Mueller Hinton Agar (MHA) – Composition, Principle, Uses and Preparation

Introduction

The name Mueller Hinton agar(MHA) is derived from the names of co-developers, microbiologist John Howard Mueller and veterinary scientist Jane Hilton at Harvard University who first published the method in 1941. This Mueller Hinton agar (MHA) was a protein-free medium for isolating the pathogenic strains of Neisseria. Later in 1966, Muller Hinton agar (MHA) was adopted as the common medium to use for routine antibiotics susceptibility testing for non fastidious bacteria (both aerobes and facultative anaerobe). It was also found that the Mueller Hinton Agar (MHA) medium was useful in identifying the sulfonamide-resistant and responsive strains of gonococci (Neisseria gonorrhoeae).

Moreover, Mueller Hinton Agar Medium is considered as the ideal medium for the Antibiotic sensitivity / Susceptibility test (AST) and also for the cultivation of microorganisms (as a growth medium), and for making dilutions of organisms to be used in the Kirby-Bauer disk diffusion method for AST.

Principle

The Mueller Hinton Agar medium commonly called as MHA medium or AST medium is the ideal medium for the Antibiotic Sensitivity test (AST) which is commonly done by using Kirby-Bauer Method and sometimes by Stokes method.Now, because of its reproducibility, it is recommended by various institutions like American Society of Microbiology (ASM), Clinical, Laboratory Standards Institute (CLSI) and  FDA, World Health Organization (WHO)  for antibiotic susceptibility testing of bacteria.

Some alterations in the composition of Mueller Hinton Agar is applicable as 5% sheep blood and Mueller Hinton Agar (MHA) with Hemoglobin has been recommended for antimicrobial susceptibility testing of Streptococcus pneumoniae and Hemophilus influenzae.

Ingredients of MHA are beef infusion solids, starch, casein hydrolysate and agar. Beef infusion solids and casein hydrolysate provide nitrogen, vitamins, carbon, amino acids, sulphur and other essential nutrients. Starch act as a “protective colloid” which absorbs any toxic metabolites produced in the medium. Starch hydrolysis yields dextrose, which serves as a source of energy where as agar is the solidifying agent.

Preparation Of Mueller Hinton Agar

Composition of Mueller Hinton Agar (MHA)

Material Required For Preparation

• Sterile Conical Flask / Erlenmeyer Flask
• Spatula
• Beef Extract
• Casein acid hydrolysate
• Starch
• Agar-Agar
• Measuring Cylinder
• 1N HCl
• 1N NaOH
• pH Strip
• Weighing Scale
• Distilled Water

Preparation Procedure

1. Weigh the quantity of Beef Extract, Casein acid hydrolysate, Agar and Starch using the weighing scale for 1000 ml of Mueller Hinton Agar.
2. Take a clean and dry Conical Flask/ Erlenmeyer flask.
3. Pour 500 ml of distilled water to the flask and add the weighed quantity of Beef Extract, Starch, and Casein acid Hydrolysate and mix thoroughly.
4. Now add the weighed quantity of Agar-Agar.
5. Mix well the content, Heat it with continuous agitation and boil it for 1-2 minutes to dissolve the constituents.
6. Now add more distilled water to the medium and make the volume 1000 ml and mix thoroughly to make the solution homogeneous.
7. Check the pH of the solution using pH strip, it should be 7.3 ± 0.1 i.e. 7.2 – 7.4 and If required, adjust the pH by adding either 1N HCl (acid) or 1N NaOH (base) as per the case.
8. Mix well the content and apply the Non-absorbent cotton plug to the flask.
9. Autoclave the content at 121 °C and 15 psi pressure for 15 minutes.
10. Allow the content to cool down to 40-45 °C and pour in the empty sterile media plates on a flat horizontal surface under the strict aseptic atmosphere (preferably in Laminar Air Flow) and allowed it to cool at room temperature.
11. The prepared MHA medium can be stored at 4 to 8°C and is stable for approximately 70 days from the date of preparation.
12. Use the prepared media plates to inoculate the specimen to be cultured and then place in the incubator at optimum temperature.

Modifications of Muller Hinton agar

• Mueller Hinton agar medium supplemented with 5% sheep blood and nicotinamide adenine dinucleotide (NAD) and is recommended for determining the antimicrobial susceptibility of Streptococcus species, Neisseria, Campylobacter.
• Haemophilus test medium (HTM)is the preferred medium for the AST of H. influenzae using modified Kirby Bauer disc diffusion. HTM medium consists of following inredients: thymidine free MHA supplemented with 15 μg/ml NAD, 15 μg/ml bovine hemin, and 5 mg/ml yeast extract.
• Mueller Hinton chocolate agar: For Haemophilus influenzae
•  Mueller Hinton Agar No. 2: Thymine and thymidine inhibit sulfonamide and trimethoprim activity and calcium and magnesium interferes with activity of aminoglycoside antibiotics. To overcome this problems MHA No. 2 is manufactured to contain low levels of thymine, thymidine and controlled levels of calcium and magnesium.

