Add 1 vial X209 to 1 litre medium
Rehydrate contents of vial with 5mls of Ethyl or Methyl alcohol. Addaseptically to sterilised medium cooled to 47˚C, mix gently and pour.
References:Jervis, B. 1973. Rose Bengal Chlortetracycline agar with other mediafor the selective isolation and enumeration of moulds and yeasts infoods. J. Appl. Bact. 36 Pages 723-727.
X089OXYTETRACYCLINE for O.G.Y.E. medium.
For use with LAB 89 Oxytetracycline Glucose Yeast Extract Agar forthe enumeration of yeasts and moulds from foodstuffs. Highlyproteinaceous foods and incubation above 30˚C will inactivateoxytetracycline.
Final Concentration mg/litre
Oxytetracycline 100
Add 1 vial X089 to 500mls medium
Rehydrate contents of vial with 5mls sterile deionised water. Addaseptically to sterilised medium cooled to 47˚C, mix gently and pour.
References:Mossel, D. A. A., et al. 1970. O.G.Y.E. for the selective enumerationof moulds and yeasts in food and clinical material. J. Appl. Bact. 35:454-457.
YersiniaX120
C.I.N. - Cefsulodin, Irgasan, Novobiocin for the isolation ofYersinia spp. from clinical and environmental material.
For addition to LAB 120 Yersinia C.I.N. Agar Base used in theselective isolation of Y. enterocolitica.
Final Concentration mg/litre
Cefsulodin 15
Irgasan 4
Novobiocin 2.5
Add 1 vial X120 to 500mls medium
Rehydrate contents of vial with 5mls of 30% sterile alcohol. Addaseptically to sterilised medium cooled to 47˚C, mix gently and pour.
References:Schiemann, D. A. 1979. Synthesis of a selective medium of Yersiniaenterocolitica. Can. J. Microbiol. 25 (2): 1298.
Schiemann, D. A. 1980. Isolation of toxigenic Yersinia enterocoliticafrom retail pork products. J. Food Prot. 43: 360.
Schiemann, D. A. 1982. Development of a two-step enrichmentprocedure for recovery of Yersinia enterocolitica from food. Appl.Microbiol. 43 (1): 14.
104
Agars, Peptones, Extracts &Other Media Constituents
SourcingThe Lab M range of media constituents are selected on the basis ofquality and performance from the world’s leading suppliers. It is adeliberate policy not to invest in our own peptone manufacturingfacility in order to allow our microbiologist freedom to choose thebest ingredients available on the international market.
AgarsA range of agars are offered to suit all microbiological applications.Koch originally used gelatin to solidify culture media, but thesuperior properties of agar resulted in its universal adoption as thegelling agent of choice. Careful selection of agars is vital as they caninteract with nutrient components in a beneficial or deleteriousmanner.
Peptones and ExtractsLike agars, peptones and extracts are biologically variable productsrequiring careful selection. They provide the amino acids andpeptides required by micro-organisms for growth as well as othervital growth factors such as minerals, vitamins and nucleic acidfractions.
To ensure we use only the best available peptones and extracts thesematerials are exhaustively tested. Growth parameters are obtained byclassical microbiological techniques and by automated growth rateanalysis. Chemical and physical properties are also closelymonitored. LAB M can select specific peptones for special purposessuch as vaccine production and fermentation processes. If moreinformation is required on special services please contact LAB M oryour local agent.
Acid Hydrolysed CaseinMC 7
A soluble protein hyrolysate obtained by digesting casein with hotacid. It is almost free from growth factors, vitamins and antagonists,and these qualities make it suitable for use as a protein source inmedia for antibiotic and vitamin assays.
Typical Analysis
Appearance cream/white powder
Solubility in water at 5% total
Clarity clear and colourless
pH of 2% solution 6.0 ± 0.5
Total Nitrogen 8.3% ± 0.5
Total Amino Nitrogen 6.1% ± 0.5
Agar No. 1MC 2
A high clarity agar with good gelling properties and a lowconcentration of metal ions. This agar is suitable for allbacteriological purposes including sensitivity testing and pour platetechniques. A firm gel is obtained at working concentrations of 1.0 to1.5%. No significant precipitation is observed on reheating orprolonged holding at 65˚C.
