Composition

* Dehydrated powder, hygroscopic in nature, store in a dry place in tightly- sealed containers 25°C and protected from direct Sunlight.

Instructions for Use

Dissolve 22gms in 1000ml distilled water. Gently heat to boiling with gentle swirling and dissolve the medium completely. Dispense into desired container. Sterilize by autoclaving at 10 psi (115°C) for 10 minutes. Do not overheat. Cool to 45 – 500C.

Appearance: Light to medium amber colour, slightly opalescent gel

pH (at 250 C): 7.1 ± 0.2

Principle

MUELLER HINTON BROTH NO. 2 is used for susceptibility testing of rapidly growing bacteria using antibiotic discs by Kirby Bauer technique. The medium consist of Beef extract and Casein enzymatic hydrolysate which provides nitrogen, vitamins, carbon, and amino acids. Starch soluble is added to absorb any toxic metabolites produced. It is suitable medium for testing the susceptibility of microorganisms to Sulphonamides and Trimethoprim. Antagonism to sulphonamide activity is demonstrated by para-aminobenzoic acid (PABA) and its analogs. Reduced activity of Trimethoprim, resulting in smaller growth inhibition zones and inner zonal growth, is demonstrated on medium possessing high levels of thymide. The PABA and thymine/thymidine content of Mueller Hinton Broth No. 2 are reduced to a minimum, reducing the inactivation of sulphonamides and Trimethoprim. It has become the standard medium for the Bauer Kirby method and its performance is specified by the NCCLS guidelines also. For inoculation: these antimicrobial agents are prepared by two fold serial dilutions technique and inoculated with the test culture to give a final concentration of 5 X 105 CFU/ml. For incubation: at 35 – 37°C, after a given time period observe the tubes; the turbidity in the tubes denotes growth of microbes. Whereas, the lowest concentration of antimicrobial agent showing no growth is the MIC of that organism for that agent. The microorganism is susceptible, intermediate growth or resistant in the response to the antimicrobial agents by comparing them to MIC. The antimicrobial agent susceptibility is derived by using the amount of inoculum size, pH of the broth, antimicrobial stability or susceptibility, and their mechanisms of resistance the test microorganisms.

Interpretation

Cultural characteristics observed after inoculating (103CFU/ml), on incubation at 35 ± 2°C for 18 – 24 hours.

References

1. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial disk susceptibility tests. Approved standard M2-A6. National Committee for Clinical Laboratory Standards, Wayne, PA. (1997).
2. Mueller, J. H., and J. Hinton. A protein-free medium for primary isolation of gonococcus and meningococcus. Proc. Soc. Exp. Biol. Med. 48:3330-3333. (1941).
3. Gordon and Hine. Br. Med. J. 678. (1916).
4. Bauer, A. L., W. M. M. Kirby, J. C. Sherris, and M. Turck. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45:493-496. (1966).
5. World Health Organization. Standardization of methods for conducting microbic sensitivity tests. Technical Report Series No. 210, Geneva. (1961).
6. Wood, G. L., and J. A. Washington. Antibacterial susceptibility tests: dilution and disk diffusion methods, p. 1327-1341. In Murray, P.R., E. J. Baron, M. A. Pfaller, F. C.Tenover, and R. H. Yolken(eds.). Manual of clinical microbiology, 6^th ed. American Society for Microbiology, Washington, D.C. (1995).
7. Wikler, M. A., et al. Performance standards for antimicrobial disk susceptibility tests; approved standard, 9th ed. Clinical and Laboratory Standards Institute document M2-A9. Clinical and Laboratory Standards Institute, Wayne, PA. (2006).