TRYPTIC SOY BROTH WITH 6.5% SODIUM CHLORIDE

Cat. no. K126 Tryptic Soy Broth (TSB) with 6.5% Sodium Chloride, 15x103mm Tube, 5ml 20 tubes/box
Cat. no. D07 Tryptic Soy Broth (TSB) with 6.5% Sodium Chloride, 15x45mm Tube, (without tube label), 1.5ml 100 tubes/box

INTENDED USE

Hardy Diagnostics Tryptic Soy Broth (TSB) with 6.5% NaCl is a growth medium recommended for the differentiation of enterococci from the group D streptococci. This medium is also useful as an enrichment step for increasing sensitivity in the detection of MRSA in high risk patients.

SUMMARY

Historically, streptococci have been classified by phenotypic characteristics, such as hemolytic reactions, biochemical tests and Lancefield serological groups. Based on these criteria, enterococci were initially classified as group D streptococci. However, in the mid-1980s, molecular tests showed significant genetic differences between these two groups. Consequently, enterococci are now placed in the genus of Enterococcus and the non-enterococcal group D streptococci remain classified as streptococci.(6-9)

The most clinically significant distinction between enterococci and group D streptococci is their difference in antimicrobial resistance. In general enterococci are more resistant to penicillins, cephalosporins, and aminoglycosides, permitting them to survive in hospital environments and cause nosocomial infections in patients receiving broad-spectrum antibiotics.(10,11) The last two decades have seen particularly virulent strains of Enterococcus resistant to vancomycin (commonly called Vancomycin-Resistant Enterococcus or VRE) emerge in nosocomial infections, especially in the U.S.(10,11)

Further studies on these microbes show differences in physiological characteristics. For example, most species of enterococci will grow in medium containing 6.5% NaCl. However, group D streptococci are inhibited when grown under conditions with high salt concentrations.(7,8)

Additionally, enrichment broths are commonly used to increase sensitivity testing for MRSA. Research shows an increase in isolation rates when using an enrichment broth prior to plating high-risk patient samples.(19) In this capacity, enrichment broths are primarily used as multibroths, where multiple swabs from the same patient are inoculated into a single broth. This method provides substantial savings in media costs and time needed to determine infection when compared to conventional testing methods.(20)

Hardy Diagnostics Tryptic Soy Broth (TSB) with 6.5% NaCl contains enzymatic digest of casein and enzymatic digest of soybean meal, which provide amino acids and complex nitrogenous compounds that promote microbial growth. Dextrose acts as a carbon energy source that facilitates growth. Dipotassium phosphate acts as a buffering agent. The addition of sodium chloride at a concentration of 6.5% permits the differentiation of salt-tolerant microorganisms from salt-intolerant species. At this strength, sodium chloride acts selectively by interfering with membrane permeability and osmotic and electrokinetic equilibria.(14)

FORMULA

Ingredients per liter of deionized water:*

Sodium Chloride 65.0gm
Pancreatic Digest of Casein 17.0gm
Papaic Digest of Soybean Meal 3.0gm
Dextrose 2.5gm
Dipotassium Phosphate 2.5gm

Final pH 7.3 +/- 0.3 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-30ºC. away from direct light. Media should not be used if there are any signs of deterioration, discoloration, contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Specimens appropriate for culture may be handled using various laboratory techniques. Refer to the appropriate reference(s) for specific procedures.(1,4,16,19,20)

Observe aseptic techniques and use standard laboratory precautions.

1. Inoculate tubes lightly with suspect bacterium.

a. Using a single colony, lightly inoculate the side of the tube, just at the surface of the broth, so as not to
over-inoculate the sample. Alternatively, inoculate tubes using a 0.01ml loop with a 10-1 dilution of 18-24 hour primary cultures.

b. Swab specimens from high-risk patients may be directly inserted into the medium as in enrichment for MRSA.

2. Incubate tubes aerobically with loosed caps at 35 +/- 2ºC. for 18-24 hours.

3. Examine tubes at 18-24 hours and 42-48 hours for growth (turbidity). It may be necessary to compare tubes to an uninoculated control tube to determine if growth is present. Growth may be present with or without the formation of a precipitate.

INTERPRETATION OF RESULTS

Refer to the appropriate reference(s) for the accurate interpretation of test results.(1,4,16,19,20)

Growth in broth media indicates a positive reaction and is demonstrated by the presence of turbidity (cloudiness) when compared to an uninoculated control tube. A positive reaction may be present with or without the formation of a precipitate.