Result on Mueller Hinton Agar (MHA)

Zone of inhibition is observed around the antibiotics.

Limitations of Mueller Hinton Agar (MHA)

• It is recommended medium for susceptibility testing of pure cultures only.
• Inoculum density may affect the size of microbial growth zone of inhibition. Heavy inoculum may result in smaller zones or too less inoculum may result in larger zones.
• Fastidious organisms ( S. pneumoniae, H. influenzae )  may not grow on this medium and may require supplementation of blood.
•  Fastidious anaerobes may not grow on this medium.
• As antimicrobial susceptibility testing is carried with antibiotic disc, proper storage of the disc is desired which may affect the potency of the disc.
• Under certain circumstances, the in vitro results of antibiotic susceptibility may not show the same in vivo.

Reasons For Use Of MHA In Antibiotic Susceptibility Testing

1. Both the para-aminobenzoic acid (PABA) and thymine/thymidine content in Mueller Hinton Agar are reduced to a minimum, thus markedly reducing the inactivation of sulfonamides and trimethoprim when the media is used for testing the susceptibility of bacterial isolates to these antimicrobics.
2. It is a non-selective, non-differential medium. This means that almost all organisms plated on here will grow.
3. MHA is low in sulfonamide, trimethoprim, and tetracycline inhibitors (i.e. concentration of inhibitors thymidine and thymine is low in MHA).
4. It contains starch. Starch is known to absorb toxins released from bacteria, so that they cannot interfere with the antibiotics. It also mediates the rate of diffusion of the antibiotics through the agar.
5. It is a loose agar. This allows for better diffusion of the antibiotics than most other plates. A better diffusion leads to a truer zone of inhibition.
6. MHA shows acceptable batch-to-batch reproducibility for susceptibility testing.

Uses Of Mueller Hinton Agar (MHA)

1. MHA is the common medium to use for routine antibiotics susceptibility testing for non fastidious bacteria (both aerobes and facultative anaerobe).
2. It is the standard medium for AST by Bauer Kirby method and its performance is specified by the CLSI.
3. It is also applicable medium for antimicrobial susceptibility testing by Stoke’s method.
4. It can be used to cultivate Neisseria and Moraxella species.
5. It is also useful medium for Epsilometer test (E-test) for measuring the MIC of the bacterial isolate.
6. Antimicrobial susceptibility testing of fastidious bacteria can be tested  using modified MHA like  Mueller Hinton blood agar ( for S. pneumoniea) Mueller Hinton chocolate agar( for H. influenzae).
7. Mueller Hinton agar is  also specified in FDA Bacteriological Analytical Manual for food testing, and procedures commonly performed on aerobic and facultative anaerobic bacteria.
8. Muller Hinton Agar + 2% Glucose and 0.5 mg/ml Methylene Blue Dye (GMB) Medium: It uses for antifungal susceptibility testing (AFST) of yeasts ( for Candida species).
9. HiCrome™ Mueller Hinton Agar (for Antifungal Testing):  Chromogenic differentiation of yeast cells along with antifungal susceptibility.

Further References

1.  Dewees, et al. 1970. Effect of storage of Mueller Hinton Agar plates on zone sizes for antimicrobial testing. Appl. Microbiol.; 30:203.
2. Mueller, J.H. and J. Hinton. 1941. A protein-free medium for primary isolation of the Gonococcus and Meningococcus Proc. Soc. Exp. Diol. and Med; 48:330-333.
3. Performance Standards for Antimicrobial Disk Susceptibility Tests . M2-A. Clinical and Laboratory Standards Institute (CLSI – formerly NCCLS), Wayne, PA.
4.  Haltiner, R.C., P.C. Migneault, and R.G. Roberston. 1980. Incidence of Thymidine-Dependent Enterococci Detected on Mueller-Hinton Agar with Low Thymidine Content. Anti. Ag. and Chemo.; 18(3): 365-368.
5. Performance Standards for Antimicrobial Susceptibility Testing. M100. Clinical and Laboratory Standards Institute (CLISI), Wayne, PA.
6. Ryan, K.J., et al. 1970. Disk sensitivity testing. Hosp. Prac.; 5:91-100.
7. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI – formerly NCCLS), Wayne, PA.
8. Barry and Fay. 1973. Am. J. Clin. Pathol.; 50:196.
9. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.
10. Performance Standards for Antimicrobial Susceptibility Testing. M100. Clinical and Laboratory Standards Institute (CLISI), Wayne, PA.