bacteriological analytical manual 7th edition/1992

FI>J\ BACTERIOLOGICAL ANALYTICAL MANUAL 7TH EDITION/1992

Appendix 1. Rapid Methods for Detecting Foodborne Pathogens

PETER FENG

Introduction

This section differs from others in this manual in that it lists methods that are not necessarily used by FDA for regulatory samples. However, the detailed protocols for these methods are not presented, and the user is referred to the instructions that accompany the test kits. The reason for this departure is the incremental rate of change and innovation in rapid testing technology. It is recommended that the best of these new techniques be studied individually by user labs for their particular needs, as well as collaboratively for possible adoption as official methods by the Association of Official Analytical Chemists (AOAC).

The following text and tables classify many commercially available rapid methods by the underlying principles of the procedure. The assay principles and detailed procedures are discussed in other (specific) chapters of the manual or in reviews published elsewhere (6,20,39,42). Addresses of manufacturers and selected references on the use of these methods for analyzing foods are given in the tables. The current AOAC status of these rapid tests is indicated for methods that have been validated or evaluated by AOAC collaborative studies. For some of these methods, already sanctioned as standard screening techniques, positive results must still be confirmed by the appropriate official FDA method, which in-;a;y instances is cultural. In many other instances the rapid method has not been validated. Therefore, the listing of a method or kit in this chapter in no way constitutes FDA recommendation or approval.

Rapid Methods

The rapid detection of pathogens and other microbial contaminants in food is critical for ensuring the safety of consumers. Traditional methods to identify foodborne microorganisms often rely on time-consuming cultivation in diverse types of culture media followed by a considerable amount of biochemical testing. Recent advances in methodology make detection and identification faster, more convenient, more sensitive, and more specific than conventional assays -- at least in theory. These new "rapid methods" include miniaturized biochemical systems, antibody-based assays, DNA probes, modifications of conventional tests, and automated identification systems.

Experts in food microbiology who were surveyed in 1981 about future developments of detection methods for microorganisms (21) accurately predicted the widespread use of miniaturized biochemical identification systems. The test systems available today, a decade later, generally contain 15-20 biochemical tests and are 90-99% accurate as compared with conventional methods (6,20,23). Miniaturized systems are more versatile and efficient, simpler to use, and generally cheaper than conventional procedures. A partial list of commonly used miniaturized systems is given in Table 1. A few automated biochemical identification systems are listed in Table 2. Although most of these kits are for the identification of

427


Enterobacteriaceae, the biochemical tests in some kits are interchangeable and may be adapted to identify other Gram-negative and Gram-positive bacteria and yeasts.

Not forecast in that 1981 survey were the potential applications of immunological and genetic techniques in food microbiology (21). During the 1980s, major basic advances in these fields were transferred rapidly to applied areas, as "biotechnology" companies emerged and sought markets in the diagnostic field. DNA probe and immunologically based assays for numerous microbes or their products are now available commercially. Although most of these assays were originally intended for clinical use, many have been adapted for food analysis and for the identification and detection of foodborne pathogens. Immunological methods and tests based on DNA probes are currently used as rapid screening assays, but all positive results must still be confirmed by culturing or other conventional methodologies. Additional drawbacks in using these tests are the high cost of some kits, their limited availability (some can be obtained only from a single source), and the need for culture enrichment before some of the tests can be applied. Some commercially available DNA probe diagnostic kits are listed in Table 3. A more extensive list of immunological methods, including both monoclonal and polyclonal antibody systems in various assay formats, is given in Table 4.

Table 5 lists other methods that may be classified as "rapid." Some, e.g., the MUG test, are enzyme-based assays that allow rapid detection of Escherichia coli in food and water (7). Still others are variations or modifications of existing methodologies that have been simplified to make them easier to perform and to give more rapid results.

Advances in instrumentation in the past decade have also led to the development of automated systems for identifying foodborne microorganisms (Table 2). Two of the systems are based on biochemical identification; the third is based on the analysis of bacterial fatty acid profiles.

Whenever a method for detecting foodborne pathogens and their toxins is developed or modified, the accuracy and efficiency of the new procedure must be thoroughly tested, compared with the appropriate standard (conventional) method, and validated by collaborative study before it can be used routinely. Because the AOAC status of new methods changes constantly, the information presented here may not be the most recent or the most complete.

NOTE: The listings provided in Tables 1-5 are intended for general reference only and do not indicate endorsement or approval by FDA for use in food analysis.

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fl))\ BACTERIOLOGICAL ANALYTICAL MANUAL 7TH EDITION/1992

Table 1. Miniaturized biochemical multitest kits for detecting and identifying foodborne pathogens

Organism System Manufacturer

Enteric bacteria API-20Ea Analytab Products Inc., Plainview, NY

Listeria

:AOAC status: AOAC status:

Enterotube IIa

b Micro-ID

Enterobacteriaceae II

Micro-ID

Hoffmann-LaRoche, Nutley, NJ

Organon Teknika, Durham, NC

BBL Microbiology Systems, Cockeysville, MD


adopted final action for Salmonella only. adopted final action for all enteric bacteria.

Table 2. Automated identification systems for microorganisms

Trade name

Quantum II

GNia

MIS

Malthus M1000Sb


Assay format Manufacturer

Biochemical tests Abbott Diagnostics, Irving, TX

Biochemical tests Vitek Systems, Hazelwood, MO

Fatty acid profile Microbial ID, Inc., Newark, DE

Conductance Radiometer, Westlake, OH

adopted first action for selected bacteria. adopted first action for Salmonella.

Reference

12,20,23

20,23

12,20,23

12,23

37

Reference

36

2,12,28

12,23

429


Table 3. Nucleic acid hybridization assays

Organism

Campylobacter

Escherichia coli

Listeria

Salmonella

Trade name

SNAP

GENE-TRAK

GENE-TRAK

GENE-TRAK

GENE-TRAK

GENE-TRAK

Staphylococcus GENE-TRAK aureus

Yersinia GENE-TRAK enterocolitica

Format

Nonisotopic

Colorimetric

Colorimetric

Colorimetric

I . a sotop1.c

b Colorimetric

Colorimetric

Colorimetric


adopted final action. adopted first action.

Manufacturer

Molecular Biosystems, San Diego, CA

GENE-TRAK Systems, Framingham, MA







Reference

2,27

12,20,23 26,40,41

2,8,10,38

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Table 4. Immunoassays

Organism/ toxin Trade name

Bacillus cereus Tecra enterotoxin

Campylobacter

Escherichig coli EHEC

Listeria

Salmonella

Campy slide

Meriteccampy

Petrifilm HEC

ListeriaTek

TECRA Listeria

Bactigen

1-2 Test

PATH-STIK

SalmonellaTEKc TEKe

TECRA e f Salmonella '

Immunocapture

EQUATE

BacTrace

Assurance

Assay format a

EIA

Latex agglutination

Latex agglutination

Immunoblot

EIA

EIA

Latex agglution

Immunodiffusion

Dipstick EIA

EIA

Dipstick/EIA

EIA

EIA

EIA

Manufacturer Reference

Bioenterprises Pty, NSW, Australia


Meridian Diagnostics, Cincinnati, OH

3M Co., St. Paul, MN



Wampole Labs, Cranbury, NJ

BioControl Systems, Bothell, WA

Integrated Biosolutions Monmouth, NJ




Binax, Portland, ME

KPL, Inc., Gaithersburg, MD

Biocontrol Systems, Bothell, WA

30

30

35

29,45

43

31

24,34,41

9,19,25 14,44

18

24

11

431


Table 4 (continued)

Shigella

Staphylococcus aureus

S. aureus enterotoxins

Vibrio cholera toxin

Serobact Salmonella

Bactigen

Staphyloslide

SET-RPLA

TECRA

VET-RPLA

Latex agglu-tination

Latex agglu-tination

Agglu-tination

RPLA

EIA

EIA

RPLA

REMEL, Lenexa, KS

Wampole Labs, 19 Cranbury, NJ


Oxoid USA, 4 Columbia, MD

Bioenterprises Pty, 4 NSW, Australia

Toxin Technology, 4 Madison, WI

Oxoid USA, 1 Columbia, MD

8 Abbreviations EIA, enzyme immunoassay; FIA, fluorescent immunoassay; RPLA, reverse bpassive latex agglutination.

Enterohemorrhagic E. coli. c - ----dAOAC status: adopted first action.

Two enrichment-ErA methods: detection of Salmonella in all foods, and detection of Salmonella in low-moisture foods.

eAlso available as BioPro (International Bioproducts, Inc., Redmond, WA); and fTECRA (Vitek Systems, Hazelwood, MO).

AOAC status: adopted final action.

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Table 5. Other commercially available rapid methods

Target organism

Bacteria

Bacteria/ total coliforms

Total coliform/ E. coli

Trade name

b Redigel

b Isogrid

Petrifilmc

c Isogrid

Petrifilmc

ColiQuikd

d Colilert

Assay format a

Medium. APC

HGMF

Dry-film

HGMF/MUG

Dry-film

MUG& ONPG

MUG& ONPG

Manufacturer

RCR Scientific. Goshen. IN

QA Labs. Toronto. Canada

3M Co. • St. Paul. MN


3M Co. • St. Paul. MN

Reference

5

5

5

Hach Company. 32 Ames. IA

Access Analytical. 13 Branford. CT

MPN Difco. Detroit. MI 17.22.33

E. coli

Salmonella

Yersinia

Colitrak

MUG disk

c Isogrid

OSRT

Rambach

Dye assay

MPN

MUG

HGMF/EF18

Medium

Medium

Crystal violet

Gibco. Grand Island. NY

BioControl Systems. Bothell. WA

REMEL. Lenexa. KS


Oxoid USA. Columbia. MD

Technogram Paris. France

Polysciences Inc •• Warrington. PA

a Abbreviations: APC. aerobic plate count; HGMF. hydrophobic grid membrane

15

24

3

filter; MUG. 4-methylumbelliferyl-beta-D-glucuronide; ONPG. ~-nitrophenyl-beta-Dbgalactoside; MPN. most probable number.

AOAC status: adopted final action. c dAOAC status: adopted first action. Application for water analysis.

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References

1. Almeida, R.J., F.W. Hickman-Brenner, E.G. Sowers, N.D. Puhr, J.J. Farmer II, and I.K. Wachsmuth. 1990. Comparison of a latex agglutination assay and an enzyme-linked immunosorbent assay for detecting cholera toxin. J. Clin. Microbial. 28:128-130.

2. Andrews, W.A. 1990. Food microbiology (nondairy). General referee reports. J. Assoc. Off. Anal. Chem. 73:134-139.

3. Bhaduri, S., L.K. Conway, and R.V. Lachica. 1987. Assay of crystal violet binding for rapid identification of virulent plasmid-bearing clones of Yersinia enterocolitica. J. Clin. Microbial. 25:1039-1042.

4. Bennett, R.W., enterotoxins. Examination of DC (in press).

S. Notermans, and S.R. Tatini. 1992. Staphylococcal In: Compendium of Methods for the Microbiological Foods. American Public Health Association, Washington,

5. Chain, V.S., and D.Y.C. Fung. 1991. Comparison of Redigel, Petrifilm, spiral plate system, Isogrid, and aerobic plate count for determining the numbers of aerobic bacteria in selected foods. J. Food Prot. 54:208-211.

6. Cox, N.A., D.Y.C. Fung, J.S. Bailey, P.A. Hartman, and P.C. Vasavada. 1987. Miniaturized kits, immunoassays and DNA hybridization for recognition and identification of foodborne bacteria. Dairy Food Sanit. 7:628-631.

7. Curiale, M.S., M.J. Klatt, W.D. Gehle, and H.E. Chandonnet. 1990. Colorimetric and fluorometric substrate immunoassay for detection of Salmonella in all foods: comparative study. J. Assoc. Off. Anal. Chem. 73:961-968.

8. Curiale, M.S., J.J. Klatt, and M.A. Mozola. 1990. Colorimetric deoxyribonucleic acid hybridization assay for rapid screening of Salmonella in foods: collaborative study. J. Assoc. Off. Anal. Chem. 73:248-256.

9. Curiale, M.S., M.J. Klatt, B.J. Robison, and Comparison of colorimetric monoclonal enzyme method for detection of Salmonella in foods. Chem. 73:43-50.

L.T. Beck. 1990. immunoassay screening J. Assoc. Off. Anal.

10. Curiale, M.S., D. Mciver, S. Weathersby, and C. Planer. 1990. Detection of salmonellae and other Enterobacteriaceae by commercial deoxyribonucleic acid hybridization and enzyme immunoassay kits. J. Food Prot. 53:1037-1046.

11. D'Aoust, J.Y., E. Daley, and A.M. Sewell. 1990. Performance of the microplate BacTrace ELISA technique for detection of foodborne Salmonella. J. Food Prot. 53:841-845.

12. Dziezak, J.D. 1987. Rapid methods for microbiological analysis of foods. Food Techno!. 41(7):56-73.

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13. Edberg, S.C., M.J. Allen, D.B. Smith, and N.J. Kriz. 1990. Enumeration of total coliforms and Escherichia coli from source water by the defined substrate technology. Appl. Environ. Microbiol. 56:366-369.

14. Entis, P. 1989. Hydrophobic grid membrane filter/MUG method for total coliform and Escherichia coli enumeration in foods: collaborative study. J. Assoc. Off. Anar:-Chem. 72:936-950.

15. Entis, P. 1990. Improved hydrophibic membrane filter method, using EF-18 agar, for detection of Salmonella in foods: collaborative study. J. Assoc. Off. Anal. Chem. 73:734-742.

16. Entis, P., and P. Boleszczuk. 1990. Direct enumeration of coliforms and Escherichia coli by hydrophobic grid membrane filter in 24 hours using MUG. J. Food Prot. 53:948-952.

17. Feng, P.C.S., and P.A. Hartman. 1982. Fluorogenic assays for immediate confirmation of Escherichia coli. Appl. Environ. Microbiol. 43:1320-1329.

18. Flowers, R.S., M.J. Klatt, and S.L. Keelan. 1988. Visual immunoassay for detection of Salmonella in foods: collaborative study. J. Assoc. Off. Anal. Chem. 71:973-990.

19. Flowers, R.S., M.J. Klatt, B.J. Robison, and J.A. Mattingly. 1988. Evaluation of abbreviated enzyme immunoassay method for detection of Salmonella in low-moisture foods. J. Assoc. Off. Anal. Chem. 71:341-343.

20. Fung, D.Y.C., N.A. Cox, and J.S. Bailey. 1988. Rapid methods and automation in the microbiological examination of foods. Dairy Food Sanit. 8:292-296.

21. Gutteridge, C.S., and M.L. Arnott. 1989. Rapid methods: an over the horizon view, pp. 297-319. In: Rapid Methods in Food Microbiology: Progress in Industrial Microbiology. M.R. Adams and C.F.A. Hope (eds). Elsevier, New York.

22. Hartman, P.A. 1989. The MUG (glucuronidase) test for Escherichia coli in food and water, pp. 290-308. In: Rapid Methods and Automation~ Microbiology and Immunology. A. Balows, R.C. Tilton, and A. Turano (eds). Brixia Academic Press, Brescia, Italy.

23. Hartman, P.A., B. Swaminathan, M.S. Curiale, R. Firstenberg-Eden, A.N. Sharpe, N.A. Cox, D.Y.C. Fung, and M.C. Goldshmidt. 1992. Rapid methods and automation. In: Compendium of Methods for the Microbiological Examination of Foods. American Public Health Association, Washington, DC (in press).

24. Holbrook, R., J.M. Anderson, A.C. Baird-Parker, and S.H. Stuchbury. 1989. Comparative evaluation of the Oxoid Salmonella Rapid Test with three other rapid salmonella methods. Lett. Appl. Microbiol. 9:161-164.

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25. Ibrahim, G.F. 1986. A review of immunoassays and their application to salmonellae detection in foods. J. Food Prot. 49:299-310.

26. Izat, A.L., C.D. Driggers, M. Colberg, M.A. Reiber, and M.H. Adams. 1989. Comparison of the DNA probes to culture methods for the detection of Salmonella on poultry carcasses and processing waters. J. Food Prot. 52:564-570.

27. King, W., S. Raposa, J. Warshaw, A., Johnson, D. Halbert, and J.D., Klinger. 1989. A new colorimetric nucleic acid hybridization assay for Listeria in foods. J. Food Prot. 8:225-232.

28. Knight, M.T., D.W. Wood, J.F. Black, G. Gosney, R.O. Rigney, J.R. Agin, C.K. Gravens, and S.M. Farnham. 1990. Gram-negative identification card for identification of Salmonella, Escherichia coli and other Enterobacteriaceae isolated from foods: collaborative study. J. Assoc. Off. Anal. Chem. 73:729-733.

29. Mattingly, J.A., B. Butman, B.T. Plank, M.C. Durham, and B.J. Robison. 1988. Rapid monoclonal antibody-based enzyme immunosorbent assay for the detection of Listeria in food products. J. Assoc. Off. Anal. Chem. 71:679-681.

30. Mayer, K.W., and R.L. Kaplan. 1988. A comparison of two latex agglutination tests for the identification of Campylobacter sp. ASM Abstracts, p. 356.

31. Metzler, J., and I. Nachamkin. 1988. Evaluation of a latex agglutination test for the detection of Salmonella and Shigella spp. by using broth enrichment. J. Clin. Microbial. 26:2501-2504.

32. Mick, T., and C. Ziel. 1990. Comparison of Coliquik and confirmed lauryl tryptose with 4-methylumbelliferyl-S-D-glucuronide for the detection of total coliforms and Escherichia coli. ASM Abstracts, p. 289.

33. Moberg, L.J., M.K. Wagner, and L.A. Kellen. 1988. Fluorogenic assay for rapid detection of Escherichia coli in chilled and frozen foods: collaborative study. J. Assoc. Off. Anal. Chem. 71:589-602.

34. Oggel, J.J., D.C. Nundy, and C.J. Randall. 1990. Modified 1-2 test system as a rapid screening method for detection of Salmonella in foods and feeds. J. Food Prot. 53:656-658.

35. Okrend, A.J.G., B.E. Rose, and R. Matner. 1990. An improved screening method for the detection and isolation of Escherichia coli 0157:H7 from meat, incorporating the 3M Petrifilm test kit - HEC - for hemorrhagic Escherichia coli 0157:H7. J. Food Prot. 53:936-940.

36. Rhoden, D., and C.M. O'Hara. 1989. Evaluation of the updated QUANTUM II system for the identification of Gram-negative bacilli. J. Clin. Microbial. 27:2420-2422.

37. Robison, B., D. Kronish, L. Beck, and C. Brown. 1990. Identification of Listeria species using MICRO-ID Listeria. ASM Abstracts, p. 285.

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38. Rose, B.E., C.M. Llabres, and B. Bennett. 1991. Evaluation of a colorimetric DNA hybridization test for detection of salmonellae in meat and poultry products. J. Food Prot. 54:127-130.

39. Rose, S.A., and M.F. Stringer. 1989. Immunological methods, pp. 121-167. In: Rapid Methods in Food Microbiology: Progress in Industrial Microbiology. M.R. Adams and C.F.A. Hope (eds). Elsevier, New York.

40. Sall, B.S., M. Lombardo, B. Sheridan, and G.H. Parsons. 1988. Performance of a DNA probe-based Salmonella test in the AACC check sample program. J. Food Prot. 51:579-580.

41. St. Clair, V.J., and M.M. Klenk. 1990. Performance of three methods for the rapid identification of Salmonella in naturally contaminated foods and feeds. J. Food Prot. 53:961-964.

42. Tenover, F.C. 1988. infectious diseases.

Diagnostic deoxyribonucleic acid probes for Clin. Microbiol. Rev. 1:82-101.

43. Vanderlinde, P.B., and F.H. Grau. 1991. Detection of Listeria spp. in meat and environmental samples by an enzyme-linked immunosorbent assay (ELISA). J. Food Prot. 54:230-231.

44. Van Poucke, L.S.G, 1990. Salmonella-TEK, a rapid screening method for Salmonella species in food. Appl. Environ. Microbiol. 56:924-927.

45. Walker, S.J., P. Archer, and J. Appleyard. 1990. Comparison of the Listeria-Tek ELISA kit with cultural procedures for the detection of Listeria species in foods. Food Microbiol. 7:335-342.

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Appendix 2. Most Probable Number Determination

JAMES T. PEELER and FOSTER D. MCCLURE

Various methods may be used to estimate the number of bacteria in a particular sample. The 3 methods used most often are the direct microscopic count, the agar plate count, and determination of the most probable number (MPN). Both viable and nonviable organisms are enumerated by the direct microscopic count, whereas only viable organisms are enumerated by the agar plate count and the MPN determination.

The first accurate estimation of the number of viable bacteria was published by McCrady (12). An early use of this technique (4) compared the number of bacteria observed by using solid and liquid media and the estimation of Bacillus coli in water (11). Halvorson and Ziegler (10) published a series of articles on the statistical foundation of the MPN in bacteriology. A general review of the statistical foundations of the MPN is given by Eisenhart and Wilson (9) and Cochran (5). Woodward (16) recommended that MPN tables should be limited. Currently, two American Public Health Association (APHA) methods books (1,2) use abbreviated MPN tables developed by deMan (6-8).

The MPN is an estimate of the density of viable organisms in a sample. To obtain this estimate, the sample must be diluted in such a manner that a more dilute sample will result in fewer positive tubes, which are indicated by the presence of gas or microbial growth. To obtain the MPN, the theory of probability is applied to the test results, with the following assumptions accepted as given. 1) A random distribution of the bacteria exists in the sample. This implies that the likelihood of finding an organism in one part of the sample is the same as that for finding an organism in any other part of the same sample. 2) The bacteria exist as entities, not as groups or clusters, and they do not repel each other. 3) An organism(s) present in the sample will grow in the medium when incubated. 4) The proper conditions exist for growth, i.e., the correct medium has been supplied and the correct incubation temperature is maintained. The number of dilutions to be prepared should be based on the expected population in the sample. The most reliable results are obtained when all tubes at the lower dilution are positive (i.e., microbial growth present) and all tubes at the higher dilution are negative (i.e., microbial growth absent). If a high microbial count is expected, the sample must be diluted to the extent at which the MPN can be obtained (see Chapter 3). Tenfold dilutions are the simplest to perform, and the inoculum is frequently used in 3-, 5-, or 10-tube MPN series; an MPN can be calculated for other than tenfold dilutions. As the number of tubes inoculated for each dilution increases, the confidence limits for the MPN are narrowed. For high-density populations, the MPN is not as precise as a direct plate count and a computed MPN value is an estimate of the population density, not a direct count of living organisms. The MPN is particularly useful for low concentrations of organisms (<10/g) especially in milk, water, and foods where particulate matter may interfere with accurate colony counts. Proper choice of media can give early results for some of the biochemical characteristics, and use of selective media can provide estimates of a mixed population.

439


A. Tables of MPN and 95% confidence limits

Tables 1-3 present the deMan (6-8) estimates of statistically probable MPN values and the corresponding 95% confidence limits for 3-, 5-, and 10-tube tests. Combinations not in the table have a low probability of occurrence. If the results are not included in the table, the test should be repeated on the original sample. If this is not possible, the MPN can be obtained (3- and 5-tube combinations) from Tables 4 and 5 (3); an equation in section B may also be used to obtain an approximation of MPN combinations. Personal computers may be programmed to compute the MPN and confidence limits (13).

The 95% confidence intervals are interpreted as follows. If the analyst assumes that the true number of organisms lies between the limits, then the assumption will be correct 95% of the time. The tabulated MPN represents a range and not an absolute value.

When more than 3 dilutions of a sample are prepared, the MPN must be determined from only 3 consecutive dilutions (when using Tables 1-3). First, for all dilutions that have all tubes positive, select the highest dilution (smallest sample volume), and then use the next 2 higher dilutions (smaller volumes) (A and B of Tables 6 and 7). When none of the tested dilutions yield all tubes positive, select (if possible) the first 3 consecutive dilutions (sample volumes) for which the middle dilution contains the positive result (C of Tables 6 and 7). ExampleD illustrates the MPN calculation when a positive result occurs in a higher dilution (smaller sample volume) than the 3 selected. In this case, add the number of positive tubes in the highest dilution, not to exceed 3 or 5. When all tubes are positive, express the MPN as greater than the dilution times the MPN resulting from the combination 3-3-2 or 5-5-4, as appropriate (example E). Read 10-tube values from Table 3 in a similar manner.