Typical Analysis
Gel strength (Nikan) 650-1000g/m2
Colourimetry (1.5% soln at 65˚C) > 0.28 at 340nm
> 0.02 at 525nm
Melting point > 85˚C
Setting point 32-35˚C
pH 6.5-7.4
Moisture < 10%
Total ash < 3%
Calcium < 0.02%
Magnesium < 0.02%
Sodium chloride < 1.0%
Iron < 0.01%
Insoluble ash < 0.1%
Sulphate 1.5%
Salmonella Absent
TVC < 103/g
Spores < 2/g
Agar No. 2MC 6
A bacteriological agar which gives a firm gel at workingconcentrations of 1.0 to 1.5% which is reasonably clear. This agar isrecommended for all culture media except sensitivity testing mediaand those where absolute clarity is advantageous.
Typical Analysis
Gel strength (Nikan) 650-1000g/m2
Colourimetry (1.5% soln at 65˚C) > 0.3 at 340nm
>0.04 at 525nm
Appearance cream/white powder
Melting point >85˚C
Setting point 32-35˚C
pH 6.5-7.4
Moisture <12%
Total ash <3.5%
Calcium < 0.5%
Magnesium < 0.1%
Sodium chloride <1.0%
Iron < 0.01%
Insoluble ash < 0.1%
Sulphate < 3.0%
Salmonella Absent
TVC < 103/g
Spores < 2/g
105
Agar No.4Plant Tissue Culture Grade
MC 29Lab M Agar No.4 has been selected specifically for use as a gellingagent in plant tissue culture techniques. The product is selectedprimarily on gel strength, a parameter of particular importance forthis application, and then tested to ensure it meets the parameters setby a major plant producer. The agar contains no nutrients for plantgrowth and is designed to be incorporated into classical formulationssuch as Murashige and Skoog, as well as customer’s ownformulations.
Typical analysis
Ash 2.30%
Acid Insoluble Ash 0.16%
Calcium 0.31%
Magnesium 0.12%
Iron 0.018%
Total Nitrogen 0.15%
Recommended Concentration 0.75 - 1.5%
Melting Point 88-91˚C
Setting Point 32-33˚C
Mesh 80
pH (1.5% at 20˚C) 7.0 ± 0.2
Gel Strength (1.5% W/V) >700g/cm2
Bacteriological PeptoneMC 24
An economical source of nutrients provided by a balanced mixture ofmeat peptones and tryptone. The growth requirements of most nonfastidious organisms will be fulfilled by the range of amino acids,peptides and proteoses in this mixture.
Typical Analysis
Appearance cream powder
Solubility in water at 5% total
Clarity clear, pale straw colour
pH of 2% solution 7.2 ± 0.2
Total Nitrogen 12% ± 0.5
Amino Nitrogen 5% ± 0.5
Balanced Peptone No. 1MC 4
A rich mixture of tryptone and meat peptones which fulfills thenutritional demands of a wide variety of micro-organisms. Thispeptone is used in many LAB M culture media formulations.
Typical Analysis
Appearance beige powder
Solubility in water at 5% total
Clarity clear, pale straw colour
pH of 2% solution 7.2 ± 0.2
Total Nitrogen 12.8% ± 0.5
Amino Nitrogen 5.1% ± 0.5
Beef ExtractMC 19
This complements the nutritive properties of peptones in culturemedia and is often used as an added enrichment. Beef extract can beused as a direct replacement for meat peptones and, as it contains nocarbohydrates, can be used as a component of media for fermentationstudies.
Typical Analysis
Appearance light brown powder
Solubility in water at 5% total
Clarity clear, light brown colour
pH of 2% solution 7.0 ± 0.2
Total Nitrogen 12.0% ± 0.5
Amino Nitrogen 1.6% ± 0.5
Bile Salts No. 3MC 25
A refined bile salt, comprising mainly sodium cholate and sodiumdesoxycholate. It is used as a selective agent in culture media such asViolet Red Bile Agar for the enumeration of coliforms, MacConkeyAgar No. 3 for the isolation and differentiation of enteric organisms,and SS Agar for the isolation of enteric pathogens. This fraction ofbile is highly active, allowing maximum selection of organisms ofenteric origin at relatively low concentrations (0.15%).
