MUELLER-HINTON AGAR

Code: CM0337

An antimicrobial susceptibility testing medium which may be used in internationally recognised standard procedures.

* Adjusted as required to meet performance standards

Directions
Add 38g to 1 litre of distilled water. Bring to the boil to dissolve the medium completely. Sterilize by autoclaving at 121°C for 15 minutes.

Description
Mueller-Hinton Agar was designed to be a reproducible culture medium for the isolation of pathogenic Neisseria species (Mueller and Hinton¹). The inclusion of starch ensures that toxic factors found during growth will be absorbed and its presence is often essential to establish growth from very small inocula².

However, specific GC media have replaced Mueller-Hinton Agar for this purpose.

The major use of Mueller-Hinton Agar is for Antimicrobial Susceptibility Testing (AST). It has become the standard medium for the Bauer-Kirby method^3,4, and is specified by the Clinical & Laboratory Standards Institute (CLSI) formerly the National Committee for Clinical Laboratory Standards (NCCLS)^5,6,7 and The European Committee on Antimicrobial Susceptibility Testing (EUCAST)⁸.

Oxoid Mueller-Hinton Agar meets the requirements of WHO^9,10. Criticisms have been made about variation in performance of Mueller-Hinton Agar between and with manufacturers’ batches/lots of medium¹¹. The causes of such variation are:

1. Differences in concentration of divalent cations Mg++ and Ca++. These effects are shown as MIC variations with aminoglycosides against Pseudomonas aeruginosa and tetracycline against staphylococci^12,13,14.
2. Variation in thymine and thymidine content, which affect sulphonamide and trimethoprim MIC values^15,16.
3. The concentration of manganese, which affects resistance interpretations with glycylcylines^17.
4. The concentration of zine, which affects resistance interpretations with imipenem and potentially other carbapenems.¹⁸
5. Differences in the characteristics of the agar used in the medium, especially diffusion properties¹⁹.

In the light of such criticisms the CLSI called interested manufacturers together to discuss the standardization and stabilization of Mueller-Hinton Agar. Control methods were established whereby critical antimicrobial/organism combinations had to yield consistent zones of inhibition within 2mm of the specified diameters in the standards^7.

The result of this cooperative effort is that Mueller-Hinton Agar became a standard medium and as well as being the medium of choice for CLSI methodology it is also specified by EUCAST^8. The medium meets the criteria described in the Technical Specification ISO/TS 16782:2016^18.

Antibiotic susceptibility tests are performed in accordance with and meet the acceptance limits of the current ISO/TS 16782 using the following microorganisms:

Staphylococcus aureus ATCC®25923 WDCM00034
Staphylococcus aureus ATCC®29213 WDCM00131
Staphylococcus aureus ATCC®43300 WDCM00211
Staphylococcus aureus NCTC 12493 WDCM00212
Escherichia coli ATCC®25922 WDCM00013
Escherichia coli ATCC®35218
Pseudomonas aeruginosa ATCC®27853 WDCM00025
Enterococcus faecalis ATCC®33186 WDCM00210
Enterococcus faecalis ATCC®29212 WDCM00087
Streptococcus pneumoniae ATCC®49619
Haemophilus influenzae ATCC®49247
Haemophilus influenzae ATCC®49766

Mueller-Hinton Agar supplemented with yeast, NAD and haematin is used specifically for the susceptibility testing of Haemophilus influenzae²⁰. For further details see Haemophilus Test Medium (HTM), CM0898.

Mueller-Hinton Agar supplemented with 5% defibrinated horse blood (SR0050) and 20 mg/L β-NAD is specified by EUCAST for testing fastidious organisms.

Mueller-Hinton Agar and Broth are used as the basis of solid and liquid media containing cefoperazone, trimethoprim, piperacillin and cycloheximide for selective isolation of Arcobacter spp. from meats²¹.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared plates at 2-8°C.