Often it is necessary to calculate the MPN from initial sample volumes other than those listed in Tables 1-5. If the largest (greatest) sample volume used for the table reference is 0.01 g, multiply the MPN index listed in the table by 10. Thus, results of a 5-tube MPN determination showing 3 positive 0.01 g portions, 2 positive 0.001 g portions, and 1 positive 0.0001 g portion (3-2-1) are read from Table 2 as 17, and multiplied by 10 to arrive at 170 as the actual MPN/g for the sample. Similarly, if the greatest portion used for the table reference is 1 g rather than 0.1 g, divide the MPN derived from the table by 10. Thus, the result of a 3-tube MPN determination for Salmonella that shows 3 positive 1 g portions, 1 positive 0.1 g portion, and no positive 0.01 g portions (3-1-0) is read from Table 1 as 43 and divided by 10, arriving at 4.3 as the presumptive MPN/g for the sample.

An alternative approach to obtain the MPN/g uses the following formula (14):

(MPN/g from Table) - 100) x dilution factor of middle tube • MPN/g.

For MPN/100 g, multiply by 100.

440


B. Approximate MPN and 95% confidence limits

Because of the inherent complexity of actually computing MPNs and confidence limits, published tables are often used to determine the MPN. Usually, these tables are restricted to 3, 5, or 10 tubes per dilution. Even with the standard design, irregular data or a laboratory accident can cause the loss of one or more results from a dilution. In the latter case, a dilution series of 5, 4, 4 tubes might yield a positive code of 5-2-0.

Thomas (15) published a simple formula that does not require iteration. MPNs approximated by this formula may not exactly agree with those obtained from theory; however, deviations are usually small. (The approximation may not be acceptable for use in regulatory actions.) The formula is not restricted to the number of tubes and dilutions used and may be applied to all types of tests. The approximation is given by the following equation:

where P is the number of positive tubes, ~ is the total quantity of sample (g) in all negative tubes, and T is the total quantity of sample (g) in all tubes.

For example, consider the twofold dilution series given below:

Sample (g) No. of tubes No. of positive tubes

8 5 5 4 5 4 2 5 2 1 5 0

0.5 5 1 0.25 5 0

The number of positive tubes is P = (5 + 4 + 2 + 1) = 12; N [(8 x 0) + (4 X 1) + (2 X 3) + (1 X 5) + (0:5 X 4) + (0.25 X 5)) = 18:25; and! • 5 (8 + 4 + 2 + 1 + 0.5 + 0.25) c 78.75.

MPN/g = 12/[(18.25) (78.75)] 112 • 0.32/g or 32/100 g

Estimates of the 95% confidence limits (5) can be obtained from the antilogs base 10 of the following equation.

112 log (MPN/g) ± 1.08 [(log a)/n)]

where a is the dilution ratio, and n is the number of tubes per dilution. This expression assumes that the dilution ratio is other than 1 to 10 (e1, 2, 1 to 2). For the 1 to 10 ratio, the± quantity should be (1.14) (n) for the best estimation. If the number of tubes per dilution-(~1 ) is unequal (e.g., lab accident at a dilution) replace~ in the above expression by the harmonic mean (~) of the number of tubes per dilution (~i) for the k dilution. The harmonic mean is defined as ~ • ~/ E(l/~i) and ~ is the number of dilutions. For example, suppose 3 dilu£f~ns resulted in ~i of 5,4,4. Then~= 3/ [(1/5) + (1/4) + (1/4)] - 3/0.70- 4.3.

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FI>i\ BACTERIOLOGICAL ANALYTICAL MANUAL 7TH EDITION/1992

For the above MPN example with n = 5, the approximate 95% confidence limits are as follows:

log 0.32 ± (1.08) [(log 2)/5] 1 / 2

-0.495 ± 0.265

then the lower limit is antilog (-0.76) a 0.17/g or 17/100 g and the upper limit is antilog (-0.23) = 0.59/g or 59/100 g. By comparison, when computed with the deMan tables (7), the MPN would be 0.31/g with confidence limits of 0.16/g and 0.57/g.

References

1. American Public Health Association. 1985. Standard Methods for the Examination of Dairy Products, 15th ed. APHA. Washington, DC.

2. American Public Health Association. 1989. Standard Methods for the Examination of Water and Wastewater, 17th ed. APHA. Washington, DC.

3. Association of Official Analytical Chemists. 1990. Official Methods of Analysis, 15th ed. AOAC, Arlington, VA.

4. Butterfield, C.T. 1933. Comparison of the enumeration of bacteria by means of solid and liquid media. Public Health Rep. 48:1292-1297.

5. Cochran, W.G. 1950. Estimation of bacterial densities by means of the "Most Probable Number." Biometrics 6:105-116.

6. deMan, J.C. 1975. The probability of most probable numbers. Eur. J. Appl. Microbiol. 1:67-78.

7. deMan, J.C. 1977. MPN tables for more than one test. Eur. J. Appl. Microbiol. 4:307-316.

8. deMan, J.C. 1983. MPN tables, corrected. Eur. J. Appl. Biotechnol. 17:301-305.

9. Eisenhart, C., and P.W. Wilson. 1943. Statistical methods and control in bacteriology. Bacteriol. Rev. 7:57-137.

10. Halvorson, H.O., and N.R. Ziegler. 1933. Application of statistics to problems in bacteriology. J. Bacteriol. 25:101-121; 26:331-339, 26:559-567.

11. Hoskins, J.K. 1933. The most probable number of B. coli in water analysis. J. Am. Water Works Assoc. 25:867-879.

12. McCrady, M.H. 1915. The numerical interpretation of fermentation-tube results. J. Infect. Dis. 17:183-212.

13. Parnow, R.J. 1972. Computer program estimates of bacterial densities by means of the most probable numbers. Food Technol. 26(7):56-62.

442

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FI>~ BACTERIOLOGICAL ANALYTICAL MANUAL 7TH EDITION/1992

14. Silliker, J.H., D.A. Gabis, and A. May. 1979. ICMSF methods studies. XI. Collaborative/comparative studies on determination of coliforms using the most probable number procedure. J. Food Prot. 42:638-644.

15. Thomas, H.A. 1942. Bacterial densities from fermentation tube tests. J. Am. Water Works Assoc. 34:572-576.

16. Woodward, R.L. 1957. How probable is the most probable number? J. Am. Water Works Assoc. 49:1060-1068.

443


Table 1. Selected MPN and 95% confidence limit (7) estimates for fermentation tube tests with 3 tubes of 0.1, 0.01, and 0.001 g (ml) portions a

No. of ~ositive tubes 95% Confidence limits 0.1 0.01 0.001 MPN/g (ml) b Lower Upper

a

0 0 0 <3 0 1 0 3+ <1 17 1 0 0 4 <1 21 1 0 1 7+ 2 27 1 1 0 7 2 28 1 2 0 11+ 4 35 2 0 0 9 2 38 2 0 1 14+ 5 48 2 1 0 15 5 50 2 1 1 20+ 7 60 2 2 0 21 8 62 3 0 0 23 9 130 3 0 1 39 10 180 3 1 0 43 10 210 3 1 1 75 20 280 3 2 0 93 30 380 3 2 1 150 50 500 3 2 2 210+ 80 640 3 3 0 240 90 1400 3 3 1 460 100 2400 3 3 2 1100 300 4800 3 3 3 >1100

Normal results, obtained in 95% of tests, are not followed by a plus (+). Less likely results, obtained in only 4% of tests, are followed by a plus (+). Combinations of positive tubes not shown here occur in less than 1% of tests, and their frequent occurrence indicates that techniques are faulty or that assumptions underlying the MPN estimate are not being fulfilled. MPN estimates for combinations not shown here may be obtained by extrapolation or by Thomas' formulae (15) to the next highest combination shown; for example, a result of 2-0-2 would have an MPN of approximately 20, which is the MPN for a more likely result of 2-1-1.

bMultiply all values under MPN/g (ml) by 100 for reporting MPN/100 g (ml).

444

• •


Table 2. Selected MPN and 95% confidence limits (7) es timates for fermentation tube tests with 5 tubes of 0.1, 0.01, and 0.001 g (ml) portions a

No. of ;EOSitive tubes b

95% Confidence limits 0.1 0.01 0.001 MPN/g (ml) Lower Upper

0 0 0 <2 0 0 1 2+ <1 10 0 1 0 2 <1 10 1 0 0 2 <1 11 1 0 1 4+ 1 15 1 1 0 4 1 15 1 2 0 6+ 2 18 2 0 0 4 1 17 2 0 1 7+ 2 20 2 1 0 7 2 21 2 1 1 9+ 3 25 2 2 0 9 3 25 3 0 0 8 3 24 3 0 1 11 4 29 3 1 0 11 4 30 3 1 1 14+ 6 35 3 2 0 14 6 35 3 2 1 17+ 7 40 3 3 0 17+ 7 41 4 0 0 13 5 38 4 0 1 17 7 45 4 1 0 17 7 46 4 1 1 21 9 55 4 2 0 22 9 56 4 2 1 26+ 12 65 4 3 0 27 12 67 4 3 1 33+ 15 77 4 4 0 34+ 16 80 5 0 0 23 9 68 5 0 1 31 13 110 5 1 0 33 14 120 5 1 1 46 20 150 5 1 2 63+ 22 180 5 2 0 49 21 170 5 2 1 70 30 210 5 2 2 94+ 40 250 5 3 0 79 30 250 5 3 1 110 40 300 5 3 2 140 60 360 5 4 0 130 50 390 5 4 1 170 70 480 5 4 2 220 100 580

445


Table 2 (continued)

No. of Eositive tubes 95% Confidence limits 0.1 0.01 0.001 MPN/g (ml) b Lower Upper

a

b

5 4 3 280+ 120 690 5 4 4 350+ 160 820 5 5 0 240 100 940 5 5 1 350 100 1300 5 5 2 540 220 2000 5 5 3 920 300 2900 5 5 4 1600 600 5300 5 5 5 >1600

Normal results, obtained in 95% of tests, are not followed by a plus (+). Less likely results, obtained in only 47. of tests, are followed by a plus (+). Results not shown here occur in less than 1% of tests, and their frequent occurrence indicates that techniques are faulty or that assumptions underlying the MPN estimate are not being fulfilled. MPN estimates for combinations not shown here may be obtained by extrapolation or by Thomas' formulae (15) to the next highest combination that is shown; for example, a result of 4-0-2 would have an MPN of approximately 21, which is the MPN for a more likely result of 4-1-1.

Multiply all figures under MPN/g (ml) by 100 for reporting MPN/100 g (ml).

446


Tabl e 3. Selected MPN and 95% conf idence limits (7) estimates for fermentation tube tests wit h 10 t ubes of 0.1, 0.01, and 0.001 g (ml) a portions

No. of Eositive tubes (ml)b

95% Confidence limits 0.1 0.01 0.001 MPN/g Lower Upper

0 0 0 <1 0 0 1 1+ <1 5 0 1 0 1 <1 5 0 2 0 2+ <1 7 1 0 0 1 <1 5 1 0 1 2+ <1 7 1 1 0 2 <1 7 1 2 0 3+ 1 8 2 0 0 2 <1 7 2 0 1 3+ 1 9 2 1 0 3 1 9 2 1 1 4+ 1 10 2 2 0 4 2 10 2 3 0 5+ 2 12 3 0 0 3 1 9 3 0 1 4 2 11 3 1 0 4 2 11 3 1 1 5+ 2 13 3 2 0 5 2 13 3 2 1 6+ 3 14 3 3 0 6+ 3 14 4 0 0 4 2 12 4 0 1 6 2 13 4 1 0 6 2 14 4 1 1 7 3 15 4 2 0 7 3 15 4 2 1 8 4 17 4 3 0 8 4 17 4 4 0 9+ 5 19 5 0 0 6 2 15 5 0 1 7 3 16 5 1 0 7 3 17 5 1 1 9 4 18 5 2 0 9 4 18 5 2 1 10+ 5 20 5 3 0 10 5 20 5 3 1 11+ 6 22 5 4 0 11+ 6 22 6 0 0 8 3 18 6 0 1 9 4 20 6 1 0 9 4 20 6 1 1 11 5 22

447

pi))\ BACTERIOLOGICAL ANALYTICAL MANUAL 7TH EDITION/1992

Table 3 (continued)

No. of Eositive tubes (ml)b


6 2 0 11 5 22 6 2 1 12 6 24 6 3 0 12 6 25 6 3 1 14+ 7 27 6 4 0 14+ 7 27 6 5 0 15+ 8 29 7 0 0 10 5 22 7 0 1 12 6 24 7 0 2 13+ 7 27 7 1 0 12 6 25 7 1 1 13 7 27 7 1 2 15+ 8 30 7 2 0 13 7 27 7 2 1 15 8 30 7 2 2 17+ 9 32 7 3 0 15 8 30 7 3 1 17 9 33 7 4 0 17 9 33 7 4 1 19+ 10 36 7 5 0 19+ 10 36 8 0 0 13 6 28 8 0 1 15 7 31 8 0 2 17+ 8 34 8 1 0 15 7 31 8 1 1 17 9 34 8 1 2 19+ 10 37 8 2 0 17 9 35 8 2 1 19 10 38 8 2 2 21+ 12 42 8 3 0 19 10 39 8 3 1 21 12 42 8 3 2 24+ 13 46 8 4 0 22 12 43 8 4 1 24 13 46 8 5 0 24 13 47 8 5 1 27+ 15 51 8 6 0 27+ 15 52 9 0 0 17 8 37 9 0 1 19 10 41 9 0 2 22+ 11 46 9 1 0 19 10 42 9 1 1 22 11 47 9 1 2 25+ 13 52 9 2 0 22 12 47

.. • 448


Table 3 (continued)

No. of :eositive tubes (ml)b


9 2 1 25 13 53 9 2 2 28+ 15 58 9 3 0 25 13 54 9 3 1 29 15 60 9 3 2 32+ 18 66 9 4 0 29 16 61 9 4 1 33 18 67 9 4 2 37+ 20 74 9 5 0 33 18 69 9 5 1 37 20 76 9 5 2 42+ 23 83 9 6 0 38 21 77 9 6 1 43+ 24 85 9 7 0 44+ 24 87

10 0 0 23 12 58 10 0 1 27 14 67 10 0 2 31+ 16 77 10 1 0 27 14 69 10 1 1 32 17 79 10 1 2 38 20 92 10 2 0 33 17 83 10 2 1 39 20 96 10 2 2 50 20 110 10 2 3 50+ 30 120 10 3 0 40 20 100 10 3 1 50 20 120 10 3 2 60+ 30 130 10 3 3 70+ 30 150 10 4 0 50 30 120 10 4 1 60 30 140 10 4 2 70 30 160 10 4 3 80+ 40 170 10 5 0 60 30 150 10 5 1 70 40 170 10 5 2 90 40 190 10 5 3 100 50 210 10 6 0 80 40 180 10 6 1 90 50 200 10 6 2 110 50 230 10 6 3 120 60 250 10 6 4 140+ 70 270 10 7 0 100 50 220 10 7 1 120 60 250 10 7 2 140 70 280

44 9


Table 3 (continued)

No. of Eositive tubes 95% Confidence limits b 0.1 0.01 0.001 MPN/g (ml) Lower Upper

10 7 3 150 80 310 10 7 4 170+ 90 340 10 8 0 130 60 280 10 8 1 150 80 320 10 8 2 170 90 360 10 8 3 200 100 400 10 8 4 220 120 440 10 8 5 250+ 140 480 10 9 0 170 90 380 10 9 1 200 100 430 10 9 2 230 120 490 10 9 3 260 140 560 10 9 4 300 160 640 10 9 5 350 180 720 10 9 6 400+ 210 820 10 10 0 240 120 610 10 10 1 290 150 750 10 10 2 350 170 910 10 10 3 400 200 1100 10 10 4 500 300 1400 10 10 5 700 300 1700 10 10 6 900 400 2100 10 10 7 1120 600 2700 10 10 8 1160 BOO 3700 10 10 9 2300 1100 6000 10 10 10 >2300

a Normal results, obtained in 95% of tests, are not followed by a plus (+). Less likely results, obtained in only 4% of tests, are followed by a plus (+). Combinations of positive tubes not shown here occur in less than 1% of tests, and their frequent occurrence indicates that techniques are faulty or that assumptions underlying the MPN estimate are not being fulfilled. MPN estimates for combinations not shown here may be obtained by extrapolation or by Thomas' formulae (15) to the next highest combination that is shown; for example, a result of 7-0-3 would have an MPN of approximately 15, which is the MPN for a more likely result of 7-2-1.

bMultiply all figure under MPN/g (ml) by 100 for reporting MPN/100 g (ml).

450


Table 4. Moat Probable Numbers (MPN) per 1 g of Sample, Using 3 Tubes with Each of 0.1, 0.01, and 0.001 g Portlona

0.1

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Positive Tubes

0.01

0 0 0 0 1 1 1 1 2 2 2 2 3 3 3 3

0.001

0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3

MPN

<3 3 6 9 3 6.1 9.2

12. 6.2 9.3

12 16 9.4

13 16 19

0.1

Positive Tubes

0.01

0 0 0 0 1 1 1 1 2 2 2 2 3 3 3 3

0.001

0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3

MPN

3.6 7.2

11 15 7.3

11 15 19 11 15 20 24 16 20 24 29

0.1

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

Positive Tubes

0.01

0 0 0 0 1 1 1 1 2 2 2 2 3 3 3 3

0.001

0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3

MPN

9.1 14 20 26 15 20 27 34 21 28 35 42 29 36 44 53

0.1

3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

Positive Tubes

0.01

0 0 0 0 1 1 1 1 2 2 2 2 3 3 3 3

0.001 MPN

0 23 1 39 2 64 3 95 0 43 1 75 2 120 3 160 0 93 1 150 2 210 3 290 0 240 1 460 2 1100 3 >1100

Table 5. Most Probable Numbers per 100 ml of Sample, Planting 5 Portions in Each of 3 Dilutions In Geometric Series Number of

Positive Tubes

Number of Positive Tubes


Number of POSitive Tubes



10 1 0.1 10 1 0.1 10 1 0.1 10 0.1 10 0.1 10 1 0.1 ml ml ml MPN ml ml ml MPN ml ml ml MPN ml ml mL MPN mL mL mL MPN ml ml mL MPN

0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0

0 1 2 3 4 5

0 1 2 3 4 5

2 0 2 1 2 2 2 3 2 4 2 5

3 0 3 1 3 2 3 3 3 4 3 5

4 0 4 1 4 2 4 3 4 4 4 5

5 0 5 1 5 2 5 3 5 4 5 5

1.8 3.6 5.4 7.2 9.0

1.8 3.6 5.5 7.3 9.1

11

3.7 5.5 7.4 9.2

11 13

5.6 7.4 9.3

11 13 15

7.5 9 .4

11 13 15 17

9.4 11 13 15 17 19

0 o. 0 1 0 2 0 3 0 4 0 . 5

2 2 2 2 2 2

3 3 3 3 3 3

5 5 5 5 5 5

0 1

2 3 4 5

0 1

2 3 4 5

0 1 2 3 4 5

0 1 2 3 4 5

0 1

2 3 4 5

2.0 4.0 6.0 8.0

10 12

4.0 6.1 8.1

10 12 14

8.1 8.2

10 12 15 17

8.3 10 13 15 17 19

11 13 15 17 19 22

13 15 17 111 22 24

2 2 2 2 2 2

2 2 2 2 2 2

2 2 2 2 2 2

2 2 2 2 2 2

2 2 2 2 2 2

2 2 2 2 2 2

0 0 0 0 0 0

2 2 2 2 2 2

3 3 3 3 3 3

4 4 4 4 4 4

5 5 5 5 5 5

0 1 2 3 4 5

0 1 2 3 4 5

0 1 2 3 4 5

0 1 2 3 4 5

0 1 2 3 4 5

0 1

2 3 4

5

4.5 6.8 9.1

12 14 16

8.8 11.2

12 14 17 19

9.3 12 14 17 19 22

12 14 17 20 22 25

15 17 20 23 25 28

17 20 23 28 29 32

3 3 3 3 3 3

3 3 3 3 3 3

3 3 3 3

3

3 3 3 3 3 3

3 3 3 3 3 3

3 3 3 3 3 3

0 0 0 0 0 0

2 2 2 2 2 2

3 3 3 3 3 3

4 4 4 4 4 4

5 5 5 5 5 5

0 1 2 3 4

5

0 1

2 3 4 5

0 1

2 3 4 5

0 1 2 3 4

5

0 1

2 3 4 5

0 1

2 3 4 5

7.8 11 13 16

20 23

11 14 17 20 23 27

14 17 20 24 27 31

17 21 24 28 31 35

21 24 28 32 38 40

25 29 32 37 41 45

4 4 4 4 4 4

4 4 4 4 4 4

0 0 0 0 0 0

2 2 2 2 2 2

3 3 3 3 3 3

5 5 5 5 5 5

0 1

2 3 4

5

0 1

2 3 4 5

0 1

2 3 4

5

0 1 2 3 4 5

0 1 2 3 4

5

0 1 2 3 4

5

13 17 21 25 30 36

17 21 26 31 38 42

22 26 32 38

" 50

27 33 39 45 52 59

34 40 47 s.4

62 69

41 48 56 64 72 81

5 5 5 5 5 5

5 5 5 5 5 5

5 5 5 5 5 5

5 5 5 5 5 5

5 5 5 5 5 5

5 5 5 5 5

0 0 0 0 0 0

2 2 2 2 2 2

3 3 3 3 3 3

4 4 4 4 4 4

5 5 5 5 5

0 1

2 3 4 5

0 1

2 3 4 5

0 1 2 3 4

5

0 1

2 3 4

5

0 1

2 3 4 5

0 1

2 3 4

23 31 43 56 76 95

33 46 64 64

110 130

49 70 95

120 150 180

79 110 140 180 210 250

130 170 220 280 350 430

240 350 540 920

1,600

451


Table 6. Examples of determining MPN estimates: 3-tube series, 1 g (ml) sample per tube

S 1 ( ml) a amp e amount g or Example 0.1 0.01 0.001 0.0001 0.00001

Reported positive values

MPN b estimate/g or ml

A 3/3 3/3 2/3 0/3 0/3 3-2-0 930 B 3/3 3/3 3/3 2/3 0/3 3-2-0 9300 c 0/3 0/3 1/3 0/3 0/3 0-1-0 30 D 3/3 3/3 2/3 1/3 1/3 3-2-2 2100 E 3/3 3/3 3/3 3/3 3/3 3-3-3 >110,000

a Numerator/denominator = number of positive tubes/number of tubes inoculated.

b All figures under MPN/g or ml in this table may be multipled by 100 for reporting MPN/100 g or ml.

Table 7. Examples of determining MPN estimates: 5-tube series, 1 g (ml) sample per tube

ml)a Reported

SamEle amount (g or Positive MPN b Example 0.1 0.01 0.001 0.0001 0.00001 values estimate/g or ml

A 5/5 5/5 2/5 0/5 0/5 5-2-0 490 B 5/5 5/5 5/5 2/5 0/5 5-2-0 4900 c 0/5 0/5 1/5 0/5 0/5 0-1-0 20 D 5/5 5/5 3/5 1/5 1/5 5-2-2 1400 E 5/5 5/5 5/5 5/5 5/5 5-5-5 >160,000

aNumerator/denominator = number of positive tubes/number of tubes inoculated.

b All figures under MPN/g or ml in this table may be multipled by 100 for reporting MPN/100 g or ml.

452


Appendix 3. Media and Reagents

The media and reagents listed here are recommended by the BAM-7 authors. Formulations were reviewed and approved by B. Jeffery Rhodehamel (media) and Haim M. Solomon (reagents).

Tryptone Lactose NaCl *Triton X-100 (Rohn & Haas) Salicin Distilled water

Ml.

MEDIA

A-1 Medium

20 g 5 g 5 g

1 ml 0.5 g

1 liter

Dissolve ingredients in 1 liter distilled water. Adjust pH to 6.9 ± 0.1. Dispense 10 ml portions of single strength broth into 18 x 150 mm tubes containing inverted fermentation vials. For double strength broth, use 22 x 175 mm tubes containing inverted fermentation vials. Medium may be cloudy before sterilization. Autoclave 10 min at 121°C. Store in dark up to 7 days. (Commercially available A-1 medium is unacceptable.)