Typical Analysis
Appearance white powder
Solubility in water at 2% total
Clarity clear
pH of a 2% solution 8.0 ± 0.5
106
Malt ExtractMC 23
A water soluble extract of malted barley suitable for use in thecultivation of yeasts and moulds. Malt extract has a very highcarbohydrate content and consequently is very sensitive to overheating which will cause a darkening of the medium.
Typical Analysis
Appearance yellow/brown powder
Solubility in water at 5% total
Clarity clear, light brown colour
pH of 2% solution 5.2 ± 0.2
Maltose 55%
Other Carbohydrates 40%
Protein 5%
Mycological PeptoneMC 9
A mixture of peptones with a high carbohydrate content suitable forthe rapid growth and colonial development of yeasts and moulds.Bacterial growth is inhibited by the low pH of this peptone.
Typical Analysis
Appearance beige powder
Solubility in water at 5% total
Clarity clear, pale straw colour
pH of 2% solution 5.4 ± 0.1
Total Nitrogen 13% ± 0.5
Amino Nitrogen 1.4% ± 0.5
Proteose Peptone AMC 11
An enzymatic digest of meat adapted to encourage the production oftoxins by Corynebacterium diphtheriae, staphylococci, Salmonella,and clostridia. This peptone is highly nutritious and suitable for usein culture media for fastidious organisms such as Neisseria,Haemophilus and Pasteurella species.
Typical Analysis
Appearance cream powder
Solubility in water at 5% total
Clarity clear, light straw colour
pH of 2% solution 7.0 ± 0.2
Total Nitrogen 12.0% ± 0.5
Amino Nitrogen 5.8% ± 0.5
Skim Milk PowderMC 27
A bacteriological grade of thermophile free spray dried skim milk.Used in Milk Plate Count Agar LAB 115 and in media for diagnostictests involving the digestion or coagulation of casein and thefermentation of lactose. Recommended working concentration 10%.
Typical Analysis
Appearance white powder
Clarity opaque white suspension
Total Nitrogen 5.3% ± 0.5
Lactose 48.0% ± 0.5
Sodium DesoxycholateMC26
Sodium desoxycholate is a specific bile acid, derived fromdeconjugated bile salts. Leifson showed that desoxycholic acid hadthe most inhibitory effect on bacterial growth, and that this could beenhanced by the removal of magnesium ions by chelating withsodium citrate. These components comprise the selective agents inDCA, DCA (Hynes) and DCLS.
Typical Analysis
Appearance white powder
pH of 2% solution 8.3 ± 0.5
Solubility in water at 2% total
Moisture <5%
Heavy metals <20 ppm
Sodium cholate <2%
Soy PeptoneMC 3
Prepared using the enzyme papain to digest soyabean meal, thispeptone is a rich source of nutrients with a high carbohydrate content.Most organisms will grow rapidly in this peptone but some bacteriawill produce high levels of acid leading to auto-sterilisation unless anadequate buffering system is incorporated.
Typical Analysis
Appearance cream powder
Solubility in water at 5% total
Clarity clear, straw colour
pH of 2% solution 7.1 ± 0.2
Total Nitrogen 9.0% ± 0.5
Amino Nitrogen 1.6% ± 0.5
107
TryptoneMC 5
An enzymatic hydrolysate of casein, rich in peptones and amino acids(including tryptophane). This peptone can be utilised by mostbacteria as a growth substrate. This peptone conforms to the U.S.P.requirements for a pancreatic digest of casein.
Typical Analysis
Appearance cream powder
Solubility in water at 5% total
Clarity clear, pale straw colour
pH of 2% solution 7.2 ± 0.5
Total Nitrogen 13.0% ± 0.5
Amino Nitrogen 4.9% ± 0.5
TryptoseMC 8
A blend of peptones suitable for the cultivation of most fastidiousorganisms including Neisseria gonorrhoeae, Streptococcus milleriand Brucella spp. especially where rapid or profuse growth isrequired such as in blood culture media and blood agars.