Appearance
Dehydrated medium: Straw coloured, free-flowing powder
Prepared medium: Straw coloured gel

Quality control

* This organism is available as a Culti-Loop®
Note: Please refer to relevant standards for further quality control testing

Precautions
Incubation in a carbon dioxide enriched atmosphere is not recommended because of its pH effect on the medium. If it is imperative to use CO₂ then known control organisms should be included with the test plates to measure its effect.
Carbohydrates should not be added to Mueller-Hinton Agar because they may influence the rate of growth of the organism and the resulting pH of the medium.
The addition of lysed horse blood to the medium may further reduce the levels of thymidine and prevent the growth of thymidine-dependent organisms.

The medium may not support the growth of some fastidious strains for example microorganisms that require thymine and thymidine for growth. Close adherence to the methodology is required to obtain satisfactory results.

References
1. Mueller J. H. and Hinton Jane (1941) Proc. Soc. Exp. Biol. and Med. 48. 330-333.
2. Olsen A. M. and Scott W. J. (1946) Nature 157. 337.
3. Bauer A. W., Kirby W. M., Sherris J. C. and Turck M. (1966) Amer. J. Clin. Path. 45. 493-496.
4. Ryan K. J., Schoenknecht F. D. and Kirby W. M. (1970) Hosp. Pract. 5. 91-100.
5. CLSI Performance Standards for Antimicrobial Disk Susceptibility Testing; CLSI Supplement M100S Wayne PA: Clinical and Laboratory Standards Institute
6. CLSI Performance Standards for Antimicrobial Susceptibility Tests; Approved Standard. CLSI Document M02. Wayne PA: Clinical and Laboratory Standards Institute
7. Pollock H. M. et al. (1986) J. Clin. Microbiol. 24. 1-6.
8. European Committee on Antimicrobial Susceptibility Testing. EUCAST disc diffusion Test Methodology www.eucast.org
9. WHO (1961) Standardization of Methods for Conducting Microbic Sensitivity Tests. Geneva. Tech. Rep. Ser. No.210.
10. WHO (1977) Expert Committee on Biological Standardization. Geneva. Tech. Rep. Ser. No.610.
11. Barry A. L. and Effinger L. J. (1974) Amer. J. Clin. Path. 62. 113-117.
12. Reller L. B., Schoenknecht F. D., Kenny M. A. and Sherris J. C. (1974) J. Infect. Dis. 130. 454-463.
13. D’Amato R. F., Thornsberry C., Baker N. and Kirven L. A. (1975) Antimicrob. Agents Chemotherap. 7. 596-600.
14. D’Amato R. F. and Thornsberry C. (1979) Curr. Microbiol. 2.135-138.
15. Ferone R., Bushby S. R. M., Burchall J. J., Moore W. D. and Smith D. (1975) Antimicrob. Agents Chemotherap. 7. 91-98.
16. Ferguson R. W. and Weissfeld A. S. (1984) J. Clin. Microbiol. 19. 85-86
17. Veenemans J., Mouton J.W., Kluytmans J.A,J.W., Donnerly R. Verhulst, C., van Keulen, PHJ. Effect of manganese in test media on in vitro susceptibility oe Enterobacteriaceae and Acinetobacter baumannii to tigecycline. J.Clin.Microbiol 2012, 50 pp. 3077-3079
18. Daly J.S.m Dodge R.A., Glew R.H., Soja D.T., Deluca B.A., Herbert S. Effect of zince concentration in Mueller-Hinton agar on susceptibility of Pseudomonas aeruginosa to imipenem. J. Clin. Microbiol. 1997, 35 pp. 1027-1029
19. Bridson E. Y. and Brecker A. (1970) in `Methods in Microbiology’ Eds. Norris and Ribbons. Vol.3A Academic Press London. pp. 257-266.
20. International Organization for Standardization ISO/TS 16782:2016 Clinical laboratory testing - Criteria for acceptable lots of dehydrated Mueller-Hinton agar and broth for antimicrobial susceptibility testing.
21. Jorgensen J. H., Redding J. S., Maher L. A. and Howell A. W. (1987) J. Clin. Microbiol. 25. 2105-2113.
22. De Boer E., Tilburg J. J. H. C., Woodward D. L., Lior H. and Johnson W. M. (1996) Left. Appl. Microbiol. 23. 64-66.