*Triton X-100 may be purchased from Fisher Scientific Company, Fairlawn, NJ 07410.

M2. Acetamide Medium

Stock basal medium

KH2P04

~P04 Agar Distilled water

2.38 g 0.31 g 0.5 g

400 ml

Heat with agitation to dissolve agar. Add 1 ml PR-CV (500X concentrate).

Stock acetamide, 1\l

Acetamide Distilled water

Store over CHC1 3 in screw-cap container. temperature.

PR-CV C500X concentrate)

Phenol red Crystal violet

1 g 100 ml

Stable indefinitely at room

2 g 0.2 g

453


Distilled water 200 ml

Add 5 N NaOH until ingredients are dissolved.

Final medium. Add 0.8 ml basal medium to 13 x 100 mm tube. Add 0.2 ml acetamide solution. Steam (100°C) 10 min. Cool .

Sodium acetate NaCl MgS04 (anhydrous) Ammonium phosphate ~HP04 Bromthymol blue Agar Distilled water

M3. Acetate Agar

2 g 5 g

0.2 g 1 g 1 g

0.08 g 20 g

1 liter

Add all ingredients except Mg504 to 1 liter distilled water. Heat to boiling with stirring. Add Mg504 and adjust pH. Dispense 8 ml portions into 16 x 150 mm tubes. Autoclave 15 min at 121°C. Incline tubes to obtain 5 em slant. Final pH, 6.7. NOTE: This medium contains a slightly smaller concentration of acetate than the formula recommended in Ewing (1986).

Proteose peptone Yeast extract Dextrose K2HP04

Distilled water

M4. Acid Broth

5 g 5 g 5 g 4 g

1 liter

Dissolve ingredients and dispense 12-15 ml portions into 20 x 150 mm tubes. Autoclave 15 min at 12l°C. Final pH, 5.0.

MS. AE Sporulation Medium, Modified (for c. perfringens) (AE base is available commercially)

Polypeptone Yeast extract Na2HP04

KH2P04

10.0 10.0 4.36 0.25 1.5 0.2

g g g g g g

Ammonium acetate MgS04 • 7H2o Distilled water 1 liter

Dissolve ingredients and adjust to pH 7.5 ± 0.1, using 2 M sodium carbonate. Dispense 15 ml into 20 x 150-mm screw-cap tubes and sterilize by autoclaving for 15 min at 12l°C. After sterilization, add 0.6 ml of sterilized 10% raffinose and 0.2 ml each of filter-sterilized 0.66 M sodium carbonate and 0.32% cobalt chloride (CoC1 2.6H20) dropwise to each tube. Check pH of one or two tubes; it should be 7.8 ± 0.1. Just before use, steam medium for 10 min;

454


after cooling, add 0.2 ml of filter-sterilized 1.5% sodium ascorbate (prepared da i ly) to each tube.

M6. Agar Medium p

Beef extract 3 g KzHP04 0.25 g Peptone 5 g Polysorbate 80 1 g Tryptone 1.7 g Bromcresol purple 0.06 g Soytone 0.3 g Agar 15 g Dextrose 5.25 g Distilled water 1 liter NaCl 0.5 g

Heat with agitation to dissolve agar. Autoclave 15 min at 121°C. Final pH, 7.8 ± 0.2.

Peptone Yeast extract NaCl Distilled water

M7. AKI Medium

NaHC03 , 10% aqueous, filter-sterilized

15 g 4 g 5 g

970 ml

30 ml

On day of use, dissolve peptone, yeast extract, and NaCl in distilled water. Autoclave 15 min at 121°C. Cool. Add 30 ml freshly prepared, filtersterilized NaHC03 , and mix. Dispense aseptically into screw-capped tubes (use 15 ml for 16 x 125 mm tubes). Final pH, 7.2 ± 0.2.

Peptone NaCl Agar Distilled water

MS. Alkaline Peptone Agar

10 g 20 g 15 g

1 liter

Boil to dissolve ingredients. Adjust pH so that value after sterilization is 8 . 5 + 0.2. Autoclave 15 min at 121°C. Solidify agar in tubes as slants.

Peptone NaCl Distilled water

M9. Alkaline Peptone Salt Broth CAPS)

10 g 30 g

1 liter

Dissolve ingredients. Adjust pH so that value after sterilization is 8.5 + 0.2. Dispense 10 ml into tubes. Autoclave 10 min at 121°C.

Peptone NaC1

MlO. Alkaline Peptone Water

10 g 10 g

455


Distilled water 1 liter

Adjust pH so that value after sterilization is 8.5 ± 0.2. Dispense into screw-cap tubes. Autoclave 10 min at 121°C.

Trypticase (tryptic) soy agar Agar Yeast extract

Mll. Anaerobe Agar

L-Cysteine (dissolved in 5 ml 1 N NaOH) Distilled water 1

40 g 5 g 5 g

0.4 g liter

Heat with agitation to dissolve agar. Adjust pH to 7.5 ± 0.2. Autoclave 15 min at 121°C. Cool to 50°C.

Hemin solution. Suspend 1 g hemin in 100 ml distilled water. Autoclave 15 min at 121°C. Refrigerate at 4°C.

Vitamin K1 solution. Dissolve 1 g vitamin K1 (Sigma Chemical Co., St. Louis, MO) in 100 ml 95\ ethanol. Solution may require 2-3 days with intermittent shaking to dissolve. Refrigerate at 4°C.

Final medium. To 1 liter base add 0.5 ml hemin solution and 1 ml Vitamin K1

solution. Mix and pour 20 ml portions into 15 x 100 mm petri dishes. Medium must be reduced before inoculation by 24 h anaerobic incubation in anaerobic glove box or GasPak jar.

Agar base

Fresh eggs Yeast extract Tryptone Proteose peptone NaCl Agar Distilled water

Ml2. Anaerobic Egg Yolk Agar

Autoclave 15 min at 121°C. Final pH, 7.0 ± 0.2.

1

2 5 g 5 g

20 g 5 g

20 g liter

Treatment of eaas. Wash 2 fresh eggs with stiff brush and drain. Soak in 0.1\ mercuric chloride solution for 1 h. Pour off mercuric chloride solution and replace with 70\ ethanol. Soak 30 min. Crack eggs aseptically. Retain yolks. Drain contents of yolk sacs into sterile stoppered graduate and discard sacs. Add yolk to equal volume of sterile 0.85\ saline. Invert graduate several times to mix.

Preparation of medium. To 1 liter melted medium (48-50°C) add 80 ml yolk-

456

" .

f l))\ BACTERIOLOGICAL ANALYTICAL MANUAL 7TH EDITION/1992

saline mixture, and mix. Pour plates immediately. After solidification dry 2-3 days at ambient temperature or at 35°C for 24 h. Check plates for contamination before use. After drying, plates may be stored for a short period in refrigerator.

Ml3. Andrade's Carbohydrate Broth and Indicator

Beef extract Peptone or gelysate NaCl Distilled water

Adjust pH to 7.2 ± 0.2.

Andrade's indicator

Acid fuchsin Distilled water 1 N NaOH

Autoclave 15 min at 121°C.

3 g 10 g 10 g

1 liter

0.2 g 10 m1 16 ml

Allow to decolorize before use. Add 1-2 ml NaOH if necessary. Add 10 ml indicator to 1 liter base.

Carbohydrate stock solution. Prepare dextrose, lactose, sucrose, and mannitol in 10\ solutions. Prepare dulcitol, salicin, and other carbohydrates in 5\ solutions. Sterilize by filtration through 0.20 ~m membrane. Dilute sugar solutions 1:10 in base with Andrade's indicator to give recommended concentration. Mix gently.

M14. Antibiotic Medium No. 1 CAqar Medium A)

Gelatone or gelysate Tryptone or trypticase Yeast extract Beef extract Dextrose Agar Distilled water 1

6 g 4 g 3 g

1.5 g 1 g

15 g liter

Autoclave 15 min at 121°C. Final pH, 6.5-6.6. Commercially available in dehydrated form as Difco Penassay Seed Agar or BBL Seed Agar.

& MlS. Antibiotic Medium No. 4 CAqar Medium e)

Tryptone or trypticase Yeast extract Beef extract Dextrose Agar Distilled water

6 g 3 g

1.5 g 1 g

15 g 1 liter

457


Autoclave 15 min at 121°C. Final pH, 6.5-6.6. Commercially available in dehydrated form as Difco Yeast Beef Agar or BBL Yeast Beef Agar.

Peptone Yeast extract Tryptone NaCl Glucose

K16. Arginine-Glucose Slant (AGSl

L-Arginine (hydrochloride) Ferric ammonium citrate Sodium thiosulfate Bromocresol purple Agar Distilled water

5 g 3 g

10 g 20 g

1 g 5 g

0.5 g 0.3 g

0.02 g 13.5 g

1 liter

Dispense into Suspend ingredients in distilled water and boil to dissolve. tubes (for 13 x 100 mm tubes use 5 ml). Autoclave 10-12 min sterilization, solidify as slants. Final pH, 6.8-7.0.

at 12PC. After

Basal medium

Tryptone Beef extract Yeast extract Sodium pyruvate Glycine Lithium chloride.6H20 Agar

K17. Baird-Parker Medium

Autoclave 15 min at 121°C. Final pH, 7.8 ± 0.2. If desired for use, maintain melted medium at 48-50°C before adding enrichment. store solidified medium at 4 ± l°C for 48 h. Melt medium before

Enrichment. Bacto EY tellurite enrichment.

10 g 5 g 1 g

10 g 12 g

5 g 20 g

immediate Otherwise,

use.

Complete medium. Aseptically add 5 ml prewarmed (45-50°C) Bacto EY tellurite enrichment to 95 ml melted base. Mix well (avoiding bubbles) and pour 15-18 ml portions into sterile 15 x 100 mm petri dishes. The medium must be densely opaque. Dry plates before use. For a more complete discussion on preparation and use of this medium, consult Microorganisms in Foods, 2nd ed., International Commission on Microbiological Specifications For Foods (ICMSF), International Assocation of Microbiological Societies, University of Toronto Press, Toronto, Ontario, Canada, 1978.

458


Beef extract Peptone Esculin Oxgall Ferric citrate Agar Distilled water

Ml8. Bile Esculin Agar

1

3 g 5 g 1 g

40 g 0.5 g

15 g liter

Heat with agitation to dissolve. Dispense into tubes, autoclave 15 min at 121°C, and slant until solidified. Final pH, 6.6 ± 0.2.

M19. Bismuth Sulfite Agar (Wilson and Blair)

Polypeptone (or peptone) 10 g Beef extract 5 g Dextrose 5 g Na2HP04 (anhydrous) 4 g FeS04 (anhydrous) 0.3 g Bismuth sulfite (indicator) 8 g Brilliant green 0.025 g Agar 20 g Distilled water 1 liter

Mix thoroughly and heat with agitation. Boil about 1 min to obtain uniform suspension. (Precipitate will not dissolve.) Cool to 45-50°C. Suspend precipitate by gentle agitation, and pour 20 ml portions into sterile 15 x 100 mm petri dishes. Let plates dry about 2 h with lids partially removed; then close plates. Final pH, 7.6 ± 0.2 DO NOT AUTOCLAVE. Prepare plates on day before streaking and store in dark. Selectivity decreases in 48 h.

Tryptone Phytone or soytone NaCl Agar Distilled water

M20. Blood Agar

15 g 5 g 5 g

15 g 1 liter

Heat with agitation to dissolve agar. Autoclave 15 min at 121°C. Cool to 50°C. Add 5 ml defibrinated sheep red blood cells to 100 ml melted agar. Mix and pour 20 ml portions into sterile 15 x 100 mm petri dishes. Commercially available tryptic-soy-sheep blood agar plates are satisfactory.

M20a. Blood Agar (commercially available blood agar base may be used)

Infusion from 500 g beef heart Tryptose NaCl

1 liter 10 g

5 g

459

fl)j\ BACTERIOLOGICAL ANALYTICAL MANUAL

Agar Sheep blood, sterile, defibrinated

7TH EDITION/1992

15 g 50 ml

Suspend ingredients except blood to dissolve. Autoclave 15 min at 121°C. Cool to 45-50~. Add 50 ml blood (room temperature), and mix. Dispense into sterile petri dishes. Final pH, 6.8 ± 0.2.

M21. Blood Agar Base (Infusion Agar)

Heart muscle, infusion from Thiotone NaCl Agar Distilled water

375 g 10 g

5 g 15 g

1 liter

Heat gently to dissolve. Autoclave 20 min at 121°C. Final pH, 7.3 ± 0.2.

M22. Blood Agar Base #2 (Difco)

Proteose peptone 15 g Liver digest 2.5 g Yeast extract 5 g NaCl 5 g Agar 12 g Distilled Water 1 liter

Autoclave at 121°C for 15 min. For blood agar, reduce water to 950 ml. Add 50 ml defibrinated (whole or lysed) horse blood and FBP (4 ml to agar + blood) after autoclaving and cooling to 48°C. Final pH, 7.4 ± 0.2.

M23. Brain Heart Infusion lBHil Agar (0.7') (for staphylococcal enterotoxin)

Prepare a suitable quantity of brain heart infusion broth (M24). Adjust pH to 5.3 with 1 N HCl. Add agar to give 0.7\ concentration. Dissolve by minimal boiling. Dispense 25 ml portions into 25 x 200 mm test tubes. Autoclave 10 min at 121°C.

M24. Brain Heart Infusion (BHI) Broth and Agar

Calf brain infusion Beef heart infusion Proteose peptone or gelysate NaCl Na~P04 .12H20 Dextrose Distilled water 1

200 g 250 g

10 g 5 g

2.5 g 2 g

liter

Dissolve ingredients in distilled water with gentle heat. Dispense broth into bottles or tubes for storage. Autoclave 15 min at 121°C. Final pH, 7.4 ± 0.2. FoA. '1118~\Q s'"·J o..d.tl- W()C.\ ~ o.ch··~ve. ~ 2-~~ .f,....._l COY\c~""iv~:~· it>n. To prepare brain heart infusion agar, add 15 g agar to 1 liter BHI broth. Heat to dissolve agar before dispensing into bottles or flasks. Autoclave 15 min at 12PC.

460


Peptone Lactose Oxgall Brilliant green Distilled water

M25. Brilliant Green Lactose Bile Broth

10 g 10 g 20 g

0.0133 g 1 liter

Dissolve peptone and lactose in 500 ml distilled water. Add 20 g dehydrated oxgall dissolved in 200 ml distilled water. The pH of this solution should be 7.0-7.5. Mix and add water to make 975 ml. Adjust pH to 7.4. Add 13.3 ml 0.1\ aqueous brilliant green in distilled water. Add distilled water to make 1 liter. Dispense into fermentation tubes, making certain that fluid level covers inverted vials. Autoclave 15 min at 121°C. Final pH, 7.2 ± 0.1.

Peptone Beef extract NaCl

M26. Bromcresol Purple Broth

10 g 3 g 5 g

Bromcresol purple 0.04 g Distilled water 1 liter For Vibrio spp., add NaCl to achieve a 2-3% final concentration.

Add 5 g carbohydrate per liter of base. Dispense 2.5 ml portions into 13 x 100 mm test tubes containing inverted 6 x SO mm fermentation tubes. Autoclave 10 min at 121°C. Final pH, 7.0 ± 0.2.

Dextrose Beef extract Peptone

M27. Bromcresol Purple Dextrose Broth CBCPl

Bromcresol purple (1.6\ in ethanol) Distilled water 1

10 g 3 g 5 g

2 ml liter

Dissolve ingredients in distilled water. Dispense 12-15 ml into tubes. Autoclave 15 min at 121°C. Final pH, 7.0 ± 0.2.

M28. Brucella Semisolid Medium Cfor Campvlobacterl

Tryptone 10 g Peptamine 10 g Dextrose 1 g Yeast extract 2 g NaCl 5 g Sodium bisulfite 0.1 g Sodium citrate 0.1 g Agar 1.8 g

461


Distilled water Neutral red solution

(see b below; do not add to nitrate medium)

1 liter 10 ml

If commercially prepared broth has been opened more than 2 weeks, add extra 0.25 g sodium bisulfite.

a. Appropriate biochemicals (~ c-f below) Mix all ingredients, except neutral red and biochemicals. Bring to boil, then cool slightly and divide into 4 equal portions. Add neutral red to 3 portions (2.5 ml/250 ml medium). Add glycine to one portion, NaCl to another, cysteine-HCl to a third, and KN03 to the 4th portion without neutral red. Adjust pH of portions to 7.0 ± 0.2 and dispense 7-10 ml per 16 x 125 mm tube. Autoclave at 121°C for 15 min.

b. Neutral red CNR> solution, 0.2\. Weigh 0.2 g neutral red into 10 ml EtOH in 100 ml volumetric flask. Bring to line with distilled water. Use 10 ml/L or 2.5 ml/250 ml semisolid medium.

c. Glycine, 1\. Add 10 g/L or 2.5 g/250 ml of semisolid NR agar.

d. NaCl, 3.5\. Add 30 g/L or 7.5 g/250 ml semisolid NR agar. Brucella broth already contains 0.5\ NaCl.

e. Cysteine-HCl, 0.02\. Add 0.2 g/L or 0.05 g/250 ml of semisolid NR agar.

f. Potassium nitrate CKN03 ), 1\. Add 10 g/L or 2.5 g/250 ml semisolid agar without NR.

M29. Campylobacter Enrichment Broth

a. Nutrient broth No. 2 COxoid) with 0.6\ yeast extract

Lab-Lemco powder (Oxoid L29) Peptone NaCl Yeast extract Distilled water

10 g 10 g

5 g 6 g

950 ml

Autoclave 15 min at 121°C in graduated bottles. Use broth within 1 month of preparation (preferably less than 2 weeks). Media will adsorb 0 2 during storage, which can inhibit recovery of microaerophiles. Keep bottles tightly closed. Before use add 50 ml fresh or frozen-fresh lysed horse blood (5\), 4 ml high concentration FBP, and 4 ml of appropriate antibiotic concentrate (solutions made separately). Store powdered media tightly closed in cool, dry area to reduce oxygen infusion and peroxide formation. Final pH, 7.5 ± 0.2.

b. FBP

Ferrous sulfate Sodium metabisulfite

6.25 g 6.25 g

462


Sodium pyruvate 6.25 g Distilled water to make 100 ml

Combine ingredients in 100 ml volumetric flask and bring to line with distilled water. Dissolve pyruvate in about 10 ml distilled water to facilitate pouring. Filter through 0.45 ~m membrane and store in 15-25 ml portions in sterile tightly capped polypropylene containers up to 1 month. Freezing inhibits reaction with 02 • Can be refrozen one time. Protect from light.

c. Lysed horse blood

Use only fresh blood and lyse upon receipt. If lysed when purchased, be sure it was prepared from fresh blood and shipped promptly. If not used upon receipt, freeze within 1-2 days. To lyse and/or store, gently mix and pour about 100 ml per sterile polypropylene bottle. Place in -20°C freezer. Thaw and refreeze once more if lysing. Freezing inhibits adsorption of oxygen by hemoglobin. Use blood up to 6 months. Can be thawed and refrozen several times.

d. Antibiotic formula No. 1

NOTE: All of the following antibiotic formulas are prepared in volumetric flasks and sterilized by 0.45 ~m membrane filtration. Use 4 ml/L broth of each solution (along with blood and FBP).

Sodium cefoperazone, 30 mg/L (Sigma or Pfizer). Two portions of 15 mg/L are added at separate times. Dissolve 0.375 g in 100 ml distilled water and filter. Prepare only enough for each use. Store solution 5 days at 4°C, 3 weeks at -20°C (in polypropylene containers), and 5 months at-70°C. Store powder at -20°C. Purchase powder from Sigma or obtain free from Pfizer by writing Dr. Sidney Jacobson, Roering Division, Pfizer, 235 E. 42nd St., New York, NY 10017. Request 3-5 g sodium cefoperazone for in vitro use. Also available from Oxoid but in prepared concentration (32 mg/L) that is slightly different from FDA formula.

Trimethoprim 1acatate, 12.5 mg/L (Burroughs We1lcome, Research Triangle Park, NC). Dissolve 0.3125 gin 100 ml (or 0.156 gin 50 ml) distilled water, and filter. Store up to 1 year at 4°C.

Vancomycin, 10 mg/L (Sigma). Dissolve 0.25 gin 100 ml distilled water and filter. Store up to 2 months at 4°C. Because of short shelf life, make in smaller amounts.

Cycloheximide, 100 mg/L (Sigma). Dissolve 2.5 gin 20-30 ml 70\ ethanol in 100 ml volumetric flask. Add distilled water to line, mix well, and filter. Can be refrigerated indefinitely. Do not freeze.

e. Antibiotic formula No. 2

Sodium cefoperazone, 15 mg/L. Dissolve 0.375 g in 100 ml distilled water and filter. See precautions in formula No. 1.

463


Trimethoprim lactate. Vancomycin and cycloheximide formulations are identical to formula No. 1.

f. Antibiotic formula No. 3 (Rifampicin substituted for vancomycin) - Dairy products.

Rifampicin. 10 mg/L (Sigma). Completely dissolve 0.25 gin 50 ml ethanol. Bring volume to 100 ml with distilled water, and filter. Store at -20°C up to 1 year. Store powder at -20°C also. Unless dairy products are routinely analyzed for Campylobacter, make in smaller quantities.

Cefoperazone, trimethoprim lactate and cycloheximide formulas are identical to formula No. 2.

M30. Campylobacter Isolation Agars A and B (Stern and Line formulation)

A. Campylobacter blood-free agar <Oxoidl with 0.2\ yeast extract

Nutrient broth No. 2 25 g Bacteriological charcoal 4 g Casein hydrolysate 3 g Sodium desoxycholate 1 g Ferrous sulfate 0.25 g Sodium pyruvate 0.25 g Agar 12 g Yeast extract 2 g Distilled water 1 liter

Autoclave at 121°C for 15 min. Before dispensing, add extra 100 ml/L warmed sterile distilled water plus antibiotic solution. The extra water reduces dehydration of agar in plates. Mix gently until charcoal precipitate is suspended before pouring into plates. Protect agar from light during storage. Final pH, 7.4 ± 0.2.

Antibiotics

Sodium cefoperazone, 30 mg/L. Add 8.8 ml/1100 ml agar with extra water, using one of the 15 mg/L cefoperazone solutions prepared for antibiotic formula Nos. 1 or 2.

Cycloheximide, 100 mg/L. See instructions in formula No. 1 for enrichment broth. Add 4.4 ml/1100 ml agar with extra water.

Campy-Cefex agar

Brucella agar Ferrous sulfate Sodium pyruvate Sodium bisulfite Distilled water

44.0 g 0.5 g o. 5 g

0.25 g 950 ml

464


Autoclave at 121°C for 15 min. Final pH, 7.0 ± 0.2. Cool and add 50 ml lysed horse blood and antibiotic solutions.

Antibiotics: Sodium cefoperazone, 33 ml/L. Use 8.8 ml of 15 mg/L preparation for broth. Cycloheximide, 200 mg/L. Use 8 ml of 100 mg/L preparation.

Sodium thioglycollate Na~P04 NaCl Agar CaC12 (1\ solution) Distilled water

M31. Cary-Blair Transport Medium

1.5 g 1.1 g

5 g 5 g

9 ml 991 ml

Heat with agitation to dissolve dry ingredients. Cool to 50°C Adjust pH to 8.4. Dispense 7 ml portions into 9 ml screw-cap immediately steam exactly 15 min. Cool, and tighten caps.

and add CaC12 •

Casamino acids Yeast extract ~Po. Distilled water

M32.

tubes and

Casamino acids-yeast extract (~) Broth

30 g 4 g

0.5 g 1 liter

Dissolve ingredients. Adjust pH to 7.4 ± 0.2. Dispense 10 ml into 50 ml flasks. Autoclave 15 min at 121°C.

K~P04 Distilled water

Dissolve ingredients.

Dextrose concentrate

Dextrose Distilled

M33. CYE Medium \)~

through 0.45 ~m membrane.

30 g 4 g

0.5 g 1 liter

15 min at 121°C.

