BACTERIAL NUTRITION & CULTURE BACTERIAL NUTRITION & CULTURE MEDIAMEDIA

G.HARIPRASAD M.Sc.Med micro M.PhilLECTURERTHOOTHUKUDI GOVT. MEDICAL COLLEGE THOOTHUKUDI

BASIC NUTRITIONAL REQUIREMENTSBASIC NUTRITIONAL REQUIREMENTS

WATER.WATER.CARBON SOURCE.CARBON SOURCE.NITROGEN SOURCE.NITROGEN SOURCE.INORGANIC SALTS.INORGANIC SALTS.Phosphates.Phosphates. Sulfates.Sulfates.Sodium.Sodium. Potassium.Potassium.Magnesium.Magnesium. Iron.Iron.Manganese.Manganese. Calcium.Calcium.BACTERIAL VITAMINS.BACTERIAL VITAMINS.ThiamineThiamine riboflavinriboflavinnicotinic acidnicotinic acid pyridoxinepyridoxinefolic acidfolic acid vitamine Bvitamine B12.12.

GASEOUS REQUIREMENTSGASEOUS REQUIREMENTS

OXYGEN OXYGEN Obligate aerobesObligate aerobesFacultative anaerobes.Facultative anaerobes.Microaerophillic.Microaerophillic.Obligate anaerobes. Obligate anaerobes.

CARBONDIOXIDE:CARBONDIOXIDE:Capnophillic.Capnophillic.

TEMPERATURETEMPERATURE

PsychrophillicPsychrophillic – bacteria that grows at – bacteria that grows at lower temeperature – below 20lower temeperature – below 20ºC.ºC.

Mesophillic Mesophillic – bacteria that grows at 25ºC-– bacteria that grows at 25ºC-40ºC. optimum temp range - 37ºC.40ºC. optimum temp range - 37ºC.

ThermophillicThermophillic – bacteria that grows at high – bacteria that grows at high temperatures - 55ºC-80ºC.temperatures - 55ºC-80ºC.

H-ION CONCENTRATION (pH)H-ION CONCENTRATION (pH)

Acidophillic bacteria – pH range Acidophillic bacteria – pH range below 7.below 7.

Most pathogens grow at neutral pH Most pathogens grow at neutral pH range (7.2-7.6).range (7.2-7.6).

Some grow at alkaline pH range – Some grow at alkaline pH range – above 7above 7

CULTURE MEDIACULTURE MEDIABASIC INGREDIENTSBASIC INGREDIENTS

PROTEIN SOURCE – peptone (a complex mixture of partially digested proteins).- special brands – Neopeptone & proteose peptone- digest broths can also be used.- meat extract (Lab Lemco).MINERAL SALT – sodium chloride.AGAR- Prepared for using solid media.- Obtained from sea weeds.- Long chain polysaccharides.- Also contains varying amounts of inorganic salts and small quantities of a protein – like substance.- Hydrolised at high temperature at high acid or alkaline pH.- Melts at 98ºC and usually sets at 42ºC depending on agar concentration.- Approximately 2% agar is used to prepare solid media.- Manufacture either in long shreds or in powder form.WATER- source of hydrogen and oxygen.

TYPES OF CULTURE MEDIATYPES OF CULTURE MEDIA

BASED ON THE AGAR BASED ON THE AGAR CONCENTRATION (Consistency):CONCENTRATION (Consistency):1.1. Solid media – 2% agar.Solid media – 2% agar.2. Liquid media – absence of agar.2. Liquid media – absence of agar.3. Semi solid media – 0.2-0.5% agar3. Semi solid media – 0.2-0.5% agar

TYPES OF CULTURE MEDIATYPES OF CULTURE MEDIA

BASED ON THE INGREDIENTS:BASED ON THE INGREDIENTS:1. Simple or basal medium.1. Simple or basal medium.2. Complex medium.2. Complex medium.3. Synthetic or defined medium.3. Synthetic or defined medium.4. Semisynthetic or semidefined medium.4. Semisynthetic or semidefined medium.5. Special medium5. Special medium a. enriched medium. a. enriched medium. b. enrichment medium. b. enrichment medium. c. selective medium. c. selective medium. d. indicator or differential medium. d. indicator or differential medium. e. transport medium. e. transport medium. f. Anaerobic medium. f. Anaerobic medium. g. sugar medium. g. sugar medium.

SIMPLE OR BASAL MEDIUMSIMPLE OR BASAL MEDIUM

Example: Example: Nutrient agar & Nutrient agar & Nutrient broth.Nutrient broth.

Consists of peptone, meat Consists of peptone, meat extract, sodium chloride and extract, sodium chloride and water.water.

Used to isolate separate Used to isolate separate colonies for studyingcolonies for studying1.colony morphology.1.colony morphology.2.pigmentation.2.pigmentation.3.biochemical identification 3.biochemical identification tests.tests.

NUTRIENT AGAR

COMPLEX MEDIUMCOMPLEX MEDIUM

Exact chemical composition of Exact chemical composition of ingredients of culture medium is not ingredients of culture medium is not known.known.

Widely used for the cultivation of Widely used for the cultivation of bacteria for diagnostic purposes. bacteria for diagnostic purposes.

SYNTHETIC & SEMISYNTHETIC MEDIUMSYNTHETIC & SEMISYNTHETIC MEDIUM

SYNTHETIC OR DEFINED MEDIUM:Prepared from pure chemical substances and Prepared from pure chemical substances and the exact composition of the medium is known.the exact composition of the medium is known.Used for various special studies such as Used for various special studies such as metabolic requirements.metabolic requirements.EXAMPLE: Dubo’s mediumEXAMPLE: Dubo’s medium

SEMISYNTHETIC OR SEMIDEFINED MEDIUM:SEMISYNTHETIC OR SEMIDEFINED MEDIUM:Composition is approximately known.Composition is approximately known.