Lack of growth indicates a negative reaction when compared to an uninoculated control tube.

1. Most gram-positive enterococci will grow in TSB with 6.5% NaCl. Most strains will show turbidity within 24 hours.

2. Most gram-positive, non-enterococcal group D streptococci fail to grow in TSB with 6.5% NaCl after 48 hours.

3. This medium can be used as an enrichment broth for MRSA cultures from high-risk patients. Tubes will be positive for growth (turbid) after 24 hours. Subculture to an appropriate medium for further testing. Please consult appropriate references for more information.(4,16)

LIMITATIONS

Other gram-positive cocci besides enterococci and staphylococci (e.g. group B streptococci, aerococci and pedicocci) can grow in TSB with 6.5% NaCl broth. Therefore, it is recommended that biochemical and/or serological tests be performed on colonies from pure culture for complete identification.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Enterococcus faecalis
ATCC® 29212
A 24hr 35°C Aerobic Growth
Streptococcus bovis
ATCC® 9809
B 24hr 35°C Aerobic Inhibited

USER QUALITY CONTROL

REFERENCES

1. United States Pharmacopeial Convention. 1995. The United States Pharmacopeia, 23rd ed. The United States Pharmacopeial Convention, Rockville, MD.

2. European Parliament (Ph. Eur.). 2005. 5th ed. The European Pharmacopeia. European Pharmacopoeia Commission.

3. Curry, A. S., G. G. Joyce, and G. N. McEwen, Jr. 1993. CTFA Microbiology Guidelines. The Cosmetic, Toiletry, and Fragrance Association, Inc. Washington, D.C.

4. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

5. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

6. Ruoff, K.L. 1990. "Recent taxonomic changes in the genus Enterococcus". Eur. J. Clin. Microbiol. Infect. Dis.; Vol. 9 No. 2: 75-9.

7. Ruoff, K.L., R.A. Whiley, D. Beighton. Streptococcus In: Murray, P.R., E.J. Baron, M.A. Pfaller, F.C. Tenover, R.H. Yolken. 1999. Manual of Clinical Microbiology, 7th ed. American Society for Microbiology Press. Washington D.C.

8. Facklam, R.R., D.F. Sahm, L. Martins Teixeira. Enterococcus. In: Murray, P.R., E.J. Baron, M.A. Pfaller, F.C. Tenover, R.H. Yolken. 1999. Manual of Clinical Microbiology, 7th ed. American Society for Microbiology Press. Washington D.C.

9. Schleifer, K.H., R. Kilpper-Balz. 1984. Transfer of Streptococcus faecalis and Streptococcus faecium to the genus Enterococcus nom. rev. as Enterococcus faecalis comb. nov. and Enterococcus faecium comb. nov. Int. J. Sys. Bacteriol.; Vol 34. 31-34.

10. Fischetti, V.A., R.P. Novick, J.J. Ferretti, D.A. Portnoy, J.I. Rood. 2000. Gram-Positive Pathogens. American Society for Microbiology Press. Washington D.C.

11. Ryan, K.J., C.G. Ray. 2004. Sherris Medical Microbiology, 4th ed. McGraw Hill. New York, NY.

12. U.S. Food and Drug Administration. Bacteriological Analytical Manual. AOAC, Arlington, VA. www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/default.htm

13. Cunnif, P. 1995. Official Methods of Analysis AOAC International, 16th ed. AOAC International, Arlington, VA.

14. MacFaddin, J.F. 1985. Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, Vol I. Williams & Wilkins. Baltimore, MD.

15. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

16. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

17. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

18. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

19. Lee, S., Y.J. Park, E.J. Oh, J. Khang, J.H. Yoo, I. H. Jeong, Y.M. Kwon, K. Han. 2007 Comparison of Protocols for Surveillance of Methicillin-Resistant Staphylococcus aureus (MRSA): Medical vs. ICU Patients. Ann. Clin. Lab. Sci.; 37(3):248-50.

20. Brown, D.F.J., D.I. Edwards, P.M. Hawkey, D. Morrison, G.L. Ridgway, K.J. Lowner, and M.W.D. Wren. 2005. Guidelines for the Laboratory Diagnosis and Susceptibility Testing of Methicillin-Resistant Staphylococcus aureus (MRSA). J. Antimicrob. Chemo.; 56:1000-1018.


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