20 g

M34. Casamino Acids-Yeast Extract-Salts lCA-YEl Broth (Gorbachl

Casamino acids Yeast extract NaCl ~Po.

20 g 6 g

2.5 g a. 11 g

465


Trace salts solution (below), optional 1 ml Distilled water 1 liter

Adjust pH so that value after autoclaving is 8.5 ± 0.2. Autoclave 15 min at 121°C.

Trace salts solution <optional)

Mgso. MnC1 2

FeC12

Distilled water

50 g 5 g 5 g

1 liter

Suspend ingredients. Add enough 0.1 N H2S04 to dissolve. Sterilize by filtration through 0.45 ~m membrane. Add 1 ml to 1 liter base. Dispense 10 ml portions into 50 ml flasks.

A. Basal medium

M35. Cefsulodin-Irgasan Novobiocin (CINl Agar or Yersinia Selective Agar (YSAl

Special peptone Yeast extract Mannitol Pyruvic acid (Na salt) NaCl MgSo4 • 7H20 ( 10 mg/ml) Agar Distilled water

B. Irgasan (Ciba-Geigyl solution

Irgasan 0.40\ in 95\ ethanol May be stored at -20°C up to 4 weeks.

c. Desoxycholate solution

Sodium desoxycholate Distilled water

Bring to boil with stirring; cool to 50-55°C.

D. Sodium hydroxide, 5 N

E. Neutral red, 3 mgfml

F. Crystal violet, 0.1 mg/ml

G. Cefsulodin (Abbott Labs) 1.5 mg/ml

20 g 2 g

20 g 2 g 1 g

1 ml 12 g

756 ml

1 ml

0.5 g 200 ml

1 ml

10 ml

10 ml

10 ml

May be stored at -70°C. Thaw to room temperature just before use.

466


H. Novobiocin, 0.25 mg/ml 10 ml

I. Strontium chloride, 10\; filter-sterilized 10 ml

Preparation: Add ingredients for solution A (basal medium) to water and bring to boil with stirring. Cool to about 80°C (10 min in 50°C water bath). Add solution B (Irgasan) and mix well. Cool to 50-55°C. Add solution c (desoxycholate); solution should remain clear. Add solutions D through H. Slowly add soltuion I with stirring. Adjust pH to 7.4 with 5 N NaOH. Dispense 15-20 ml into each petri dish. Commercially prepared dehydrated Yersinia selective agar (Difco) with supplements may be substituted. Follow manufacturer ' s instructions for preparation.

M36. Cell Growth Medium

Mix the following sterile solutions aseptically:

Minimal essential medium with Hanks' salts (MEMH) L-15 medium (Leibovitz)

(containing 100 units penicillin G, 100 ~g streptomycin, and 50 ~g gentamicin per ml)

1 liter 1 liter

Mix on magnetic stirrer, filter through 0.20 ~m membrane, and dispense into sterile 2 liter Erlenmeyer flask. Final pH, 7.5. Cap and store at 5°C. Just before use add:

Fetal bovine serum NaHC03 (7.5\)

Pancreatic digest Magnesium chloride Potassium sulfate Agar

M37.

of gelatin

Cetrimide (cetyl trimethyl ammonium

Cetrimide Agar

bromide)

200 ml 50 ml

20.0 g 1.4 g

10.0 g 13.6 g 0.3 g

Suspend 45.3 g of ingredients or commercial powder (Pseudosel™, BBL; BectonDickinson) in 1 liter of purified water. Add 10 ml glycerol. Mix well. Heat to boiling, agitating frequently; maintain boiling for 1 min to dissolve powder. Autoclave at 121°C for 15 min. Final pH, 7.1 ± 0.2. Bact o cetrimide agar base plus glycerol (Difco) is a similar medium.

Fresh beef liver Peptone ~P04 Soluble starch Distilled water

M38. Chopped Liver Broth

500 g 10 g

1 g 1 g

1 liter

467


Grind liver into the water. Heat to boiling and simmer 1 h. Cool, adjust pH to 7.0, and boil 10 min. Filter through cheesecloth and press out excess liquid. Add other ingredients and adjust pH to 7.0. Add water to make 1 liter. Filter through coarse filter paper. Store broth and meat separately in freezer. To 18 x 150 or 20 x 150 mm test tubes, add chopped liver to depth of 1.2-2.5 em and 10-12 ml of broth. Autoclave 15 min at 121°C.

Sodium citrate Glucose Yeast extract

M39. Christensen Citrate Agar

Cysteine monohydrochloride Ferric ammonium citrate KH2P04

NaCl Sodium thiosulfate Phenol red Agar Distilled water

3 g 0.2 g 0.5 g 0.1 g 0.4 g

1 g 5 g

0.08 g 0.012 g

15 g 1 liter

Suspend ingredients, mix thoroughly and heat with occasional agitation. Boil about 1 min to dissolve ingredients. Fill 16 x 150 mm tubes 1/3 full and cap or plug to maintain aerobic conditions. Autoclave for 15 min at 121°C. Before media solidify, incline tubes to obtain 4-5 em slant and 2-3 em butt.

Peptone NaCl Dextrose KH2P04

H40. Cristensen's Urea Agar

Phenol red (6 ml of 1:500 solution) Agar Distilled water

Urea concentrate

Urea Distilled water

1 g 5 g 1 g 2 g

0.012 g 15 g

900 ml

20 g 100 ml

Dissolve all ingredients except urea in 900 ml water (basal medium). For halophilic Vibrio spp., add extra 15 g NaCl (final NaCl concentration, 2%). Autoclave 15 min at 121°C. Cool to 50-55°C. Dissolve urea in 100 ml water. Filter-sterilize; add aseptically to cooled basal medium. Mix; dispense to sterile tubes or Petri dishes. Slant for 2 em butt and 3 em slant.

For Vibrio spp., add NaCl to achieve a 2-3% final concentration.

468


M41.

Brain heart infusion (M20) Congo Red dye Agarose Distilled water

Congo Red BHI Agarose Medium*

37 g 75 mg

15 g 1 liter

Dissolve, autoclave at 121°C for 15 mi n and pour 20 ml per plate.

*From s. Bhaduri, USDA, Philadelphia, PA . ASM Abstracts, 1989, No. P26. u.s. Patent application ser. no. 07/493,662.

Beef heart Proteose peptone Dextrose NaCl Distilled water

M42. Cooked Meat Medium

454 g 20 g

2 g 5 g

1 liter

Follow directions as in M38, or suspend 12.5 g commercial dehydrated cooked medium in 100 ml cold distilled water. Mix and let stand 15 min to wet particles thoroughly. Or distribute 1.25 g into 20 x 150 mm test tubes, add 10 ml cold distilled water, and mix thoroughly to wet all particles. Autoclave 20 min at 121°C. Final pH, 7.2.

M43. Cooked Meat Medium (Modified)

(a) Cooked meat medium (commercially available in dehydrated form)

Beef heart Proteose peptone Dextrose NaCl

(b) Diluent <not available commercially)

Tryptone Sodium thioglycollate Soluble starch Dextrose Neutral red (1\ aqueous) Distilled water 1

454 g 20 g

2 g 5 g

10 g 1 g 1 g 2 g

5 ml liter

Adjust pH to 6.8 ± 0.2. Add 1 g dehydrated (a) and 15 ml (b) to 20 x 150 mm tubes. Let meat particles rehydrate. Autoclave 15 min at 121°C.

469

pi)~ BACTERIOLOGICAL ANALYTICAL MANUAL 7TH EDITION/1992

M44. Decarboxylase Basal Medium (Arginine, Lysine, Ornithine)

Peptone or gelysate Yeast extract Glucose

5 g 3 g 1 g

0.02 g Bromcresol purple Distilled water For Vibrio spp., add NaCl to achieve a 2-3% final

1 liter concentration.

For arginine broth, add 5 g L-arginine to 1 liter base; for lysine (Falkow) broth, add 5 g L-lysine to 1 liter base; for ornithine, add 5 g L-ornithine to 1 liter base. Adjust pH so that value after sterilization is 6.5 ± 0.2. Dispense 5 ml portions into 16 x 125 mm screw-cap tubes. Autoclave loosely capped tubes 10 min at 121°C. Screw the caps on tightly for storage and after inoculation. For control, use unsupplemented base.

M45. Duncan-Strong fDSl Sporulation Medium. Modified (for ~. perfringens)

Proteose peptone 15 Yeast extract 4 Sodium thioglycollate 1 Na~P04 • 7H20 10 Raffinose 4

g g g g g


Dissolve ingredients and sterilize by autoclaving for 15 min at 12l°C. Adjust to pH 7.8 ± 0.1, using filter-sterilized 0.66 M sodium carbonate.

M46. Bagle•s Minimal Essential Medium fMBMBl (with Barle"s salts and nonessential amino acids)

L-Alanine 8.9 mg L-V aline 46.0 L-Arginine HCl 126.0 mg D-Calcium pantothenate 1.0 L-Asparagine-~0 150.0 mg Choline chloride 1.0 L-Aspartic acid 13.3 mg Folic acid 1.0 L-Cystine.2HC1 31. 29mg Isoinositol 2.0 L-Glutamic acid 14.7 mg Nicotinamide 1.0 L-Glutamine 292.0 mg Pyridoxal HCl 1.0 L-Glycine 7.5 mg Riboflavin 0.1 L-Histidine HCLH20 42.0 mg Thiamine HCL 1.0 L-Isoleucine 52.0 mg Glucose 1000.0 L-Leucine 52.0 mg CaC12 • 2H20 265.0 L-Lysine HCl 72 . 5 mg XCl 400.0 L-Methionine 15.0 mg MgS04 • 7Hp 200.0 D-Phenylalanine 32.0 mg NaCl 6800.0 L-Proline 11.5 mg NaHC03 2200.0 L-Serine 10.5 mg NaH2P04 • H20 140.0 L-Threonine 48.0 mg Phenol red 10.0

mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg

L-Tryptophan 10.0 mg Distilled water 1 liter L-Tyrosine (disodium

salt) 52.1 mg

Dissolve ingredients in distilled water. Sterilize by filtration. Final pH, 7.2 ± 0.2.

470


NaCl KCl CaC12 .2Hp Mgso •• 7H2o NaH2P04 • H20 Glucose NaHC03

Distilled water

M47. Earle's Balanced Salts (Phenol Red-Free)

1

6.8 g 400 mg 265 mg 200 mg 140 mg l.Og 2.2 g liter

Dissolve ingredients in the water. Sterilize by filtration. Final pH, 7.2 ± 0.2.

Beef extract Peptone or gelysate NaCl Agar Distilled water

M48. EB Motility Medium

3 g 10 g

5 g 4 g

1 liter

Heat with agitation and boil 1-2 min to dissolve agar. Dispense 8 m1 portions into 16 x 150 mm screw-cap tubes. Autoclave 15 min at 121°C. Final pH, 7.4 ± 0.2.

M49. EC Broth

Trypticase or tryptose 20 g Bile salts No. 3 1.5 g Lactose 5 g KzHP04 4 g KH2P04 1.5 g NaCl 5 g Distilled water 1 liter

Distribute 8 ml portions to 16 x 150 mm test tubes containing inverted 10 x 75 mm fermentation tubes. Autoclave 15 min at 121°C. Final pH 6.9 ± 0.2.

MSO. BC- MUG Medium

Prepare as for EC broth (M49), but add 50 mg 4-methylumbelliferyl-betaD-glucuronide (MUG) per liter before autoclaving (15 min, 121°C). EC-MUG medium is available commercially.

471


MSl. Egg Yolk Emulsion, 50'

Wash fresh eggs with a stiff brush and drain. Soak eggs 1 h in 0.1\ HgC12 •

Drain solution, replace with 70\ ethanol, and soak 30 min. Drain ethanol. Crack eggs aseptically and discard whites. Remove egg yolks with sterile syringe or wide-mouth pipet. Place yolks in sterile container and mix aseptically with equal volume of sterile 0.85\ saline. Store at 4°C until use.

M52. Enrichment Broth (EB), pH 7.3 (with or without sodium pyruvate)

TSBYE supplemental with:

Acriflavin HCl (Sigma) (for nondairy foods) Acriflavin HCl (Sigma) (for milk and dairy products) Nalidixic acid (sodium salt) (Sigma) Cycloheximide (Sigma) Pyruvic acid (sodium salt) (Sigma), 10\ (w/v) aqueous

solution (for damaged Listeria spp.)

15 mg/L 10 mg/L 40 mg/L 50 mg/L

11.1 ml

Add the 3 supplementary ingredients aseptically to TSBYE after autoclaving and just before use. Make acriflavin and nalidixic supplements as 0.5\ (w/v) stock solutions in distilled water. Make cycloheximide supplement as 1.0\ (w/v) stock solution in 40\ (v/v) solution of ethanol in water. Filtersterilize all 3 supplementary ingredients. Add 0.68 ml acriflavin stock solution (0.455 ml for milk and dairy products), 1.8 ml nalidixic stock solution, and 1.15 ml cycloheximide stock solution to 225 ml TSBYE to achieve (within ± 1.6\ or less) the stated amounts in mg/L. If recovering damaged Listeria, add 2.5 ml of 10\ (w/v) filter-sterilized sodium pyruvate at zerotime, and delay adding the 3 supplements until the 6th hour of incubation at 30°C. EB for non-dairy foods is commercially available. ~: Cycloheximide is very toxic. Take care with its disposal, especially if large volumes of EB are used. Refer to a current edition of the Merck Index (Merck Co. Inc., Rahway, NJ) for information on its stability properties in relation to disposal methods.

Infusion agar Esculin Ferric citrate Distilled water

M53. Esculin Agar. Modified (CDC)

40 g 1 g

0.5 g 1 liter

Heat with agitation to dissolve agar. Cool to 55°C. Adjust pH to 7.0 ± 0.2. Dispense 4 ml portions to 13 x 100 mm tubes. Autoclave 15 min at 121°C. Slant tubes.

472

A •


Peptone Yeast extract Gelatin Agar Distilled water

M54. Gelatin Agar fGAl

4 g 1 g

15 g 15 g

1 liter

suspend ingredients with constant stirring to prevent scorching gelatin, and boil to dissolve gelatin and agar. Adjust to pH 7.2 ± 0.2. Autoclave 15 min at 12rc. cool to 45-50°C. Pour plates.

MSS. Gelatin Salt Agar (GS)

Prepare gelatin agar (M54), but add 30 g NaCl/L. Suspend ingredients and boil to dissolve gelatin and agar. Adjust to pH 7.2 ± 0.2. Autoclave 15 min at 121°C. Cool to 45-50°C. Pour plates. If necessary, to inhibit spreading by Vibrio spp. such as y. alginolyticus, use 25-30 g agarfL.

M56. Gelatinase Solution, 5'

Gelatinase 5 g Distilled water 100 ml

Suspend gelatinase in distilled water. Centrifuge 10 min at 9500 rpm and sterilize by filtration through 0.45 ~m membrane. Dispense 100 ml portion into bottles.

Gentamicin sulfate Distilled water

M57. Gentamicin Sulfate Solution

500,000 ~g 100 ml

Sterilize by filtration through 0.20 ~m membrane. Store at -20°C.

M58. Ham's P'-10 Medium

L-Alanine 8.91 mg Sodium pyruvate 110 mg L-Arginine HCl 211.00 mg Thymidine o. 727 mg L-Asparag ine-H20 15.00 mg Biotin 0.024 mg L-Aspartic acid 13.30 mg Choline chloride 0.698 mg L-Cysteine.2HC1 35.12 mg Folic acid 1.320 mg L-Glutamine 146.20 mg Isoinositol 0.541 mg L-Glutamic acid 14.70 mg Niacinamide 0.615 mg Glycine 7.51 mg D-Calcium pantothenate o. 715 mg L-Histidine HCl. H20 21.00 mg Pyridoxine HCl 0.206 mg L-Isoleucine 2.60 mg Riboflavin 0.376 mg L-Leucine 13.10 mg Thiamine HCl 1.010 mg L-Lysine HCl 29.30 mg Vitamin 8 12 1.360 mg L-Methionine 4.48 mg CaC12 • 2H20 44.10 mg D-Phenylalanine 4.96 mg CuS04 • 5H20 0.0025 mg

473


L-Proline 11.50 mg Feso •. 7H20 0.83 mg L-Serine 10.50 mg KCl 285.00 mg L-Threonine 3.57 mg KH2PO• 83.00 mg L-Tryptophan 0.60 mg Mgso •• 7H20 152.80 mg L-Tyrosine 1.81 mg NaCl 7400.00 mg L-Valine 3.50 mg NaHC03 1200.00 mg Glucose 1100.0 mg NaH2PO •• H20 290.0 mg Hypoxanthine 4.08 mg znso •. 7Hp 0.028 mg Lipoic acid 0.2 mg Distilled water 1 liter Pehnol red 1.2 mg

Dissolve ingredients in water. Sterilize by filtration. Final pH, 7.0 ± 0.2. Check sterility before use.

M59. Heart infusion agar (HIA) (Difco)

Beef heart infusion (Difco) Tryptose NaCl Agar Distilled water

Or

Blood Agar Base No. 2 (Difco) Proteose peptone Liver digest Yeast extract NaCl Agar

500 g 10 g

5 g 15 g

1 liter

15 g 2.5 g

5 g 5 g

12 g

Autoclave either base at 121°C for 15 min. If preparing blood agar, reduce water to 950 ml. Add 50 ml defibrinated (whole or lysed) horse blood and FBP (4 ml to agar + blood) after autoclaving and cooling to 48°C. Final pH, 7.4 ± 0.2.

M60. Heart Infusion (HI) Broth and Agar (HIA) (for Vibrio) (commercially available blood agar base may be used)

Infusion from 500 g beef heart Tryptose NaCl

1 liter 10 g

5 g

Dissolve ingredients and dispense into tubes. For halophilic Vibrio spp. add an extra 15 g NaCl/liter. Autoclave 15 min at 121°C. Final pH, 7.4 ± 0.2.

For heart infusion agar, add 15 g agar/L and boil to dissolve before dispensing and sterilizing.


474


M61. Hektoen Enteric (BEl Agar

Peptone 12 g Sodium thiosulfate 5 g Yeast extract 3 g Ferric ammonium citrate 1.5 g Bile salts No. 3 9 g Bromthymol blue 0.065 g Lactose 12 g Acid fuchsin 0.1 g Sucrose 12 g Agar 14.0 g Salicin 2 g Distilled water 1 liter NaCl 5 g

Heat to boiling with frequent agitation to dissolve. Boil no longer than 1 min. Do not overheat. Cool in water bath. Pour 20 ml portions into sterile 15 x 100 mm petri dishes. Let dry 2 h with lids partially removed. Final pH, 7.6 ± 0.2. Do not store more than 1 day.

Tryptone Bile salts No. 3 NaCl Sorbitol MUG reagent Bromcresol purple Agar

M62. Hemorrhagic coli (HCl Agar

Distilled water (deionized)

20.0 g 1.12 g 5.0 g

20.0 g 0.1 g

0.015 g 15.0 g

1 liter

Dissolve ingredients in distilled water by heating with stirring. Autoclave 15 min at 121°C. Final pH, 7.2 ± 0.2. NOTE: The MUG reagent is not essential for the enzyme-labeled monoclonal antibody procedure.

For DNA probe: If colonies are not to be isolated by growth and metabolic characteristics, MUG reagent may be omitted. Plates may be kept 3-4 weeks if wrapped and stored at 4°C. Colonies do not attach well on filters if plates are too dry. If HC agar is to be utilized fully, see Chapter 24, ref. 103. MUG reagent may be purchased from HACH Company, P.O. Box 389, Loveland, co.

Peptone Yeast extract NaCl Dextrose Bromcresol purple Agar Distilled water

M63. Hugh-Leifson Glucose Broth lHLGB)

2 g 0.5 g

30 g 10 g

0.015 g 3 g

1 liter

Heat with agitation to dissolve agar. Adjust pH to 7.4 ± 0.2. Autoclave 15 min at 121°C.

475

pi)~ BACTERIOLOGICAL ANALYT.ICAL MANUAL 7TH EDITION/1992

L-Tryptophan NaCl ~P04 KH2P04

Distilled water

M64. Indole Medium

1 g 1 g

3.13 g 0.27 g 200 ml

Dissolve ingredients. Dispense 1 ml portions to 13 x 100 mm screw-cap tubes. Autoclave 15 min at 121°C. Final pH, 7.2 ± 0.2.

M65. Indole Medium (CDC)

Tryptone or trypticase 20 g Distilled water 1 liter

Adjust pH to 7.3 ± 0.2. Dispense 4 ml portions to 13 x 100 mm tubes. Autoclave 15 min at 121°C. Final pH, 7.2 ± 0.2.

Trypticase Na~P04 Dextrose KN03

Agar Distilled water

M66. Indole Nitrite Medium (Tryptic Nitrate)

1

20 g 2 g 1 g 1 g 1 g

liter

Heat with agitation to dissolve ingredients. Mix and dispense 11 ml portions into 16 x 150 mm tubes. Autoclave 15 min at 118°C. Final pH, 7.2 ± 0.2.

M67. Irgasan-Ticarcillin-Chlorate CITCl Broth

Tryptone 10 g Yeast extract 1 g MgC12 • 6H20 60 g NaCl 5 g Potassium chlorate 1 g Malachite green, 0.2\ 5 ml Distilled water 1 liter

Adjust to pH 7.6 ± 0.2 and autoclave at 121°C for 15 min. Add the following:

Ticarcillin (1 mgfml) 1 ml Irgasan DP300 (1 mgfml) 1 ml

Fresh whole milk Ferrous sulfate. 7H20 Distilled water

M68. Iron Milk Medium (Modified)

1 liter 1 g

50 ml

476


Dissolve ferrous sulfate in 50 ml distilled water. Add slowly to 1 liter milk and mix with magnetic stirrer. Dispense 11 ml medium into 16 x 150 mm culture tubes. Autoclave 12 min at 118°C. Prepare fresh medium before use.

Proteose peptone No. 3 Glycerol, C.P. K~P04 Mgso. Agar Distilled water

M69. King's B Medium

20 g 10 ml l.Sg l.Sg

15 g 1 liter

Add all ingredients except MgS04 • Heat with agitation to dissolve agar. Adjust pH to 7.2 ± 0.2. Add MgS04 slowly and mix. Dispense 4 ml portions to 13 x 100 tubes. Autoclave 15 min at 121°C. Incline tubes to give half butt and half slant.

M70. King's 0/F Basal Medium

Trypticase (or casitone) Phenol red, 1.5\ solution Agar Distilled water

2 g 2 ml

3 g 1 liter

Heat with agitation to dissolve agar. Adjust pH to 7.3 ± 0.2. Dispense 100 ml portions to flasks. Autoclave 15 min at 121°C. Cool to 50°C.

Carbohydrates, 10\. Dissolve 10 g quantities of carbohydrates in 100 ml distilled water. Sterilize by filtration through 0.22 ~m membrane. Add 10 ml concentrate to 90 ml melted base and mix. Aseptically dispense 3 ml portions to sterile 13 x 100 mm tubes.

M71. Kligler Iron Agar

Polypeptone peptone Lactose Dextrose NaCl Ferric ammonium citrate Sodium thiosulfate Agar Phenol red Distilled water

Heat with agitation to dissolve. and autoclave 15 min at 121°C. 7.4 ± 0.2.

20 g 20 g

1 g 5 g

0.5 g 0.5 g

15 g 0.025 g 1 liter

Dispense into 13 x 100 mm screw-cap tubes Cool and slant to form deep butts. Final pH,


477


NaNH4HP04 • 4H20 K~P04 MgS04 • 7H20 Sodium citrate. 2H20 Distilled water

M72. Koser's Citrate Broth

l.Sg 1 g

0.2 g 3 g

1 liter

Dispense into screw-cap tubes as desired. Autoclave 15 min at 121°C. Final pH, 6.7 ± 0.2. This formulation is listed in Official Methods of Analvsis of the AOAC, and Standard Methods for the Examination of Wastewater of the APHA. It differs from the composition of commercially available dehydrated media. The latter are satiafactory.