SPECIAL MEDIUMSPECIAL MEDIUMENRICHED MEDIUMENRICHED MEDIUM

Substances such as blood, egg, serum are Substances such as blood, egg, serum are added to the basal medium.added to the basal medium.Used to grow bacteria which are more Used to grow bacteria which are more exacting in their nutritional needs.exacting in their nutritional needs.EXAMPLE: EXAMPLE: Blood agarBlood agar – hemolytic properties. – hemolytic properties. Chocolate agar - Chocolate agar - to supply blood factors to supply blood factors like X factor (Hemin) and Y factor (NAD)like X factor (Hemin) and Y factor (NAD)

BLOOD AGAR

CHOCOLATE AGAR

GAMMA HEMOLYSIS

ALPHA HEMOLYSIS WITH GREENISH DISCOLORATION(no clear zone around colonies)

BETA HEMOLYSIS(clear zone around colonies)

BETA HEMOLYSIS UNINOCULATED BLOOD AGAR

BROWN PIGMENT ON BLOOD AGAR

PORPHYROMONAS / PREVOTELLA SP., - IMPORTANT DENTAL PATHOGEN

CHOCOLATE AGAR (HEATED BLOOD AGAR)

SPECIAL MEDIUMSPECIAL MEDIUMENRICHMENT MEDIUMENRICHMENT MEDIUM

Obtaining the desired bacteria in Obtaining the desired bacteria in more numbers from the mixed more numbers from the mixed culture.culture.

Contains substances that Contains substances that stimulates the desired bacteria stimulates the desired bacteria or has inhibitory substances that or has inhibitory substances that inhibits the growth of unwanted inhibits the growth of unwanted bacteria.bacteria.

EXAMPLE: EXAMPLE: Selenite F broth.

Tetrathionate broth.

TCBS AGAR – FOR VIBRIO CHARCOAL AGAR – FOR BORDETELLA

DCA – FOR SALMONELLA MSA – FOR STAPH

EOSIN METHYLENE BLUE – E.COLI

LOWENSTEIN –JENSEN (LJ) LOWENSTEIN –JENSEN (LJ) MEDIUMMEDIUM

SELECTIVE MEDIUM FOR MYCOBACTERIUM TUBERCULOSIS

SPECIAL MEDIUMSPECIAL MEDIUMSELECTIVE MEDIUMSELECTIVE MEDIUM

This media enables a greater number of This media enables a greater number of the required bacteria to form colonies than the required bacteria to form colonies than the other bacteria.the other bacteria.EXAMPLE: EXAMPLE: LJ medium for the selective for the selective isolation of M.tuberculosis.isolation of M.tuberculosis.Desoxy cholate agar for Salmonella and for Salmonella and ShigellaShigellaXylose lysine deoxycholate agar for Xylose lysine deoxycholate agar for Salmonella and Shigella.Salmonella and Shigella.

SPECIAL MEDIUMSPECIAL MEDIUMDIFFERENTIAL MEDIUMDIFFERENTIAL MEDIUM

A medium which has A medium which has substances incorporated in substances incorporated in it, enabling it to bring out it, enabling it to bring out differing characteristics of differing characteristics of bacteria and thus helping to bacteria and thus helping to distinguish between them.distinguish between them.EXAMPLE: EXAMPLE: MacConkey agar – consists of peptone, – consists of peptone, lactose, agar, neutral red lactose, agar, neutral red and taurocholate – and taurocholate – differentiates between differentiates between lactose fermentors and non-lactose fermentors and non-laactose fermentors.laactose fermentors.

LACTOSE FERMENTERS (PINK COLONIES)

LACTOSE FERMENTERS

NON - LACTOSE FERMENTERS

MACCONKEY AGAR

NON-LACTOSE FERMENTER

(PALE OR COLORLESS)

LACTOSE FERMENTER

(PINK COLOUR)

MAC CONKEY AGAR

NUTRIENT AGAR BLOOD AGAR

PINK COLOUR

(TRANSPARENT)

DARK RED COLOUR

(OPAQUE)

PALE YELLOW COLOUR

(TRANSPARENT)

SPECIAL MEDIUMSPECIAL MEDIUMTRANSPORT MEDIUMTRANSPORT MEDIUM

This media is used in case of delicate organisms which may not survive the time taken for transporting the specimen to the lab or may be overgrown by nonpathogens.EXAMPLE: Stuart’s medium – a non nutrient soft agar gel containing- a reducing agent to prevent oxidation. – Charcoal to neutralise certain bacterial inhibitors (for Gonococci).- buffered glycerol saline – for enteric bacilli.

SPECIAL MEDIUMSPECIAL MEDIUMANAEROBIC MEDIUMANAEROBIC MEDIUM

Media used to grow anaerobic organisms.Media used to grow anaerobic organisms.

EXAMPLE: EXAMPLE: Robertson’s cooked meat medium – – contains cooked meatcontains cooked meat Thioglycollate broth – Thioglycollate broth – Thioglycollate Thioglycollate

Medium may be overlayed with paraffin Medium may be overlayed with paraffin wax to prevent oxygen entrywax to prevent oxygen entry

ROBERTSON COOKED MEAT BROTH

THIOGLYCOLLATE BROTH