Typical Analysis
Appearance beige powder
Solubility in water at 5% total
Clarity clear, light straw colour
pH of 2% solution 7.2 ± 0.2
Total Nitrogen 12.5% ± 0.5
Amino Nitrogen 4.9% ± 0.5
Yeast Extract PowderMC 1
Prepared by the autolysis of Saccharomyces cerevisae underthermostatically controlled conditions to protect the B vitamins andother heat labile constituents. This extract provides a mixture ofamino acids, peptides, vitamins and carbohydrates making it suitablefor many applications.
Typical Analysis
Appearance yellow powder
Solubility in water at 5% total
Clarity clear, pale yellow
pH of 2% solution 7.0 ± 0.2
Total Nitrogen 10.5% ± 0.5
Amino Nitrogen 5.3% ± 0.5
108
109
TY
PIC
AL
AN
ALY
SIS
MC
7
MC
24M
C 4
MC
9M
C 1
1M
C 3
MC
5M
C 8
MC
1A
cid
Hyd
roly
sed
Bac
teri
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ical
Bal
ance
dM
ycol
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ose
Soy
Pep
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Try
pton
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rypt
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Yea
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epto
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epto
ne N
o. 1
Pep
tone
Pep
tone
AE
xtra
ct
Tota
l Nitr
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8.3%
12.0
%12
.5%
12.6
%12
.0%
9.0-
9.4%
12.8
%12
.7%
10.5
%
Tota
l Am
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8%4.
8%4.
9%5.
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Am
ino
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otal
N.
73.4
%41
.7%
40.8
%11
.1%
48.0
%17
.7%
37.5
%38
.7%
50.4
%
Tota
l Am
ino
Aci
d A
ssay
(mg/
g)
Lysi
ne53
.562
.075
.338
.948
.243
.179
.777
.549
.0
His
tidin
e18
.721
.026
.812
.715
.616
.231
.229
.014
.0
Arg
inin
e22
.132
.041
.055
.050
.639
.237
.539
.227
.0
Asp
artic
Aci
d44
.169
.058
.469
.963
.974
.462
.860
.652
.0
Thr
eoni
ne23
.540
.031
.117
.727
.521
.736
.934
.033
.0
Seri
ne30
.040
.040
.326
.236
.227
.050
.345
.334
.0
Glu
tam
ic A
cid
130.
016
0.0
138.
910
3.5
100.
211
0.0
184.
016
1.5
73.0
Prol
ine
52.2
46.0
61.0
74.5
55.6
28.0
82.1
71.6
26.0
Gly
cine
11.1
29.0
44.5
105.
983
.38
22.6
15.6
30.0
25.0
Ala
nine
19.0
39.0
38.1
49.9
52.3
23.1
26.9
32.5
51.0
Cys
tine
1.1
1.0
1.1
2.7
8.4
5.3
2.2
1.6
6.0
Val
ine
35.2
45.0
45.3
22.9
35.2
23.7
59.2
52.3
37.0
Met
hion
ine
10.4
8.0
19.1
7.0
12.3
6.2
25.0
22.1
9.0
Isol
euci
ne27
.933
.043
.818
.723
.326
.858
.551
.173
.0
Leu
cine
30.9
65.0
65.3
32.0
55.5
38.6
83.5
74.4
73.0
Tyro
sine
12.7
12.0
9.8
12.8
13.3
16.8
14.7
12.2
12.0
Phen
ylal
anin
e17
.034
.031
.820
.227
.222
.742
.437
.125
.0
Try
ptop
hane
_3.
04.
91.
94.
63.
76.
65.
79.
0
LAB M Peptones
110
NOTES
111
NOTES
112
NOTES
LAB M Limited
Topley House
52 Wash Lane . Bury
Lancashire BL9 6AU . UK
Tel: +44 (0)161-797 5729
Fax: +44 (0)161-762 9322
E-mail: [email protected] A member company of International Diagnostics Group plc
Cover Picture:
Campylobacter jejuni on
Campylobacter Blood Free
Selective Medium
(modified CCDA improved).