M73. L-15 Medium (Modified) Leibovitz

D-Gallactose 90 mg L-Tyrosine 300 mg Phenol red, Na 10 mg DL-Valine 200 mg Sodium pyruvate 550 mg D-Calcium pantothenate 1 mg DL-a-Alanine 450 mg Choline chloride 1 mg L-Arginine (free base) 500 mg Folic acid 1 mg L-Asparag ine. H20 250 mg i-Inositol 2 mg L-Cysteine (free base) 120 mg Nicotinamide 1 mg L-Glutamine 300 mg Pyridoxine HCl 1 mg Glycine 200 mg Riboflavin-5-phosphate, Na 0.1 mg L-Gistidine (free base) 250 mg Thiamin monophosphate.2H20 1.0 mg L-Isoleucine 250 mg CaC12 (anhydrous) 140 mg L-Leucine HC1 125 mg KC1 400 mg DL-Methionine 150 mg KH2P04 60 mg L-Phenylalanine 250 mg MgC12 (anhydrous) 93.68 mg L-Serine 200 mg NaCl 8000 mg DL-Threonine 600 mg Na~P04 (anhydrous) 190 mg L-Tryptophan 20 mg Distilled water 1 liter

Filter through 0.20 ~m membrane and dispense into 2 liter Erlenmeyer flask. Final pH, 7.5. Cap and store at 5°C.

M74. Lactose Broth

Beef extract 3 g Peptone 5 g Lactose 5 g Distilled water 1 liter

Dispense 225 ml portions into 500 ml Erlenmeyer flasks. After autoclaving 15 min at 121°C and just before use, aseptically adjust volume to 225 ml. Final pH, 6.9 ± 0.2.

M75. Lactose-Gelatin Medium (for c. perfringensl

Tryptose 15 g Yeast extract 10 9

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Lactose Phenol red (as solut i on) Gelatin Distilled water

10 g 0.05 g

120 g 1 liter

Heat to dissolve tryptose, yeast extract, and lactose in 400 ml water. Suspend gelatin in 600 ml water and heat at 50-60°C with agitation to dissolve. Mix 2 solutions. Adjust pH to 7.5 ± 0.2. Add phenol red and mix. Dispense 10 ml portions into 16 x 150 mm s crew-cap tubes. Autoclave 10 min at 121°C. If not used within 8 h, deaerate by heating at 50-70°C for 2-3 h before use.

Tryptose or trypticase Lactose K~PO• KH2P04

NaCl Sodium lauryl sulfate Distilled water

M76. Lauryl Tryptose (LST) Broth

1

20 9 5 g

2.75 g 2.75 g

5 g 0.1 g liter

Dispense 10 ml portions to 20 x 150 mm tubes containing inverted 10 x 75 mm fermentation tubes. Autoclave 15 min at 121°C. Final pH, 6.8 ± 0.2.

M77. Laury! Tryptose MUG (LST-MUG) Broth

Tryptose or trypticase Lactose K~PO•

KH2PO• NaCl Sodium lauryl sulfate 4-methylumbelliferyl-B-D-glucuronide (MUG)* Distilled water 1

20 g 5 g

2.75 g 2.75 g

5 g 0.1 g 50 mg liter

Prepare lauryl tryptose broth and add MUG. Dissolve with gentle heat if necessary. Dispense 10 ml portions into 20 x 150 mm test tubes containing inverted 10 x 75 mm fermentation tubes. Autoclave 15 min at 121°C. Final pH, 6.8 ± 0.2.

*MUG may be obtained from several sources, e.g., HACH Co., Loveland, co.

M78. Letheen Agar (Modified)

Letheen agar (Difco or BBL) Trypticase peptone Thiotone peptone Yeast extract NaCl Sodium bisulfite Agar

32 g 5 g

10 g 2 g 5 g

0.1 g 5 g

479



Heat with agitation to dissolve agar. Autoclave 15 min at 121°C. Aseptically dispense 20 ml into 15 x 100 mm petri dishes. Final pH, 7.2 ± 0.2.

Letheen broth Trypticase peptone Thiotone peptone Yeast extract Sodium bisulfite Distilled water

M79. Letheen Broth fKodifiedl

1

26.7 g 5 g

10 g 2 g

0.1 g liter

Dispense 90 ml into screw-cap bottle. Autoclave 15 min at 121°C. Final pH, 7.2 ± 0.2.

Peptone Lactose K~P04 Agar Eosin Y Methylene blue Distilled water

MBO. Levine's Eosin-Methylene Blue fL-EMBl Agar

10 g 10 g

2 g 15 g

0.4 g 0.065 g 1 liter

Boil to dissolve peptone, phosphate, and agar in 1 liter of water. Add water to make original volume. Dispense in 100 or 200 ml portions and autoclave 15 min at not over 121°C. Final pH, 7.1 ± 0.2. Before use, melt, and to each 100 ml portion add (a) 5 ml sterile 20\ lactose solution; (b) 2 ml aqueous 2\ eosin Y solution; and (c) 4.3 ml 0.15\ aqueous methylene blue solution. When using complete dehydrated product, boil to dissolve all ingredients in 1 liter water. Dispense in 100 or 200 ml portions and autoclave 15 min at 121°C. Final pH, 7.1 ± 0.2.

M81. Lithium Ch1oride-Phenylethanol-Moxalactam fLPMl Medium

Phenylethanol agar (Difco) Glycine anhydride (NOTE: not glycine) Lithium chloride Moxalactam stock solution, 1\ in phosphate buffer, pH 6.0 Distilled water

Sterilize medium (without moxalactam) at 121°C for 15 min. and add filter-sterilized moxalactam solution.

35.5 g 10 g

5 g 2 ml

cool to 48-50°C

Moxalactam stock solution consists of 1 g moxalactam salt (ammonium or sodium) in 100 ml 0.1 M potassium phosphate buffer, pH 6.0. Store filter-sterilized stock solution frozen in 2 ml aliquots.

Moxalactam (Eli Lilly Co.) is retailed by Sigma Chemical Co. The LPM medium

480


is most effective in the Henry illumination system when poured thin, i.e., 12-15 ml per standard petri dish. To avoid drying of thin agar, refrigerate and use plates rapidly. LPM basal medium is commercially available as a powder.

M82. LPM Plus Esculin and Ferric Iron

Esculin 1.0 g Ferric ammonium citrate 0.5 g

Add these components to those for LPM (M81). Sterilize medium, temper, and add filter-sterilized moxalactam, as described for LPM medium.

M83. Liver-Veal Agar

Liver, infusion from 50 g Casein, isoelectric 2 g Veal, infusion from 500 g NaCl 5 g Proteose peptone 20 g Sodium nitrate 2 g Neopeptone 1.3 g Gelatin 20 g Tryptone 1.3 g Agar 15 g Dextrose 5 g Distilled water 1 liter starch, soluble 10 g

Heat with agitation to dissolve. Autoclave 15 min at 121°C. Final pH, 7.3 ± 0.2.

Fresh eggs, yolks only Liver veal agar Distilled water

M84. Liver-Veal-Egg Yolk Agar

2 or 3 48.5 g 500 ml

Heat with agitation to dissolve. Autoclave 15 min at 121°C. Cool to 50°C.

Egg yolk emulsion. To each 500 ml of melted liver veal agar, add 40 ml egg yolk-saline suspension (see M51 for instructions). Mix thoroughly and pour into sterile 15 x 100 mm petri dishes. Dry plates at room temperature for 2 days or at 35°C for 24 h. Check plates for sterility and store sterile plates in refrigerator. In certain instances the medium may be used without addition of egg yolk emulsion.

Yeast extract, 0.3\ Peptone NaCl Agar Distilled water

M85. Long-term Preservation Medium

3 g 10 g 30 g

3 g 1 liter

Heat to dissolve ingredients. Dispense 4 ml portions to 13 x 100 mm screw-cap tubes. Autoclave 15 min at 121°C. Cool and tighten caps for storage. No pH adjustment is necessary.

481

fl)j\ BACTERIOLOGICAL ANALYTICAL MANUAL 7TH EDITION/1992

Peptone Yeast extract Glucose L-Lysine L-Arginine

H86. Lysine Arginine Iron Agar fLAIAl

Ferric ammonium citrate Sodium thiosulfate Bromcresol purple Agar

5 g 3 g 1 g

10 g 10 g

0.5 g 0.04 g 0.02 g

15 g

Adjust pH to 6.8. Heat to boiling and dispense 5 ml into each 13 x 100 mm screw-cap culture tube. Autoclave at 121°C for 12 min. Cool tubes in slanted position. (This medium may also be prepared by supplementing Difco lysine iron agar (LIA) with 10 g L-arginine per liter . )

M87. Lysine Decarboxylase Broth fFalkowl (for Salmonella)

Gelysate or peptone Yeast extract Glucose L-Lysine Bromcresol purple Distilled water

5 g 3 g 1 g 5 g

0.02 g 1 liter

Heat until dissolved. Dispense 5 ml portions into 16 x 125 mm screw-cap tubes. Autoclave loosely capped tubes 15 min at 121°C. Screw the caps on tightly for storage and after inoculation. Final pH, 6.5-6.8.

L-Lysine HCl Dextrose KH2PO• Distilled water

H88. Lysine Decarboxylase fLDCl Medium (for Gram-negative nonfermentative bacteria)

0.5 g 0.5 g 0.5 g

100 ml

Dissolve ingredients. Adjust pH to 4.6 ± 0.2. Autoclave 15 min at 121°C. Aseptically dispense 1 ml portions to sterile 13 x 100 mm tubes.

H89. Lysine Iron Agar (Edwards and Fife)

Gelysate or peptone 5 g Yeast extract 3 g Glucose 1 g L-Lysine hydrochloride 10 g Ferric ammonium citrate 0.5 g Sodium thiosulfate (anhydrous) 0.04 g Bromcresol purple 0.02 g Agar 15 g

482

~

FI)J\ BACTERIOLOGICAL ANALYTICAL MANUAL 7TH EDITION/1992


Heat to dissolve ingredients. Dispense 4 ml portions into 13 x 100 mm screwcap tubes. Autoclave 12 min at 121°C. Let solidify in slanted position to form 4 em butts and 2.5 em slants. Final pH, 6.7 ± 0.2.

K90. Lysozyme Broth

Base. Prepare nutrient broth as recommended. Dispense 99 ml portions to 170 ml bottles. Autoclave 15 min at 121°C. Cool to room temperature before use.

Lysozyme solution. Dissolve 0.1 g lysozyme in 65 ml sterile 0.01 N HCl. Heat to boiling for 20 min. Dilute to 100 ml with sterile 0.01 N HCl. Alternatively, dissolve 0.1 g lysozyme in 100 ml distilled water. Sterilize by filtration through 0.45 ~m membrane. Test for sterility before use. Add 1 ml lysozyme solution to 99 ml nutrient broth. Mix and dispense 2.5 ml portions to sterile 13 x 100 mm tubes.

K91. KacConkey Agar

Proteose peptone or polypeptone Peptone or gelysate Lactose Bile salts No. 3 NaCl Neutral red Crystal violet Agar Distilled water

3 g 17 g 10 g

1.5 g 5 g

0.03 g 0.001 g

13.5 g 1 liter

Suspend ingredients and heat with agitation to dissolve. Boil 1-2 min. Autoclave 15 min at 121°C, cool to 45-50°C, and pour 20 ml portions into sterile 15 x 100 mm petri dishes. Dry at room temperature with lids closed. DO NOT USE WET PLATES. Final pH, 7.1 ± 0.2.

Yeast extract (NH4 hS04

~P04 KH2P04

NaCl Sodium malonate Glucose Bromthymol blue Distilled water

K92. Malonate Broth

1 g 2 g

0.6 g 0.4 g

2 g 3 g

0.25 g 0.025 g 1 liter

Dissolve by heating, if necessary. Dispense 3 ml portions into 13 x 100 mm test tubes. Autoclave 15 min at 121°C. Final pH, 6.7 ± 0.2.

483


Malt extract Agar Distilled water

M93. Malt Extract Agar

30 g 20 g

1 liter

Boil to d i ssolve ingredients. Autoclave 2S min at 121°C. Dispense 20-2S ml into steri le 1S x 100 mm petri dishes. Final pH, S.S ± 0.2.

For cosmetics, cool medium to 47-S0°C after autoclaving. Add 40 ppm chlortetracycline. Mix thoroughly and dispense 20 ml portions into 1S x 100 mm petri dishes.

M94. Malt Extract Broth «Difco)

Malt extract base 6. 0 g Maltose, technical 1. 8 g Dextrose 6.0 g Yeast extract 1.2 g

Final pH, 4.7 ± 0.2.

Beef extract Peptone Mannitol NaCl Phenol red Agar Distilled water

M9S. Mannitol-Egg Yolk-Polymyxin Agar

1 g 10 g 10 g 10 g

0.02S g 1S g

900 ml

Heat with agitation to dissolve agar. Adjust pH so that value after sterilization is 7.2 ± 0.2. Dispense 22S ml portions to SOC ml Erlenmeyer flasks. Autoclave 1S min at 121°C. Cool to S0°C.

Polymyxin B solution, 0.1\. Dissolve SOO,OOO units sterile polymyxin B sulfate powder in SO ml sterile distilled water. Store in 85 ml bottle at 4°C.

Egg yolk emulsion, SO\ (~ MSl)

Final medium. To 22S ml melted base add 2.S ml polymyxin B solution and 12.S ml egg yolk emulsion. Mix and dispense 18 ml portions to sterile 1S x 100 mm petri dishes. Dry plates at room temperature for 24 h before use.

484


Phytone Polypeptone Beef extract D-Mannitol Maltose NaCl Agar lOOOX Dye stock solution* Distilled water

M96. Mannitol Maltose Agar

1

5 g 5 g 5 g

10 g 10 g 20 g 13 g 1 ml

liter

Suspend ingredients and boil to dissolve. Adjust to pH 7.8 ± 0.2. Autoclave 15 min at 12rc.

*lOOOX Dye stock solution. See formulation under modified cellobiosepolymyxin B-colistin (mCPC) agar (M98).

M97. Mannitol Salt Agar

Beef extract 1 g Polypeptone 10 g NaCl 75 g Mannitol 10 g Phenol red 0.025 g Agar 15 g Distilled water 1 liter

Heat with agitation to dissolve agar and boil 1 min. Dispense 20 ml portions into 15 x 100 mm petri dishes. Autoclave 15 min at 121°C. Final pH, 7.4 ± 0.2.

M98. Modified Cellobiose-Polymyxin B-Colistin lmCPCl Agar

Solution 1

Peptone Beef extract NaCl lOOOX Dye stock solution* Agar Distilled water

Adjust to pH 7.6. Boil to dissolve agar. Cool to 48-55°C.

*lOOOX Dye stock solution

Bromthymol blue cresol red Ethanol, 95\

10 g 5 g

20 g 1 ml 15 g

900 ml

4.0 g 4.0 g

100 ml

For consistent medium color, use dye stock solution rather than repeatedly

485


weighing out dry dyes. Dissolve dyes in ethanol for 4\ (w/v) stock solution. Using 1 ml of this solution per liter of mCPC agar gives 40 mg bromthymol blue and 40 mg cresol red per liter.

Solution 2

Cellobiose Colistin Polymyxin B Distilled water

10 g 400,000 units 100,000 units

100 ml

Dissolve cellobiose in distilled water by heating gently. Cool. Add antibiotics. Add Solution 2 to cooled Solution 1, mix, and dispense into petri dishes. Final color, dark green to green-brown.

NOTE: This medium, like TCGS, is very inhibitory and does not require autoclaving.

M99. Motility-Indole-ornithine (MIO) Medium

Yeast extract 3 g Peptone 10 g Tryptone 10 g L-Ornithine HCl 5 g Dextrose 1 g Agar 2 g Bromcresol purple 0.02 g Distilled water 1 liter

Dispense 4 ml portions into 13 x 100 mm tubes. Autoclave 15 min at 121°C. Final pH, 6.5 ± 0.2.

Trypticase Yeast extract Dextrose Na2HP04

Agar Distilled water

M100. Motility Medium (for B. cereus)

1

10 g 2.5 g

5 g 2.5 g

3 g liter

Heat with agitation to dissolve agar. bottles. Autoclave 15 min at 121°C. Aseptically dispense 2 ml portions to room temperature 2 days before use.

Dispense 100 ml portions to 170 ml Final pH, 7.4 ± 0.2. Cool to 50°C. sterile 13 x 100 mm tubes. Store at

Tryptose Infusion agar

M101. Motility Nitrate Medium (for cosmetics) (for Gram-negative nonfermentative bacteria)

KN03 (nitrite-free)

10 g 8 g 1 g

486



Heat with agitation to dissolve agar. Dispense 4 ml portions into 13 x 100 mm screw-cap tubes. Autoclave 15 min at 121°C.

M102. Motility-Nitrate Medium, Buffered (for C. perfingenal

Beef extract 3 g Peptone (Difco) 5 g KN03 1 g Na~P04 2.5 g Agar 3 g Galactose 5 g Glycerin (reagent grade) 5 ml Distilled water 1 liter

Dissolve all ingredients except agar. Adjust pH to 7.3 ± 0.1. Add agar and heat to dissolve. Dispense 11 ml portions into 16 x 150 mm tubes. Autoclave 15 min at 121°C. If not used with 4 h, heat 10 min in boiling water or flowing steam. Chill in cold water.

Ml03. Motility Teat Medium (Semisolid)

Beef extract Peptone or gelysate NaCl Agar Distilled water

3 g 10 g

5 g 4 g

1 liter

Heat with agitation and boil 1-2 min to dissolve agar. Dispense 8 ml portions into 16 x 150 screw-cap tubes. Autoclave 15 min at 121°C. Final pH, 7.4 ± 0.2. For Vibrio spp., add NaCl to achieve a 2-3% final concentration. For Salmonella: Dispense 20 ml portions into 20 x 150 mm screw-cap tubes, replacing caps loosely. Autoclave 15 min at 121°C. Cool to 45°C after autoclaving. Tighten caps, and refrigerate at 5-8°C. To use, remelt in boiling water or flowing steam, and cool to 45°C. Aseptically dispense 20 ml portions into sterile 15 x 100 mm petri plates. Cover plates and let solidify. Use same day as prepared. Final pH, 7.4 ± 0.2.

M104. MR-VP Broth

Buffered peptone 7 g Glucose 5 g ~P04 5 g Distilled water 1 liter

Dissolve ingredients in 800 ml water with gentle heat. and dilute to 1 liter. Autoclave 12-15 min at 121°C.

Filter, cool to 20°C, Final pH, 6.9 ± 0.2.

For use with v. parahaemolyticus: Add 30 g NaCl per liter.


487

fi)~ BACTERIOLOGICAL ANALYTICAL MANUAL 7TH EDITION/1992

For Salmonella: Dispense 10 ml into 16 x 150 mm test tubes, and autoclave 12-15 min at 121°C.

Peptone Mucic acid Bromthymol blue Distilled water

MlOS. Mucate Broth

10 g 10 g

0.024 g 1 liter

Dissolve peptone. Dissolve mucic acid by slowly adding 5 N NaOH and stirring. Dispense 5 ml portions into 13 x 100 mm screw-cap tubes. Autoclave 10 min at 121°C. Final pH, 7.4 ± 0.1.

Peptone Bromthymol blue Distilled water

Ml06. Mucate Control Broth

10 g 0.024 g 1 liter

Dissolve ingredients. Dispense 5 ml portions into 13 x 100 mm screw-cap tubes. Autoclave 10 min at 121°C. Final pH, 7.4 ± 0.1.

Ml07. Mueller-Hinton Agar

Beef, infusion from Acidicase peptone (BBL) or casamino acids (Difco) Starch Agar Distilled water 1

300 g 17.5 g 1.5 g

17 g liter

For Vibrio spp., add NaCl to achieve a 2-3% final concentration. Heat to boiling for 1 min. Autoclave 15 min at 116°C. Final pH, 7.3 ± 0.2.

Beef extract Peptone KN03 (nitrite-free) Distilled water

Ml08. Nitrate Broth

3 g 5 g 1 g

1 liter

Dissolve ingredients. Dispense 5 ml portions into 16 x 125 mm tubes. Autoclave 15 min at 121°C. Final pH, 7.0 ± 0.2.

Infusion broth KN03 (nitrite-free) Distilled water

Ml09. Nitrate Broth. Enriched (CDC)

25 g 2 g

1 liter

Dispense 4 ml portions into 13 x 100 mm tubes with inverted Durham tubes. Autoclave 15 min at 12l°C. Final pH, ~.3 ± 0.2.

1·'3

488


MllO. Nitrate Reduction Medium and Reagents

Culture medium. Prepare nitrate broth (M108) from nutrient broth (Mll4) containing 1.0 g/L potassium nitrate.

Reagent A. Dissolve 0.5 g alpha-naphthylamine (a carcinogen) in 100 ml 5 N acetic acid by gently heating. Prepare 5 N acetic acid by adding distilled water to 28.7 ml glacial acetic acid (17.4 N) to give final volume of 100 ml.

Reagent B. Dissolve 0.8 g sulfanilic acid in 100 ml 5 N acetic acid by gently heating.

Reagent c. Dissolve 1 g alpha-naphthol in 200 ml acetic acid.

Zinc powder.

Cadmium reagent. Place zinc rods in 20\ solution of cadmium sulfate for several hours. Draw off precipitated cadmium and add to 1 N HCl.

Mlll. Nonfat Dry Milk (Reconstituted)

Nonfat dry milk 100 g Distilled water 1 liter

For Salmonella: Suspend 100 g dehydrated nonfat dry milk in 1 liter distilled water. Swirl until dissolved. Autoclave 15 min at 121°C.

For monkey kidney cell culture: Dispense 500 ml into 1 liter Erlenmeyer flasks.

Beef extract Peptone Agar Distilled water

Mll2. Nutrient Agar

3 g 5 g

15 g 1 liter

Heat to boiling to dissolve ingredients. Dispense into tubes or flasks. Autoclave 15 min at 121°C. Final pH, 6.8 ± 0.2. If used as base for blood agar, add 8 g NaCl to prevent hemolysis of blood cells.

M113. Nutrient Agar (for B. cereus)

For slants, prepare nutrient agar and dispense 6.5 ml portions into 16 x 125 mm screw-cap tubes. Autoclave 15 min at 121°C. Slant tubes until medium solidifies. For plates, dispense 100-500 ml portions into bottles or flasks and autoclave. Cool to 50°C and dispense 18-20 ml into sterile 15 x 100 mm petri dishes. Dry plates for 24-48 h at room temperature before use.

489


M114. Nutrient Broth

Beef extract 3 g Petone 5 g Distilled water 1 liter

Heat to dissolve. Dispense 10 ml portions into tubes or 225 ml portions into 500 ml Erlenmeyer flasks. Autoclave 15 min at 121°C. Final pH, 6.8 ± 0.2.

Infusion broth Gelatin Distilled water

Ml15. Nutrient Gelatin (CDC) (for Gram-negative nonfermentative bacteria)

25 g 120 g

1 liter

Heat with agitation to dissolve. Cool to 55°C and adjust pH to 7.4 ± 0.2. Dispense 4 ml portions into 13 x 100 mm screw-cap tubes. Autoclave 15 min at 121°C. For use with y. parahaemolyticus, add 30 g NaCl.

M116. OF Glucose Medium, Semisolid

Tryptone (trypticase) 2 g NaCl 5 g Dipotassium phosphate 0.3 g Bromthymol blue dye 0.08 g Agar 2 g Glucose 10 g Distilled water 1 liter For Vibrio spp., add NaCl to achieve a 2-3% final concentration. Boil ingredients to dissolve. For halophilic Vibrio spp., add 15 g NaCl (2\ NaCl, final concentration) to medium. Dispense 5 ml into 13 x 100 mm tubes and autoclave 15 min at 121°C. Final pH, 6.8 ± 0.2.

To use OF medium with sugars other than glucose, prepare medium without glucose in 900 ml water, sterilize as above, and cool to 45-50°C. Add 100 ml of 10\ solution of filter-sterilized sugar to basal medium. Aseptically dispense 5 ml into sterile 13 x 100 mm tubes.

For Campylobacter, prepare half the tubes with glucose and half without.

Peptone NaCl ~P04 Bromthymol blue Agar Distilled water

M117. Oxidative-Fermentative (OF) Test Medium

2 g 5 g

0.3 g 0.03 g

3 g 1 liter

For Vibri o spp., add NaCl to achieve a 2-3% final concentration.

490


Heat with agitation to dissolve agar. Dispense 3 ml portions to 13 x 100 mm tubes. Autoclave 15 min at 12l°C. Cool to 50°C.

Carbohydrate stock solution. Dissolve 10 g carbohydrate in 90 ml distilled water. Sterilize by filtration through 0.22 ~m membrane. Add 0.3 ml stock solution to 2.7 ml base in tube. Mix gently and cool at room temperature. Inoculate tubes in duplicate. Layer one tube with sterile mineral oil. Incubate 48 h at 35°C.

Columbia blood agar base Esculin Ferric ammonium citrate Lithium chloride Cycloheximide Colistin sulfate Acriflavin Cefotetan Fosfomycin Distilled water

Ml18. Oxford Medium

39.0 g 1.0 g 0.5 g

15.0 g 0.4 g

0.02 g 0.005 g 0.002 g 0.010 g 1 liter

Add the 55.5 g of the first 4 components (basal medium) to 1 L distilled water. Bring gently to boil to dissolve completely. Sterilize by autoclaving at 121°C for 15 min. Cool to 50°C and aseptically add supplement (~below), mix, and pour into sterile petri dishes. To prepare supplement, dissolve cycloheximide, colistin sulfate, acriflavin, cefotetan, and fosfomycin in 10 ml of 1:1 mixture of ethanol and distilled water. Filter-sterilize supplement before use. Oxford basal medium and supplement mixture are available commercially.

Penicillin G Streptomycin Distilled water

Ml19. Penicillin-Streptomycin Solution (antibiotic concentrate)

500,000 IU 500,000 ~g

100 ml

Dissolve antibiotics in water and sterilize by filtration. Store at 5°C.

For V. cholerae, use commercially available penicillin G-streptomycin sulfate solution. Add 5 ml solution per 500 ml medium. Store at -20°C.

M120. Peptone Sorbitol Bile Broth

Na~P04 8.23 g NaH2P04 1.2 g Bile salts No. 3 1.5 g NaCl 5 g Sorbitol 10 g

491

Fl)J\ BACTERIOLOGICAL ANALYTICAL MANUAL 7TH EDITION/1992

Peptone Distilled water

5 g 1 liter

Dispense 100 ml into Wheaton bottles. Autoclave 15 min at 121°C. Final pH, 7.6 ± 0.2.

Kl21. Phenol Red carbohydrate Broth

Trypticase or proteose peptone No. 3 NaCl Beef extract (optional) Phenol red (7.2 ml of 0.25\ phenol red

solution) Distilled water Carbohydrate*

10 g 5 g 1 g

0.018 g 1 liter

*Dissolve either 5 g dulcitol, 10 g lactose, or 10 g sucrose (as specified in the Salmonella test) in this basal broth. Dispense 2.5 ml portions into 13 x 100 mm test tubes containing inverted 6 x 50 mm fermentation tubes. Autoclave 10 min at 118°C. Final pH, 7.4 ± 0.2. Alternatively, dissolve ingredients, omitting carbohydrate, in 800 ml distilled water with heat and occasional agitation. Dispense 2.0 ml portions into 13 x 100 mm test tubes containing inverted fermentation tubes. Autoclave 15 min at 118°C and let cool. Dissolve carbohydrate in 200 ml distilled water and sterilize by passing solution through bacteria-retaining filter. Aseptically add 0.5 ml sterile filtrate to each tube of sterilized broth after cooling to less than 45°C. Shake gently to mix. Final pH, 7.4 ± 0.2.

Proteose peptone No. 3 NaCl Beef extract (optional) Dextrose

Kl22. Phenol Red Glucose Broth

Phenol red (7.2 ml of 0.25\ solution) Distilled water

10 g 5 g 1 g 5 g

0.018 g 1 liter

Dispense 2.5 ml portions into 13 x 100 mm tubes. Autoclave 10 min at 118°C. Final pH, 7.4 ± 0.2.

Kl23. Phenylalanine Deaminase Agar

Yeast extract 3 g L-Phenylalanine or 1 g DL-Phenylalanine 2 g Na~P04 1 g NaCl 5 g Agar 12 g Distilled water 1 liter

Heat gently to dissolve agar. Tube and autoclave 10 min at 121°C. Incline tubes to obtain long slant. Final pH 7.3 ± 0.2.

492


Tryptone Yeast extract Dextrose Agar Distilled water

K124. Plate Count Agar (Standard Methods)

5 g 2.5 g

1 g 15 g

1 liter

Heat to dissolve ingredients. Dispense into suitable tubes or flasks. Autoclave 15 min at 121°C. Final pH 7.0 ± 0.2.

For viable yeasts and molds, dispense 20-25 ml portions into sterile 15 x 100 mm petri dishes.

Na:zHPO• NaH2PO• Peptone D-Mannitol Distilled water

K125. PKP Broth

Adjust pH to 7.6. Autoclave at 121°C for 15 min.

K126. Potassium Cyanide fXCNl Broth

Potassium cyanide Proteose peptone No. 3 or polypeptone NaCl KH2PO• Na:zHPO• Distilled water

7.9 g 1.1 g 2.5 g 2.5 g

1 liter

0.5 g 3 g 5 g

0.22 5 g 5.64 g

1 liter

Dissolve ingredients and autoclave 15 min at 121°C. Cool and refrigerate at 5-8°C. Final pH, 7.6 ± 0.2. Dissolve 0.5 g KCN stock solution in 100 ml sterile distilled water cooled to 5-8°C. Using bulb pipetter, add 15 ml cold KCN stock solution to 1 liter cold, sterile base. DO NOT PIPET BY MOUTH. Mix and aseptically dispense 1.0-1.5 ml portions to 13 x 100 mrn sterile tubes. Using aseptic technique, stopper tubes with No. 2 corks impregnated with paraffin. Prepare corks by boiling in paraffin about 5 min. Place corks in tubes so that paraffin does not flow into broth but forms a seal between rim of tubes and cork. Store tubes at 5-8°C no longer than 2 weeks before use.

Potato infusion Dextrose Agar Distilled water

K127. Potato Dextrose Agar

200 ml 20 g 20 g

1 liter

To prepare potato infusion, boil 200 g sliced, unpeeled potatoes in 1 liter distilled water for 30 min. Filter through cheesecloth, saving effluent,

493


which is potato infusion. Mix in other ingredients and boil to dissolve. Autoclave 15 min at 121°C. Dispense 20-25 ml portions into sterile 15 x 100 mm petri dishes. Final pH, 5.6 ± 0.2. Medium should not be re-melted more than once.

Ml28. Pseudomonas Agar F (for fluorescein production)

Use Difco product, if available.

Proteose peptone No. 3 (Difco) Tryptone (Difco) Dipotassium phosphate Magnesium sulfate Glycerol (Difco) Agar Distilled water

Add medium powder and glycerol to water; mix. ingredients. Autoclave at 121°C for 15 min.

20.0 g 10.0 g 1.5 g

0.73 g 10.0 g 15 . 0 g

1 liter

Heat to boiling to dissolve Final pH, 7.0.

Ml29. Pseudomonas Agar P (for pyocyanine production)

Peptone (Difco) Potassium sulfate Magnesium chloride Glycerol (Difco) Agar Distilled or deionized water

Prepare as described for Pseudomonas agar F.

Proteose peptone No. 3 Beef extract NaCl Bromcresol purple Distilled water

Ml30. Purple Carbohydrate Broth

1

1

20.0 g 10.0 g 1.4 g

10.0 g 15.0 g liter

10 g 1 g 5 g

0.02 g liter

Prepare as for phenol red carbohydrate broth (M121). Final pH, 6.8 ± 0.2.

Ml30a. Purple Carbohydrate Fermentation Broth Base

Purple broth base (Becton-Dickinson) Distilled water

15 g 900 ml

Add purple broth base to distilled water. Dispense 9 ml into 16 x 125 mm tubes containing Durham tubes. Autoclave at 121°C for 15 min. Prepare all carbohydrates, except esculin, as sterile 5\ solutions. Filter-sterilize or autoclave, as appropriate. Add 1 ml carbohydrate solution to 9 ml broth base to yield 0.5\ carbohydrate in broth.

494


Add esculin directly into base broth to ma ke a 0.5% solution and autoclave 15 min at ll5°C. At room temperature a 5% s olution of esculin is a gel that cannot be pipetted.

Tryptic soy agar (Ml52) Yeast extract Pyrazine-carboxamide

M131.

0.2 M Tria-maleate, pH 6.0

Pyrazinamidase Agar*

30 g 3 g 1 g

1 liter

Heat to boiling; dispense 5 ml in 16 x 125 mm tubes. Autoclave at 121°C for 15 min. Cool slanted.

*See Chapter 8, Yersinia, ref . 26.

Broth base

Tryptone NaCl I<H2P04 Distilled water

M132. Rappaport-Vassiliadis Medium

Magnesium chloride solution

MgC12 • 6H20

Distilled water

Malachite green oxalate solution

Malachite green oxalate Distilled water

5 g 8 g

1.6g 1 liter

400 g 1 liter

0.4 g 100 ml

To prepare the complete medium, combine 1000 ml broth base, 100 ml magnesium chloride solution, and 10 ml malachite green oxalate solution (total volume of complete medium is 1110 ml). Broth base must be prepared on same day that components are combined to make complete medium. Magnesium chloride solution may be stored in dark bottle at room temperature up to 1 year. To prepare solution, dissolve entire contents of MgC12 .6H20 from newly opened container according to formula, because this salt is very hygroscopic. Malachite green oxalate solution may be stored in dark bottle at room temperature up to 6 months. Merck analytically pure malachite green oxalate is recommended because other brands may not be equally effective. Dispense 10 ml volumes of complete medium into 16 x 150 mm test tubes. Autoclave 15 min at ll5°C. Final pH, 5.5 ± 0.2. Store in refrigerator and use within 1 month.

Ml33. Sabouraud's Dextrose Broth and Agar

Polypeptone or neopeptone 10 g Dextrose 40 g

495

fl)~ BACTERIOLOGICAL ANALYTICAL MANUAL 7TH BDITION/1992


Dissolve completely and dispense 40 ml portions into screw-cap bottles. Final pH, 5.8. Autoclave 15 min at 118-121°C. Do not exceed 121°C.

For Sabouraud's dextrose agar, prepare broth as above and add 15-20 g agar, depending on gel strength desired. Final pH, 5.6 ± 0.2. Dispense into tubes for slants and bottles or flasks for pouring plates. Autoclave 15 min at 118-1210C.

Medium 1

Tryptone or polypeptone Lactose

M134. Selenite Cystine Broth

Sodium acid selenite (NaHSe03 )

Na~P04 L-Cystine Distilled water

5 g 4 g 4 g

10 g 0.01 g

1 liter

Heat to boiling to dissolve. Dispense 10 ml portions into sterile 16 x 150 mm test tubes. Heat 10 min in flowing steam. po NOT AUTOCLAVE. Final pH, 7.0 ± 0.2. The medium is not sterile. Use same day as prepared.

Medium 1 <North-Bartram modification)

Polypeptone Lactose Sodium acid selenite (NaHSe03 )

Na~P04 KH2P04

L-Cystine Distilled water

5 g 4 g 4 g

5.5 g 4.5 g

0.01 g 1 liter

Heat with agitation to dissolve. Dispense 10 ml portions into sterile 16 x 150 mm test tubes. Heat 10 min in flowing steam. DO NOT AUTOCLAVE. Use same day as prepared.

Ml35. Sheep Blood Agar

Blood agar base (Oxoid No. 2) 95 ml Sheep blood, defibrinated 5 ml

Rehydrate and sterilize base as recommended by manufacturer. Agar and blood should both be at 45-46°C before blood is added and plates are poured. Commercial pre-poured sheep blood agar plates may be used.

496

fl))\ BACTERIOLOGICAL ANALYTICAL MANUAL 7TH BDITION/1992

Tryptone

~PO• KH2P04

NaCl Glucose Tween 80 Distilled water

Autoclave 15 min at 121°C.

M136. Shigella Broth

Final pH, 7.0 ± 0.2.

20 g 2 g 2 g 5 g 1 g

1.5 m1 1 liter

Novobiocin solution. Weigh 50 mg novobiocin into 1 liter distilled water. Sterilize by filtration through 0.45 ~m membrane. Add 2.5 ml concentrate to 225 ml base.

M137. SIM Motility Medium

Rehydrate and add 6 ml medium per 16 x 125 mm screw-cap tube. Sterilize according to manufacturer's instructions.

Sodium citrate.2H20 NaCl ~P04 NH4H2P04

Mgso4

Bromthymol blue Agar Distilled water

M138. Simmons Citrate Agar

1

2 g 5 g 1 g 1 g

0.2 g 0.08 g

15 g liter

Heat gently with occasional agitation • . Boil 1-2 min until agar dissolves. Fill 13 x 100 or 16 x 150 mm screw-cap tubes 1/3 full. Autoclave 15 min at 121°C. Before medium solidifies, incline tubes to obtain 4-5 em slants and 2-3 em butts. Fianl pH, 6.9 ± 0.2.

Ml39. Sorbitol-MacConkey Agar

Peptone or gelysate Protease peptone No. 3 or polypeptone Sorbitol Bile salts, purified NaCl Agar Neutral red Crystal violet Distilled water

17.0 g 3.0 g

10.0 g 1.5 g 5.0 g

13.5 g 0.03 g

0.001 g 1 liter

497


Dissolve ingredients in distilled water by heating with stirring. Autoclave 15 min at 121°C. Final pH, 7.1 ± 0.2.

H140 . Sporulation Broth (for c. perfringens)

Polypeptone Yeast extract Starch, soluble MgS04 (anhydrous) Sodium thioglycollate Na~P04 Distilled water 1

15 g 3 g 3 g

0.1 g 1 g

11 g liter

Adjust pH to 7.8 ± 0.1. Dispense 15 ml portions into 20 x 150 mm screw-cap tubes. Autoclave 15 min at 121°C.

H141. Spray's Fermentation Medium (for c. perfringens>

Tryptone 10 g Neopeptone 10 g Agar 2 g Sodium thioglycollate 0:025

o.::z.s g

Dissolve all ingredients except agar and adjust pH to 7.4 ± 0.2. Add agar and heat with agitation to dissolve. Dispense 9 ml portions into 16 x 125 mm tubes. Autoclave 15 min at 12l°C. Before use, heat in boiling water or flowing steam for 10 min. Add 1 ml of sterile 10% carbohydrate solution to 9 ml base.

Gelatin Trypticase peptone Yeast extract Lactose Mannitol NaCl K~P04 Agar Distilled water

Nutrient agar Potato starch Distilled water

H142. Staphylococcus Agar No. 110

H143. Starch Agar

30 g 10 g

2.5 g 2 g

10 g 75 g

5 g 15 g

1 liter

23 g 10 g

1 liter

Heat to dissolve agar in 500 ml water. Dissolve starch in 250 ml water. Combine and dilute to 1 liter. Autoclave 30 min at 121°C.

498

pl)~ BACTERIOLOGICAL ANALYTICAL MANUAL

Trypticase NaCl

M144.

Agar (if solid medium is preferred) Distilled water

7TH EDITION/1992

10 g 10 g 20 g

1 liter

Heat if necessary to dissolve ingredients. Dispense into 16 x 125 mm screwcap tubes (if tubed medium is required). Autoclave 15 min at 121°C. Slant tubes until cool or let medium cool to 50°C and pour into 15 x 100 mm petri dishes. Final pH, 7.1 ± 0.2.

Tetrathionate broth base

Polypeptone Bile salts Calcium carbonate Sodium thiosulfate.5H20 Distilled water

M145. Tetrathionate Broth

5 g 1 g

10 g 30 g

1 liter

Suspend ingredients in 1 liter distilled water, mix, and heat to boiling. (Precipitate will not dissolve completely . ) Cool to less than 45°C. Store at 5-8°C. Final pH, 8.4 ± 0.2.

Iodine-Potassium Iodide <I-KI> soluton

Potassium iodide Iodine, resublimed Distilled water, sterile

Dissolve potassium iodide in 5 ml sterile distilled water. stir to dissolve. Dilute to 20 ml.

Brilliant green solution

Brilliant green dye, sterile Distilled water, sterile

5 g 6 g

20 ml

Add iodine and

0.1 g 100 ml

On day of use, add 20 ml I-KI solution and 10 ml brilliant green solution to 1 liter base. Resuspend precipitate by gentle agitation and aseptically dispense 10 ml portions into 20 x 150 or 16 x 150 mm sterile test tubes. Do not heat medium after addition of I-KI and dye solutions.

L-Cystine Agar (granulated) NaCl Dextrose Yeast extract

M146. Thioglycollate Medium <Fluid) (FTC)

0.5 g 0.75 g 2.5 g

5 g 5 g

499

FI>J\ BACTERIOLOGICAL ANALYTICAL MANUAL

Tryptone Sodium thioglycollate or thioglycollic acid Resazurin, sodium solution (1:1000), fresh Distilled water

7TH EDITION/1992

15 g 0.5 g

1 ml 1 liter

Mix L-cystine, NaCl, dextrose, yeast extract and tryptone with 1 liter water. Heat in Arnold steamer or water bath unt i l ingredients are dissolved. Dissolve sodium thioglycollate or thioglycollic acid in solution and adjust pH so that value after sterilization is 7.1 ± 0.2. Add sodium resazurin solution, mix, and autoclave 20 min at 121°C. If commercial media are used, dispense 10 ml portions to 16 x 150 mm tubes and autoclave 15 min at 121°C.

Ml47. Thiosulfate-Citrate-Bile Salts-Sucrose <TCBS) Agar

Yeast extract 5 g NaCl 10 g Peptone 10 g Ferric citrate 1 g Sucrose 20 g Bromthymol blue 0. 04 g Sodium thiosulfate.5H20 10 g Thymol blue 0.04 g Sodium citrate. 2H20 10 g Agar 15 g Sodium chelate 3 g Distilled water 1 liter Oxgall 5 g

Heat to dissolve ingredients. Boil 1-2 min. DO NOT AUTOCLAVE. Dispense 20 ml into 15 x 100 mm petri dishes. Final pH, 8.6.

Ml48. Toluidine Blue-DNA Agar

Deoxyribonucleic acid (DNA) Agar CaC12 (anhydrous) NaCl Toluidine blue 0 Tria (hydroxymethyl) aminomethane Distilled water

0.3 g 10 g

1.1 mg 10 g

0.083 g 6.1 g

1 liter

Dissolve tria (hydroxymethyl) aminomethane in 1 liter distilled water. Adjust pH to 9.0. Add the remaining ingredients except toluidine blue o and heat to boiling to dissolve. Dissolve toluidine blue 0 in medium. Dispense to rubber-stoppered flasks. Sterilization is not necessary if used immediately. The sterile medium is stable at room temperature for 4 months and is satisfactory after several melting cycles.

Ml49. Triple Sugar Iron (TSI) Agar

Medium 1 Medium 2

Polypeptone NaCl Lactose Sucrose Glucose Fe (NH4 ) 2 ( S04 ) 2 • 6H20

20 g 5 g

10 g 10 g

1 g 0.2 g

Beef extract Yeast extract Peptone Proteose peptone Glucose Lactose

3 g 3 g

15 g 5 g 1 g

10 g

500


Na2S20 3 0.2 g Sucrose 10 g Phenol red 0.025 g FeS04 0.2 g Agar 13 g NaCl 5 g Distilled water 1 liter Na2S20 3 0.3 g

Phenol red 0.024 g Agar 12 g Distilled water 1 liter

For ~ibx:io spp., add NaCl to achieve a 2-3% final concentr< These two media are interchangeable for general use.

Suspend ingredients of Medium 1 in distilled water, mix thoroughly, and heat with occasional agitation. Boil about 1 min to dissolve ingredients. Fill 16 x 150 rnrn tubes 1/3 full and cap or plug to maintain aerobic conditions. Autoclave Medium 1 for 15 min 118°C. Prepare Mediurn~in the same manner as Medium 1, except autoclave 15 min at 121°C. Before the media solidify, incline tubes to obtain 4-5 ern slant and 2-3 ern butt. Final pH, 7.3 ± 0.2 for Medium 1 and 7.4 ± 0.2 for Medium 2.

Ml50.

Trypticase soy broth Bile salts No. 3 Dipotassium phosphate Novobiocin Distilled water

Trypticase Novobiocin CTNl Broth

30 g l.Sg l.Sg 20 rng

1 liter

Dissolve all ingredients except novobiocin by heating and stirring; autoclave at 122°C for 15 min. Filter-sterilize the novobiocin and add aseptically to cooled medium.

MlSl. Trypticase-Peptone-Glucose-Yeast Extract Broth lTPGYl

Trypticase Peptone Yeast extract Dextrose Sodium thioglycollate Distilled water 1

50 g 5 g

20 g 4 g 1 g

liter

Dissolve solid ingredients and dispense 15 rnl in 20 x 150 rnrn tubes. Autoclave tubes 10 min at 121°C. Final pH, 7.0 ± 0.2. Refrigerate at 5°C.

Ml5la. Trypticase-Peptone-Glucose-Yeast Extract Broth with Trypsin (TPGYTl

Trypticase Peptone Yeast extract Dextrose Sodium thioglycollate Distilled water 1

50 g 5 g

20 g 4 g 1 g

liter

501


Dissolve solid ingredients of base and dispense: 15 ml in 20 x 150 mm tubes or 100 ml in 170 ml prescription bottles. Autoclave tubes 10 min at 121°C and bottles 15 min at 121°C. Final pH, 7.0 ± 0.2. Refrigerate at 5°C. Add trypsin immediately before use.

Trypsin solution

Trypsin (1:250) Distilled water

1.5g 100 ml

Stir trypsin in water to suspend. Let particles settle and supernatant through 0.45 ~m membrane. Before use, steam or min to expel dissolved oxygen. Add 1 ml trypsin to each 15 ml trypsin to 100 ml of broth.

filter-sterilize boil base for 10 ml of broth or 6.7

Trypticase peptone Phytone peptone NaCl

H152. Trypticase (Tryptic) Soy Agar

15 g 5 g 5 g

Agar 15 g Distilled water 1 liter For Vibrio spp., add NaCl to achieve a 2-3% final concentration. Heat with agitation to dissolve agar. Boil 1 min. Dispense into suitable tubes or flasks. Autoclave 15 min at 121°C. Final pH, 7.3 ± 0.2. For use with y. parahaemolyticus, add 25 g NaCl.

H153. Trypticase Soy Agar with 0.6, Yeast Extract (TSAYE)

Trypticase soy agar Yeast extract Distilled water

Trypticase peptone Phytone peptone NaCl

H154. Trypticase (Tryptic) Soy Broth

40 g 6 g

1 liter

17 g 3 g 5 g

K~P04 2. 5 g Glucose 2.5 g Distilled water 1 liter For Vibrio spp., add NaCl to achieve a 2-3% final concentration. Heat with gentle agitation to dissolve. Dispense 225 ml into 500 ml Erlenmeyer flasks. Autoclave 15 min at 121°C. Final pH, 7.3 ± 0.2.

For trypticase soy broth without dextrose, prepare as above, but omit 2.5 g dextrose.

H154a. Trypticase (Tryptic) Soy Broth with 10' NaCl and 1' Sodium Pyruvate

Trypticase or tryptose (pancreatic digest of casein) Phytone (papaic digest of soya meal)

17 g 3 g

502

FI)J\ BACTERIOLOGICAL ANALYTICAL MANUAL 7TH EDITION/1992

NaCl 100 g ~P04 2.5 g Dextrose 2 . 5 g Sodium pyruvate 10 g Distilled water 1 liter

Dehydrated trypticase or tryptic soy broth is satisfactory with 95 g NaCl and 10 g sodium pyruvate added per liter. Adjust to pH 7.3. Heat gently if necessary. Dispense 10 ml into 16 x 150 mm tubes. Autoclave 15 min at 121°C. Final pH 7.3 ± 0.2. Store up to 1 month at 4 ± 1°C.

M155. Trypticase (Tryptic) Soy Broth (TSBl with Glycerol

Trypticase peptone (tryptone) Phytone peptone (soytone) NaCl ~P04 Glycerol Distilled water

17 g 3 g

15 g 2.5 g

240 ml 760 ml

Suspend ingredients in distilled water and heat to dissolve. Dispense into bottles or vials. Autoclave 15 min at 121°C. Final pH, 7.3 ± 0.2 .

Ml56. Trypticase Soy Broth Modified CmTSB)

Trypticase soy broth 30.0 g Bile salts No. 3 1. 5 g Dipotassium phosphate Novobiocin solution (R-50) Deionized water

1.5g 0.2 ml

1 liter

Autoclave mTSB without novobiocin and let cool to room temperature. Add novobiocin just before adding food.

Ml57. Trypticase Soy Broth with 0.6, Yeast Extract CTSBYE)

Trypticase soy broth Yeast extract Distilled water

Ml58. Trypticase Soy-Polymyxin Broth

30 g 6 g

1 liter

Prepare trypticase soy broth (M154) and dispense 15 ml portions into 20 x 150 mm tubes. Autoclave 15 min at 121°C.

Polymyxin B solution, 0.15\. Dissolve 500,000 units sterile polymyxin B sulfate powder in 33.3 ml sterile distilled water. Store solution in 85 ml bottle at 4°C. Before use, add 0.1 ml sterile 0.15\ polymyxin B sulfate solution to 15 ml medium, and mix.

503

Fl)~ BACTERIOLOGICAL ANALYTICAL MANUAL 7TH EDITION/1992

M159. Trypticase Soy-Sheep Blood Agar

Prepare trypticase soy agar (M152). Sterilize as recommended and cool to 50°C. Add 5 ml defibrinated sheep blood to 100 ml agar. Mix and dispense 20 ml portions to 15 x 100 mm petri dishes. (Commercial trypticase soy-blood agar plates are satisfactory.)

M160. Trypticase Soy-Tryptose Broth

Trypticase soy broth (commercial, dehydrated) Tryptose broth (commercial, dehydrated) Yeast extract Distilled water

15 g 13.5 g

3 g 1 liter

Dissolve ingredients in 1 liter water. Heat gently to dissolve. Dispense 5 ml portions into 16 x 150 mm test tubes. Autoclave 15 min at 121°C. Final pH , 7 • 2 ± 0 • 2 •

M161. Tryptone Broth and Tryptone Salt Broths T1N0 , T1N1 , T1N3 , T1N6 , T1N8 , T1N10

Trypticase or tryptone NaCl Distilled water

10 g O, 10, 30, 60, 80, or 100 g

1 liter

Dissolve ingredients in distilled water. For T1N0 , add no NaCl; for T1N1 , use 10 g NaCl [1\ (w/v) NaCl). For T1N3 , use 30 g NaCl/L [3\ (w/v) NaCl], etc. Dispense into 16 x 125 mm screw-cap tubes. Tighten caps to maintain correct salt concentration in tube. Autoclave 15 min at 121°C. Final pH, 7.2 ± 0.2.

Tryptone K~P04 KH2P04

NaCl Polysorbate 80 Distilled water

M162. Tryptone Phosphate fTPl Broth (for enteropathogenic ~- coli)


M163.

Trypticase or tryptone NaCl Agar Distilled water

Tryptone Salt (T1N,l Agar and T1!z Agar

20 g 2 g 2 g 5 g

15 ml 1 liter

10 g 10 g 20 g

1 liter

Suspend ingredients and boil to dissolve agar. For slants, dispense into tubes. Autoclave 15 min at 121°C. Solidify tubes as slants. Cool medium for plates to 45-50°C and pour into sterile petri dishes. For T1N2 agar, use 20 g NaCl rather than the 10 g specified for T1N1 agar.

504

f})j\ BACTERIOLOGICAL ANALYTICAL MANUAL 7TH EDITION/1992

H164.

Tryptone or trypticase Distilled water

Tryptone (Tryptophane> Broth. 1%

10 g 1 liter

Dissolve and disepnse 5 ml portions into 16 x 125 or 16 x 150 mm test tubes . Autoclave 15 min at 121°C. Final pH, 6.9 ± 0.2.

Hl65. Tryptone Yeast Extract Agar

Tryptone 10 g Yeast extract 1 g *Carbohydrate 10 g Bromcresol purple 0.04 g Agar 2 g Distilled water 1 liter

Dissolve agar with heat and gentle agitation. Adjust pH to 7.0 ± 0.2. Fill 16 x 125 mm tubes 2/3 full. Autoclave 20 min at 115°C. Before use, steam medium 10-15 min. Solidify by placing tubes in ice water.

*Glucose and mannitol are the carbohydrates used for identification of ~· aureus.

Tryptose Beef extract NaCl Agar Distilled water

H166. Tryptose Blood Agar Base

10 g 3 g 5 g

15 g 1 liter

Suspend ingredients in distilled water, mix thoroughly, and heat with occasional agitation. Boil about 1 min. Fill 16 x 150 mm tubes 1/3 full and cap or plug to maintain aerobic conditions. Autoclave 15 min at 121°C. Before media solidify, incline tubes to obtain 4-5 em slant and 2-3 em butt.

H167. Tryptose Broth and Agar (for serology)

Difco Bacto tryptose NaCl Dextrose Agar Distilled water

For broth, omit agar from formulation.

1

20 g 5 g 1 g

15 g liter

505


Tryptose Dextrose NaCl Na:zHP04

Distilled water

Ml68. Tryptose Phosphate Broth CTPB) (for cell culture)

20 g 2 g 5 g

2.5 g 1 liter

Sterilize by filtration through 0.20 ~m membrane. Available from Flow Laboratories, Inc., McLean, VA 22102.

Tryptose Yeast extract Soytone

Ml69. Tryptose-Sulfite-Cycloserine CTSC) Agar

Ferric ammonium citrate (NF Brown Pearls) Sodium metabisulfite Agar Distilled water

15 g 5 g 5 g 1 g 1 g

20 g 900 ml

Heat with agitation to dissolve. Adjust pH to 7.6 ± 0.2. Dispense 250 ml portions to 500 ml flasks. Autoclave 15 min at 121°C. Maintain medium at 50°C before use. Dehydrated SFP agar base (Difco) is satisfactory for base.

D-cvcloserine solution . Dissolve 1 g D-cycloserine (white crystalline powder) in 200 ml of d i stilled water. Sterilize by filtration and store at 4°C until use. D-cycloserine powder is available from Sigma Chemical Co., St. Louis, MO.

Final medium. For pour plates, add 20 ml of D-cycloserine solution to 250 ml base. To prepare prepoured plates containing egg yolk, also add 20 ml of 50% egg yolk emulsion (M51). Mix well and dispense 18 ml into 15 x 100 mm petri dishes. Cover plates with a towel and let dry overnight at room temperature before use.

Ml70.

Base. Prepare nutrient agar (M112). bottles. Autoclave 15 min at 121°C.

Tyrosine Agar

Dispense 100 ml portions into 170 ml Cool to 48°C.

Tyrosine suspension. x 150 mm culture tube. 121°C.

Suspend 0.5 g L-tyrosine in 10 ml distilled water in 20 Mix thoroughly with Vortex mixer. Autoclave 15 min at

Final medium. Combine 100 ml base with sterile tyrosine suspension. Mix thoroughly by gently inverting bottle 2 or 3 times. Aseptically dispense 3.5 ml into 13 x 100 mm tubes with frequent mixing. Slant tubes and cool rapidly to prevent separation of tyrosine.

506


Urea Yeast extract KH2P04

Phenol red

M171. Urea Broth

20 g 0.1 g 9.1 g

0.01 g Distilled water 1 liter NC( 4 t\ ro,. '~. 5,j Dissolve ingredients in distilled water. DO NOT HEAT. Sterilize by filtration through 0.45 ~m membrane. Aseptically dispense 1.5-3.0 ml portions to 13 x 100 mm sterile test tubes. Final pH, 6.8 ± 0.2.

Urea Yeast extract KH2P04

N<l<A. *HP04

Phenol red Distilled water

M172. Urea Broth (Rapid)

Prepare as for urea broth (M171) above.

M173.

Veal, infusion from Proteose peptone No. 3 NaCl Agar Distilled water

Veal Infusion Agar and Broth

20 g 0. 1 g

0 .09 1 g 0.095 g 0.01 g

1 liter

500 g 10 g

5 g 15 g

1 liter

Heat with agitation to dissolve agar. Dispense 7 ml portions into 16 x 150 mm tubes. Autoclave 15 min at 121°C. Incline tubes to obtain 6 em slant. Final pH , 7 . 3 ± 0 • 2 •

For veal infusion broth, prepare as above, but omit the 15 g agar. Autoclave 15 min at 121°C. Final pH, 7.4 ± 0.2.

M174. Violet Red Bile Agar (VRBAl

Yeast extract 3.0 g Peptone or gelysate 7.0 g NaCl 5.0 g Bile salts or bile salts No. 3 1.5 g Lactose 10.0 g Neutral red 0.03 g Crystal violet 0.002 g Agar 15.0 g Distilled water 1.0 liter

suspend ingredients in distilled water and let stand for a few min. Mix thoroughly and adjust to pH 7.4 ± 0.2. Heat with agitation and boil for 2

507


min. Do not sterilize. Before use, cool to 45°C and use as a plating medium. After solidification, add a cover layer above the agar of approximately 3.0 to 4.0 ml to prevent surface growth and spreading of colonies.

Kl75. Violet Red Bile-HUG Agar <VRBA-KUG)

Add 0.1 g 4-methylumbelliferyl-beta-D-glucuronide (MUG) to the ingredients for 1 liter of VRBA (Ml74), and continue as for preparation of VRBA.

Trypticase Yeast extract Mannitol K:zHP04

Lithium chloride Glycine Phenol red Agar Distilled water

Kl76. Vogel-Johnson (VJl Agar

10 g 5 g

10 g 5 g 5 g

10 g 0.025 g

15 g 1 liter

Heat with agitation to dissolve agar. Autoclave 15 min at 121°C. Cool to 50°C. Add 20 ml Chapman tellurite solution, commercially available from Difco Laboratories, Detroit, MI. Mix and pour into plates. Final pH, 6.2 ± 0.2.

K177. Voqes-Proskauer Medium <Modified)

Proteose peptone 7 g NaCl 5 g Dextrose 5 g Distilled water 1 liter

Dissolve ingredients in water and adjust pH if necessary. Dispense 5 ml portions into 20 x 150 mm tubes. Autoclave 10 min at 12l°C. Final pH, 6.5 ± 0.2.

Yeast extract Peptone NaCl K:zHP04

Mannitol Crystal violet Agar Distilled water

Kl78. Wagatsuma Agar

Human or rabbit red blood cells, fresh (24 h), with anticoagulant

3 g 10 g 70 g

5 g 10 g

0.001 g 15 g

1 liter

50 ml

Mix fresh (within 24 h of drawing) human or rabbit blood with same or larger volume of physiological saline. Centrifuge cells at about 4000 x g at 4°C for

508

FI>J\ BACTERIOLOGICAL AN~,~L~Y~T~IC~AL~~MANU~~AL~----------~'T~H~E=D=I~T~I=O=N~/~1=9~9=2

15 min. Pour off saline and wash 2 more times. After 3rd wash, pour off saline and resuspend cells to original volume with saline.

Suspend ingredients, except blood, in distilled water and boil to dissolve agar. Adjust to pH 8.0 ± 0.2. Steam 30 min. DO NOT AUTOCLAVE. Cool to 45-500C. Add 50 ml of washed red blood cells to the cooled medium. Mix and pour into sterile petri dishes. Dry plates thoroughly and use promptly. Medium can be made in smaller volumes (requiring less blood) when few plates are needed.

Ml79. Xylose Lysine Desoxycholate (XLD) Agar

Yeast extract 3 g L-lysine 5 g Xylose 3.75 g Lactose 7.5 g Sucrose 7.5 g Sodium desoxycholate 2.5 g

Ferric ammonium citrate Sodium thiosulfate NaCl Agar Phenol red Distilled water

0.8 g 6.8 g

5 g 15 g

0.08 g 1 liter

Heat with agitation just until medium boils. Do not overheat. Pour into plates when medium has cooled to 50°C. Let dry about 2 h with covers partially removed. Then close plates. Final pH, 7.4 ± 0.2. Do not store more than 1 day.

Ml80. Y-1 Adrenal Cell Growth Medium

Ham's F-10 medium Fetal bovine serum Penicillin-streptomycin solution (M119)

90 ml 10 ml

1 ml

y:. cholerae

500 ml 75 ml

5 ml

These ingredients are commercially available. Filter-sterilize. Store at 4-50C.

Proteose peptone Yeast extract NaCl Agar Distilled water

M181. Yeast Extract (YE) Agar

Adjust pH to 7.2-7.4. Autoclave at 121°C for 15 min.

10 g 3.0 g 5.0 g

15.0 g 1 liter

509


REAGENTS

Rl. 4 M Almlonium Acetate

Ammonium acetate 308. 4 g Distilled water to make 1 liter

R2. 0.25 M Almlonium Acetate

Ammonium acetate 19.3 g Distilled water to make 1 liter

R3. Basic Fuchsin Staining Solution

Dissolve 0.5 g basic fuchsin dye in 20 ml 95\ ethanol. Dilute to 100 ml with distilled water. Filter if necessary with Whatman No. 31 filter paper to remove any undissolved dye. (TB Carbolfuchsin ZN staining solution, available from Difco Laboratories, is satisfactory.)

Na2C03

NaHC03

Distilled water

R4. 0.1 M Bicarbonate Buffer <pH 9.6)

store at room temperature for not more than 2 weeks.

R5. Bovine Serum Albumin (BSA) (1 mq/ml)

Nuclease-free bovine serum albumin Distilled water

1. 59 g 2.93 g

1 liter

10 mg 10 ml

Place 0.5 ml portions into 1.5 ml plastic conical centrifuge tubes. Store frozen.

R6. 1\ Bovine Serum Albumin in Cholera Toxin ELISA Buffer

Bovine serum ablumin (BSA) ELISA buffer for (cholera toxin), pH 7.4

Dissolve BSA in ELISA buffer. Aliquot and store at -20°C.

R7. 1\ Bovine Serum Albumin in PBS

Bovine serum albumin (BSA) Phosphate-buffered saline, pH 7.4

Dissolve BSA in PBS buffer. Aliquot and store at -20°C.

1 g 100 ml

1 g 100 ml

510


RS.

Brilliant green dye Distilled water (sterile)

Brilliant Green Dye Solution. 1'

1 g 10 ml

Dissolve 1 g dye in sterile water. Dilute to 100 ml. Before use, test all batches of dye for toxicity with known positive and negative test microorganisms.

R9. Bromcresol Purple Dye Solution, 0.2,

Bromcresol purple dye Distilled water (sterile)

Dissolve 0.2 g dye in sterile water and dilute to 100 ml.

Bromthymol blue 0.01 N NaOH

R10. Bromthymol Blue Indicator, 0.04'

0.2 g 100 ml

0.2 g 32 ml

Dissolve bromthymol blue in NaOH. Dilute to 500 ml with distilled water.

R11. Butterfield's Phosphate-Buffered Dilution Water

Stock solution

~~~ Distilled water

34 g 500 ml

Adjust pH to 7.2 with 1 N NaOH. Bring volume to 1 liter with distilled water. Sterilize 15 min at 121°C. Store in refrigerator.

Dilution blanks

Take 1.25 ml of above stock solution and bring volume to 1 liter with distilled water. Dispense into bottles to 90 or 99 ± 1 ml. Sterilize 15 min at 121°C.

R12. Catalase Test

Pour 1 ml 3\ hydrogen peroxide over growth on slant culture. Gas bubbles indicate positive test. Alternatively, emulsify colony in 1 drop 30\ hydrogen peroxide on glass slide. Immediate bubbling is positive catalase test. If colony is taken from blood agar plate, any carry-over of red blood cells can give false-positive reaction.

R13. 0.05 M Citric Acid CpH 4.0)

Citric acid (monohydrate) 9.6 g Double distilled water to make 1 liter

511

. . FI>~ BACTERIOLOGICAL ANALYTICAL MANUAL 7TH EDITION/1992

Dissolve citric acid in 900 ml distilled wa ter. Adjust pH to 4.0 with 6 M NaOH and dilute to 1 liter. Store in refrigerator.

Solution A

Basic fuchsin, special 95\ ethanol

R14. Clark's Flagellar Stain

Mix and let stand overnight at room temperature.

Solution B

Tannic acid NaCl Distilled water

1.2 g 100 ml

3 . 0 g 1 . 5 g

200 ml

Mix solutions A and B. Adjust pH to 5.0 with 1 N NaOH or 1 N HCl, if necessary. Refrigerate 2-3 days before use. Stain is stable 1 month at 4°C or may be stored frozen indefinitely (50 ml portions). To use, thaw stain, remix, and store at 4°C. Optimum staining time for each batch varies 5-15 min. To determine staining time (after 2-3 days refrigeration at 4°C), stain a known flagellated organism on 3 or more cleaned slides for various times (e.g., 5, 10, 15 min). Mark best staining time on all containers.

IMPORTANT: Stain will not work unless slides are clean. To clean slides, soak them 4 days at room temperature in cleaning solution (either acid dichromate or 3\ concentrated HCl in 95\ ethanol). Rinse 10 times in fresh tap water and twice in distilled water. Air-dry at room temperature. Store in covered container.

Staining procedure

To prepare suspension, pick small amount of growth from 18-24 h plate (equivalent to 1 mm colony). Do not pick up agar. Suspend gently in 3 ml distilled water. (Flagella can be knocked off.) Suspension should be faintly opalescent.

To prepare slide, pass cleaned slide through blue part of burner flame several times to remove residual dirt. Cool slide, flamed side up, on paper towel. Mark wax line across slide to give area 2.5 x 4.5 em. Place large loopful of suspension in center of slide adjacent to wax line. Tilt slide, letting drop run down center of slide to end. If drop does not run evenly, slide is dirty. Discard it. Air-dry slide on level surface.

R15. Coating Solution for v. vu1nificus BIA

Phosphate-buffered saline (PBS), pH 7.4 Triton X-100 (a polyoxyethylene ether)

Mix Triton X-100 with PBS, pH 7.4.

100 ml 20 ~1

512

fl)~ BACTERIOLOGICAL ANALYTICAL MANUAL 7TH BDITION/1992

R16. Crystal Violet Stain (for Bacterial

1. Crystal violet in dilute alcohol

Crystal violet (90\ dye content) Ethanol (95\) Distilled water

2. Ammonium oxalate crystal violet (Hucker's) (See R32l

2 g 20 ml 80 ml

Either solution is generally considered suitable as a simple stain to observe morphology.

R17. SOX Denhardt's Solution

Ficoll (Av. MW 400,000) Polyvinyl pyrrolidone (Av. MW 360,000) Bovine serum albumin

2 g 2 g 2 g

Add distilled water to make 200 ml. Store at -20°C in 10 ml aliquots.

R18. Disinfectants (for preparation of canned foods for microbiological analysis)

1. Alcoholic solution of iodine

Potassium iodide Iodine Ethanol (70\)

2. Sodium hypochlorite solution*

Sodium hypochlorite Distilled water

10 g 10 g

500 ml

5.0-5.25 g 100 ml

*Laundry bleach, which is 5.25\ sodium hypochlorite (NaOCl), may be used.

R19. Dulbecco•s Phosphate-Buffered Saline lDPBSl

NaCl KCl Na~P04 KH2P04

CaC12

MgC12 • 6H20

Distilled water • .....

8.0 g 200 mg 1.15 g 200 mg 100 mg 100 mg

1 liter

Dissolve ingredients in water. Sterilize by filtration. Final pH, 7.2.

R20. 0.5 M EDTA

Na~DTA 186. 12 g

Dissolve in 800-900 ml dH20. Adjust pH to 8.0 with 10 N NaOH. Add distilled water to make 1 liter.

513


R21. EIA (V. vulnificusl Wash Solution

NaCl 87.65 g Tween 20 5.0 ml

Dissolve ingredients in 10 liters of deionized water.

R22. ELISA Buffer (for Cholera Toxin Test)

Bovine serum albumin NaCl KH2P04

Na:zHP04

KCl Distilled water Tween 20

1

1.0 g 8.0 g 0.2 g 2.9 g 0.2 g titer

0.5 ml

Adjust pH to 7.4 and add 0.5 ml Tween 20. Store frozen. Thaw before use.

R23. Ethanol Solution, 70%

Ethanol, 95% 700 ml Distilled water 250 ml

Evans Blue dye NaCl

R24. Evans Blue Dye Solution (commercially available)

Distilled water to make 100 ml (final volume)

CAUTION: Evans Blue dye is a suspected carcinogen.

FeCl3

Distilled water

R25. Ferric Chloride, 10%

R26. Formalinized Phosphate-Buffered Saline (for V. vulnificus flagellar serology>

Formaldehyde solution (36-38%) Phosphate-buffered saline (PBS), sterile

2 g 0.5 g

10 g 90 ml

6 ml 1 liter

Add formaldehyde solution to room temperature PBS, pH 7.4. Do not autoclave after addition of formaldehyde. Store in glass-stoppered bottle in dark at room temperature or in refrigerator.

514


R27. Formalinized Physiological Saline Solution

Formaldehyde solution (36-38%) NaCl Distilled water

6 ml 8.5 ml

1 liter

Dissolve 8.5 g NaCl in 1 liter distilled water. Autoclave 15 min at 121°C. Cool to room temperature. Add 6 ml formaldehyde solution. Do not autoclave after addition of formaldehyde.

R28. Gel Diffusion Agar, 1.2%

NaCl Sodium barbital Merthiolate (crystalline) Noble special agar (Difco) Distilled water 1

8.5 g 8.0 g 0.1 g

12.0 g liter

Dissolve NaCl, sodium barbital, and merthiolate in 900 ml distilled water. Adjust pH to 7.4 with 1 N HCl and/or 1 N NaOH. Bring volume to 1 liter. Add Noble agar. Melt agar mixture in Arnold steamer. Filter in steamer, while hot, through 2 layers of analytical grade filter paper (e.g., No. 588, Schleicher and Schuell or equivalent). Dispense in small (15-25 ml) portions into 4 oz prescription bottles. Do not remelt more than twice.

Gelatin Na2HP04

Distilled water

R29. Gel-Phosphate Buffer

2 g 4 g

1 liter

Use gentle heat to dissolve ingredients. Sterilize 20 min at 121°C. Final pH, 6. 2.

RJO. Giemsa Stain

Giemsa stain (Matheson Coleman & Bell, Norwood, OH 45212)

Glycerol Methanol (absolute)

1 g 66 ml 66 ml

Distilled stain in glycerol by heating 1.5-2.0 h at 55-60°C. Add methanol. Store stain in tightly stoppered bottle at 22°C for at least 2 weeks. Dilute stock solution with distilled water (1+9) before use.

R31.

Glycerin (reagent grade) K2HP04 (anhydrous) KH2P04 (anhydrous) NaCl

Glycerin-Salt Solution (Buffered)

100 ml 12.4 g

4 g 4.2 g

515


Distilled water 900 ml

Distilled NaCl and bring volume to 900 ml with water. Add glycerin and phosphates. Adjust pH to 7.2. Autoclave 15 min at 121°C. For double strength (20\) glycerin solution, use 200 ml glycerin and 800 ml distilled water.

R32. Gram Stain (commercial staining solutions are satisfactory)

Hucker's crystal violet

Solution A

Crystal violet (90\ dye content) Ethanol, 95\

Solution B

Ammonium oxalate Distilled water

2 g 20 ml

0.8 g 80 ml

Mix solutions A and B. Store 24 h and filter through coarse filter paper.

Gram's iodine

Iodine Potassium iodide (KI) Distilled water

1 g 2 g

300 ml

Place KI in mortar, add iodine, and grind with pestle for 5-10 s. Add 1 ml water and grind; then add 5 ml of water and grind, then 10 ml and grind. Pour this solution into reagent bottle. Rinse mortar and pestle with amount of water needed to bring total volume to 300 ml.

Solution A

Malachite green Distilled water

R32a. Endospore Stain (Schaeffer-Fultonl

Filter to remove undissolved dye.

Solution B

Safranin 0 Distilled water

10 g 100 ml

0.25 g 100 ml

- . . Hucker's counterstain (stock solutionl--Safranin 0 (cert~f~ed), 2.5 g; 95% ethanol 100 Add 10 ml stock solution to 90 ml distilled water. ' ml.

staining procedure: Fix air-dried films of food sample in moderate heat (~chapter 2). Stain films 1 min with crystal violet-ammonium 516 oxalate. Wash briefly in tap water and drain. Apply Gram's iodine, 1 min. Wash in tap water and drain. Decolorize with 95% ethanol until washings are no longer blue (about 30 s), or flood slides with ethanol, pour off immediately, and reflood with ethanol for 10 s. Wash briefly with water, drain, and apply Hucker's counterstain (safranin solution) for 10-30 s. Wash briefly with water, drain, blot or air-dry, and


R33. Hippurate Solution, 1~

Dissolve 0.1 g sodium hippurate in 10 ml distilled water. Filter-sterilize and store refrigerated or in 0.4 ml aliquots at -20°C. Commercial preparations are also available.

Solution A (horseradish)

R34. Horseradish Peroxidase (color development solution)

HRP color development reagent Ice cold methanol

Mix to dissolve. Protect from light and prepare fresh.

Solution B

Ice cold hydrogen peroxide, 30\ Tria-buffered saline

60 mg 20 ml

60 JJl 100 ml

Prepare fresh before use. Mix ice cold solution A with room temperature solution B. Use immediately.

R35. Hybridization Mixture (6X SSC, SX Denhardt's, 0.01 M EDTA, pH 8.0)

20x sse SOX Denhardt's solution 0.5 M EDTA Distilled water

HCl (concentrated)

R36.

Distilled water to make 1 liter

2.0 M Tria, pH 7.6 1.0 M MgCll 0.5 M Dithiothreitol 10 mM Spermidine 0 . 5 M EDTA, pH 8.0 Distilled water Store at 4°C

R37.

1 N Hydrochloric Acid

lOX Kinase Buffer

R38. Kovacs' Reagent

R-Dimethylaminobenzaldehyde Amyl alcohol (normal only) HCl (concentrated)

15.0 ml 5.0 ml 0.1 ml

23.9 ml

89 ml

2.5 ml 1.0 ml 1.0 ml 1.0 ml

20 JJl 4.5 ml

5 g 75 ml 25 ml

517


Dissolve ~-dimethylaminobenzaldehyde in normal amyl alcohol. Slowly add HCl. Store at 4°C. To test for indole, add 0.2-0.3 ml reagent to 5 ml of 24 h bacteria culture in tryptone broth. Dark red color in surface layer is positive test for indole.

R39.

Lithium hydroxide (anhydrous) Distilled water

Potassium iodide (KI) Iodine Distilled water

R40.

0.1 H Lithium Hydroxide

Lugol's Iodine Solution

2.395 g 1 liter

10 g 5 g

100 ml

Dissolve KI in about 20-30 ml of distilled water. Add iodine and heat gently with constant mixing until iodine is dissolved. Dilute to 100 ml with distilled water. Store in amber glass-stoppered bottle in the dark.

R41. Kay-Grunwald Stain

May-Grunwald stain (Matheson Coleman & Bell, Norwood, OH 45212)

Methanol (absolute) 2.5 g

1 liter

Weigh stain into 50 ml methanol, dissolve by grinding, and dilute to 1 liter with methanol. Stir 16 h at 37°C. Hold stain 1 month at 22°C (room temperature). Filter before use.

R42. McFarland Nephelometer

Make suspensions of barium sulfate as follows:

1. Prepare 1.0% solution of CP (chemically pure) sulfuric acid. 2. Prepare 1.0% solution of CP barium chloride. 3. Prepare 10 standards as follows:

Add (ml 1% BaC12 solution) to (ml 1% H2S04 solution) 1 99 2 98 3 97 4 96 5 95 6 94 7 93 8 92 9 91

10 90

4. Seal about 3 ml of each standard suspension of barium sulfate precipitate in small test tube. Select test tubes carefully for uniformity of wall

518

FI>J\ BACTERIOLOGICAL ANALYTICAL UAL 7TH EDITION/1992

thickness, diameter, and color. (The se preparations are available commercially from several laboratory supply firms.)

R43. Mercuric Chloride Solution, 0.1,

Mercuric chloride 1.0 g Distilled water 1 liter

Methyl red Ethanol, 95%

R44.

Distilled water to make 500 ml

Methyl Red Indicator

0.10 g 300 ml

Dissolve methyl red in 300 ml ethanol. Bring volume to 500 ml >iLh distilled water.

R45. Methylene Blue Stain (Loeffler's)

Solution A

Methylene blue (90\ dye content) Ethanol (95%)

Solution B

Diluted potassium hydroxide (0.01%)

Mix solutions A and B.

R46. Mineral Oil

0.3 g 30 ml

100 ml

Autoclave 30 min at 121°C. Use screw-cap containers, about 1/2 full with 20-50 ml.

R47. Ninhydrin Reagent (commercially available)

Dissolve 3.5 g ninhydrin in 100 ml of 1:1 mixture of acetone and butanol. Store refrigerated.

R48. Nitrite Detection Reagents

A. Sulfanilic acid reagent

Sulfanilic acid 5 N acetic acid

B. N-Cl-naphthyllethylenediamine reagent

N-(1-naphthyl)ethylenediarnine dihydrochloride 5 N acetic acid

1 g 125 ml

0.25 g 200 ml

519


c. alpha-Naphthol reagent

alpha-Naphthol 5 N acetic acid

1 g 200 ml

To prepare 5 N acetic acid, add 28.75 ml glacial acetic acid to 71.25 ml distilled water.

Store reagents in glass-stoppered brown bottles. To perform test, add 0.1-0.5 ml each of reagent A and either reagent B or reagent c (as specified in method) to culture grown in liquid or semisolid medium. Development of red-violet color with reagents A and B or orange color with reagents A and c indicates that nitrate has been reduced to nitrite. Since color produced with reagents A and B may fade or disappear within a few minutes, record reaction as soon as color appears. If no color develops, test for presence of nitrate by adding small amount of zinc dust. If color develops, nitrate has not been reduced.

Nitrate reduction test for enteropathogenic E. coli. To 3 ml of 18-24 h culture in indole-nitrite medium, add 2 drops each of reagents A and B. Red-violet color indicates that nitrate has been reduced to nitrite. Check negative tests by adding small amount of zinc dust; if red-violet color does not appear, nitrate has been reduced.

D. Alternative test reagents. 5-Amino-2-naphthylene sulfonic acid (Cleve's acid) and N,N-dimethyl-1-naphthylamine have been recommended as substitutes for preparation of reagent B. Absolute ethanol may be substituted for acetic acid in reagent C. However, comparative evaluations should be conducted before substitution of these alternative reagents.

CAUTION: The alpha-naphthylamine reagent recommended in previous editions of the Bacteriological Analytical Manual should not be used because of the possible hazard to laboratory personnel. Any supplies of alphanaphthylamine on hand should be inventoried and secured by supervisory personnel because of the strict rules governing the use of carcinogenic substances in laboratories of the u.s. Department of Health and Human Services.

Aniline oil Ethanol (95%)

R49. North Aniline lOill-Methy1ene Blue Stain

HCl (concentrated) Methylene blue (saturated solution) Distilled water to make 100 ml

3 ml 10 ml

1.5 ml 30 ml

Mix aniline oil with ethanol. Slowly add HCl with constant agitation. Add saturated methylene blue solution, dilute to 100 ml with water, and filter.

520

fl)~ BACTERIOLOGICAL ANALYTICAL MANUAL

R50.

Novobiocin (sodium salt) Deionized water

Novobiocin Solution (100 mq/ml)

7TB EDITION/1992

100 mg 1.0 ml

Dissolve novobiocin. Filter-sterilize, using 0.2 ~m filter and ayringe. May be stored several months in dark bottle at 4~.

R51. 0/129 Disks (2,4-Diamino-6,7-diisopropyl pteridine)

2,4-Diamino-6,7-diisopropyl pteridine (0/129) or phosphate salt Distilled water, sterilized Filter paper disk blanks, 5-6 mm or 1/4 inch, sterilized Micropipettor, calibrated to deliver 10 ~1

Mark disks to differentiate 10 ~g from 150 ~g disks. Sterilize filter paper disks in glass petri plates. Dissolve 0/129 or 0/129-P04 (the phosphate salt dissolves more easily) in sterile water. For 150 ~g disks, place 10 ~1 of 15 mg/ml 0/129 or 20.8 mg/ml 0/129-P04 on each disk. For 10 ~g disks, make 1:15 dilutions of the 0/129 solutions and place 10 ~1 of 1.0 mg/ml 0/129 or 1.4 mg/ml 0/129-P04 on each disk. Dry disks and store desiccated and protected from light in refrigerator. 0/129 disks are commercially available.

R52. 0157 Monoclonal Antibody Solution

Dispense 10 ~1 ascitic fluid into 1 ml 1\ gelatin-Tria-buffered saline (TBS). Add 3 ~1 horseradish peroxidase-protein A conjugate, and stir mixture at 4°C for 1 h. Then dilute to 10 ml with 1\ gelatin-TBS. Sufficient for 1 hydrophobic grid membrane filter.

RSJ. ONPG Test

Monosodium phosphate solution, 1.0 M, pH 7

N aH2P04 • H20 Distilled water NaOH solution, 30\ (w/v)

6.9 g 45 ml

3 ml

Dissolve NaH2P04.H20 in distilled water. Add 30\ NaOH solution and adjust to pH 7. Bring volume to 50 ml with distilled water and store in refrigerator (about 4°C) .

0.0133 M o-Nitrophenyl-beta-D-galactoside CONPG)

ONPG Distilled water, 37°C Monosodium phosphate solution, 1.0 M, pH 7

80 mg 15 ml

5 ml

Dissolve ONPG in distilled water at 37°C. Add 1.0 M NaH2P04 solution. Solution should be colorless. Store in refrigerator (about 4~). Before use,

521

~ . -


warm appropriate portion (sufficient for number of tests) of ONPG solution to 37°C.

Procedure

Inoculate cultures to be tested onto triple sugar iron agar slants and incubate for 18 hat 37°C (or other appropriate temperature, if required). Nutrient (or other) agar slants containing 1.0\ lactose may also be used. In sterile 13 x 100 mm tube containing 0.25 ml of physiological saline, emulsify large loopful of culture growth to form heavy suspension. Add 1 drop of toluene to each tube and shake well to liberate enzyme. Let tubes stand 5 min at 37°C. Add 0.25 ml buffered 0.0133 M ONPG solution to each suspension to be tested. Incubate tubes at 35-37°C. Examine tubes at intervals up to 24 h. Yellow color is positive result.

ONPG disks are available commercially. Emulsify growth from 18 h lactosecontaining medium in 0.2 ml sterile physiological saline in sterile 13 x 100 mm tube. Add disk and agitate gently. Incubate and read tubes as above.

R54. Oxidase Reagent

N,N,N',N'-Tetramethyl-£-phenylenediamine.2HC1 Distilled water

1 g 100 ml

This is the preferred reagent. Use freshly prepared. However, reagent can be used up to 7 days if stored in a dark glass bottle under refrigeration.

Apply freshly prepared solution directly to young culture (24 h) on either agar plate or slant. Oxidase-positive colonies develop a pink color and progressively turn dark purple. If cultures are to be preserved, complete the transfer from plates to which reagent has been added within 3 min, since reagent is toxic to organisms.

Ootional method: Transfer small amount of culture to reagent-impregnated filter paper. Dark purple color within 10 s indicates positive test. Use platinum wire or sterile wooden stick (toothpicks or applicator sticks), since wire containing iron (e.g., nichrome wire in ordinary inoculating needles and loops) gives false-positive reactions.

Alternatively, the test may be performed with a 1\ solution of N,N-dimethyl-£phenylenediamine hydrochloride. Apply solution directly to culture plate or slant.

R55. Penicillinase (Beta-Lactamasel

Commercially available from Difco Laboratories, Box 1058A, Detroit, MI 48232; BBL, Division of Bioquest, P.O. Box 234, Cockeysville, MD 21030; ICN Nutritional Biochemicals, 26201 Miles Road, Cleveland, OH 44128; SchwartzMann, Orangeburg, NY 10962; and Calbiochem, 10933 N. Torrey Pines Road, La Jolla, CA 93037. Obtain Penicillin G reference standard from U.S.P. reference standards, 12601 Twin Brook Parkway, Rockville, MD 20852 .

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R56. Peptone Diluent, 0.1,

Peptone 1 g Distilled water 1 liter


R57. Peroxidase Substrate for Membrane ELISA

Solution A:

4-Chloro-1-naphthol Methanol (cold)

Dissolve 4-chloro-1-naphthol in cold methanol.

Solution B:

Hydrogen peroxide, 30% Tria-buffered saline (TBS)

Store at -20°C.

60 mg 20 ml

60 /-11 100 ml

Add 30\ H202 to TBS at room temperature. Immediately before use, mix ice cold Solution A with room temperature Solution B.

ABTS

R58. Peroxidase Substrate Solution <ABTSl (2,2•-Azino-di-[3-ethyl benzthiazoline sulfonate (6)]

(ABTS) substrate)

Citric acid (0.05 M) Hydrogen peroxide, 30%

10 mg 10 ml 30 I-ll

Prepare 5 min before use. plate.

One recipe is sufficient for one 96-well microwell

R59. Phosphate-Buffered Saline (PBS), pH 7.4

NaCl 7.650 g Na~P04 , anhydrous 0. 724 g KH2P04 0. 210 g Distilled water 1 liter

Dissolve ingredients in distilled water. Adjust pH to 7.4 (with 1 N NaOH). Autoclave 15 min at 121°C. This buffer may be used for any step in the Vibrio spp. methods when PBS is needed.

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R60. 0.01 M Phosphate-Buffe red Saline fpH 7.5)

Stock solution (0.1 M>

Na~P04 (anhydrous) NaH2P04 • H20 NaCl Distilled water

12.0 g 2.2 g

85.0 g 1 liter

Dissolve ingredients in distilled water and bring volume stock solution 1+9 in double distilled water. Mix well. with 0.1 N HCl or 0.1 N NaOH if necessary. Commercially dehydrated form from Difco Laboratories and BBL.

to 1 liter. Dilute Adjust pH to 7.5

available in

R61. 0.02 M Phosphate Saline Buffer fpH 7.3-7.4)

Prepare stock solutions of 0.2 M mono- and disodium phosphate in 8.5\ salt solutions and dilute 1:10 for preparation of 0.02 M phosphate saline buffer.

Stock stolution 1

Na2HP04 (anhydrous) (reagent grade) NaCl (reagent grade) Distilled water to make 1 liter

Stock solution 2

Na~P04 (anhydrous) (reagent grade) NaCl (reagent grade) Distilled water to make 1 liter

28.4 g 85.0 g

27.6 g 85.0 g

To obtain 0.02 M phosphate-buffered saline (0.85\), make 1:10 dilutions of each stock solution. For example:

Stock solution 1 Distilled water Approximate pH, 8.2

50 ml 450 ml

Stock solution 2 Distilled water Approximate pH, 5.6

10 ml 90 ml

Using pH meter, titer diluted solution 1 to pH 7.3-7.4 by adding about 65 ml of diluted solution 2. Use resulting 0.02 M phosphate saline buffer solution in the lysostaphin susceptibility test on §. aureus.

NOTE: Do not titer 0.2 M phosphate buffer to pH 7.3-7.4 and then dilute to 0.02 M strength. This results in a drop in pH of approximately 0.25. Addition of 0.85\ salt after pH adjustment also results in a drop of approximately 0.2.

Na~P04 NaH2P04

NaCl

R62.

Distilled water

Phosphate Saline Solution (for Y-1 LT Assay)

1.07 g 0.24 g 8.9 g

1 liter

Dissolve ingredients in water. Adjust pH to 7.5.

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FI>~ BACTERIOLOGICAL ANALYTICAL MANUAL 7TH EDITION/1992 R63. Physiological Saline Solution 0.85' (Sterile)

NaCl 8.5 g Distilled water 1 liter

Dissolve 8.5 g NaCl in water. Autoclave 15 min at 121°C. Cool to room temperature.

R64. Polymyxin B Disks, 50 Units

Polymyxin B sulfate Distilled water, sterilized Filter paper disk blanks, 5-6 mm or 1/4 inch, sterilized Kicropipettor, to deliver 10 ~1

Sterilize filiter paper disks in glass petri plates. Dissolve polymyxin B in sterile water to yield a final concentration of 5000 unitsfml. Place 10 ~1 of the 5000 unitsfml solution on each disk. Dry the disks. Store desiccated and protected from light in refrigerator.

Example: Polymyxin B is sold by activity in USP units per mg. Check reagent bottle for activity of the lot in use. For polymyxin B with antibiotic activity of 8090 USP units per mg, dissolve 0.618 mg in 1 ml sterile water.

Calculation: ------~5~0~0~0~U~n~i~t~s~/~m~l~----- = 0.618 mg/ml 8090 Unitsfmg

R65. Potassium Hydroxide Solution, 40'

KOH 40 g Distilled water to make 100 ml

R66. Saline Solution, 0.5, (Sterile)

NaCl 5 g Distilled water 1 liter

Dissolve NaCl in water. Autoclave at 121°C for 15 min.

R67. Salts-Phosphate Buffered Saline Solution (Salts-PBS)

NaCl KCL MgC12 CaC12 • 2H2

NaH2P04 • H20 NaH2P~ 7H20 Distilled water

Adjust pH to 7.4.

1

R68. Scintillation Fluid

121 g 15.5 g 12.7 g 10.2 g 2.0 g 3.9 g liter

2,5 Diphenyloxazole 5.0 g Toluene 1 liter

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. . FI>~ BACTERIOLOGICAL ANALYTICAL MANUAL 7TH EDITION/1992

R69. Slide Preserving Solution

Prepare 1% acetic acid solution (10 ml glacial acetic acid, reagent grade + 990 ml distilled water). Add 1 ml glycerin to each 100 ml of solution.

R70. Sodium Bicarbonate Solution, 10%

Sodium bicarbonate 100 g Distilled water to make 1 liter

Sterilize by filtration.

R71. Sodium Chloride Dilution Water, 2% and 3%

2% solution

NaCl Distilled water

3% solution

NaCl Distilled water

20 g 1 liter

30 g 1 liter

Dissolve NaCl in water. Autoclave 15 min at 121°C. Final pH, 7.0.

R72. 0.2 M Sodium Chloride Solution

NaCl 11.7 g Distilled water to make 1 liter

Dispense in suitable containers. Autoclave 15 min at 121°C.

R73. 1 N Sodium Hydroxide Solution

NaOH 40 g Distilled water to make 1 liter

Use for adjusting pH of culture media.

R74. 10 N Sodium Hydroxide Solution

NaOH 400 g Distilled water to make 1 liter

R7S. Sonicated Calf-Thymus or Salmon-Sperm DNA

~A 1g Distilled water 1 liter

Stir 3-4 h to dissolve. May be heated to 60°C. Sonciate until MW is about 300,000 - 500,000 daltons. Store frozen in 1 ml aliquots.

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R76. Spicer-Edwards EN Complex Antibody Solution

Add 0.1 ml Spicer-Edwards EN Complex (Difco) to 0.07 ml horseradish peroxidase-protei n A conjugate in 1 ml 1\ gelatin-Tria-buffered saline (TBS), and stir at 4°C for 1 h. Dilute to 40 ml 1\ gelatin-TBS. Use within a few hours. Sufficient for 4 hydrophobic grid membrane filters.

R77.

NaCl (reagent grade) Sodium citrate

Standard Saline Citrate (SSC) Solution f20Xl

175.3 g 88.3

Dissolve in 800 ml deionized water and adjust to pH 7 with 10 N NaOH. Bring volume to 1 liter.

6X sse

NaCl (reagent grade) Sodium citrate

52.6 g 26.5 g

Dissolve in 800 ml deionized water and adjust to pH 7 with 10 N NaOH. Bring volume to 1 liter.

3X sse

6X sse Deionized water

2X sse

6X sse Deionized water

R78. Tergitol Anionic 7

500 ml 500 ml

333 ml 667 ml

This reagent is a sodium sulfate derivative of 3,9-diethyl tridecanol-6. Its recommended use is for wetting and emulsifying when the electrolyte is below 1\ in textiles, emulsion polymers, rubber lattices, leather, and pharmaceuticals. Tergitol-7 is an anionic wetting agent manufactured by Union Carbide Corp., Chemicals and Plastics, 270 Park Avenue, New York, NY 10017.

R79. Thiazine Red R Stain (for §. aureus enterotoxin gel diffusion technique)

Dissolve Thiazine Red R (Color Index No. 14780), 0.1\, in 1.0\ acetic acid. Thiazine Red R is available from Matheson Coleman & Bell, Norwood, OH 45212.

R80. 1.0 M Tria fpH 8.0)

Tria 121.14 g Distilled water to make 1 liter

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Dissolve in several hundred ml distilled water. Adjust pH to 8.0 with concentrated HCl. Bring volume to 1 liter.

R81. Tria-Buffered Saline (TBS) tpB 7.5)

Tris 2.42 g NaCl 29.24 g Double distilled water to make 1 liter

Dissolve ingredients. Adjust pH to 7.5 with HCl and bring volume to 1 liter.

1\ solution

Gelatin TBS, pH 7.5

3\ solution

Gelatin TBS, pH 7.5

R82. Tria-Buffered Saline fTBS), with Gelatin

1 g 100 ml

3 g 100 ml

Add gelatin to TBS at 40°C. Stir to dissolve. Cool to room temperature before use.

R83. Tria-Buffered Saline (TBS)-Tween

Tween 20 50 ~1 TBS, pH 7.5 100 ml

Dissolve Tween 20 in TBS.

R84. Tria-Buffered Saline (TBS), 1' or 3' Gelatin, or Tween 20

Tris NaCl Distilled water

2.42 g 29.24 g 1 liter

Dissolve ingredients in distilled water by heating and stirring. Adjust pH to 7.5 with HCl. Autoclave 15 min at 121°C.

For 1\ and 3\ gelatin-TBS, add 10 g and 30 g gelatin, respectively, to ingredients before autoclaving. Adjust final pH to 7.5 with HCl.

For Tween-TBS, add 0.5 ml Tween 20 to ingredient and adjust pH to 7.5 before autoclaving.

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Triton X-100TM 1.0 M Tria (R80) 0.5 M Na~DTA (R20)

R85. Tris-EDTA-Triton X-100TM(TETl Buffer (Triton Lytic Mix)


R86. Triton X-100

1.0 ml 5.0 ml

12.5 ml

This reagent is the registered trademark for octylphenoxy polyethoxy ethanol. Its recommended uses include wetting agent, detergent, dispersant, emulsifier in household and industrial cleaners, textile processing, wool scouring, and emulsifying agent for insecticides and herbicides. Triton X-100 is a nonionic preparation manufactured by Rohm and Haas Company, Independence Mall West, Philadelphia, PA 19105. It is also sold in small quantities by Fisher Scientific Co., 711 Forbes Avenue, Pittsburgh, PA 15219.

R87. Trypsin-EDTA Solution, 1x

Trypsin EDTA Solution lX: Tryps i n (1:250*), 0.5 g; EDTA, disodium salt, 0.2 g; 0.9% NaCl solution, 100 ml. Dissolve trypsin and EDTA in NaCl solution. Filter-ster i lize throu gh 0.22 ~m membrane. *l g Trypsin (1:250) will digest 250 g casein substrate under standard conditions.

RBB. Verocytotoxin Antiserum

Grow I· coli 0157:H7 in trypticase soy broth . at 37°C for 18 h; add 0.5\ formalin and keep at 37°C for 2 weeks. Inoculate formalized culture into rabbits through ear vein. Give a total of 5 inoculations (0.5, 1.0, 2.0, 4.0 and 4.0 ml), one each, at 5-day intervals. Remove blood after 6 weeks and obtain serum. Heat serum at 56°C for 30 min and adsorb antiserum 6 times with about 1012 cells of heat-treated (121°C, 1 h) I· coli Ol57:H7 (l ml packed cells to 20 ml antiserum). Use antibody solution at 1:5000 dilution.

Solution 1

alpha-Naphthol Alcohol (absolute)

Solution 2

Potassium hydroxide

R89. Voges-Proskauer (VP) Test Reagents


5 g 100 ml

40 g

Voges-Proskauer lVP> test. Transfer 1 ml of 48 h culture to test tube and add 0.6 ml solution 1 and 0.2 ml solution 2. Shake after adding each solution. To intensify and speed reaction, add a few creatine crystals to mixture. Let stand at room temperature. Read results 4 h after adding reagents. Development of eosin pink color is positive test.

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bacteriological analytical manual 7th edition/1992

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