Mueller-Hinton agar (MHA): Introduction, Composition, Principle, Preparation Requirements, Testing Procedure, Colony Characteristics, Uses, Keynotes, and MHA Footages

History and Introduction of Mueller-Hinton Agar (MHA)

The name Mueller- Hinton agar(MHA) is from the surname of co-developers microbiologist John Howard Mueller and veterinary scientist Jane Hilton at Harvard University as a culture for gonococcus and meningococcus, who published the method in 1941. It is a non-selective and non-differential medium. In 1966, Bauer et al. adopted MHA for antimicrobial susceptibility testing. Later 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 anaerobes). It is also used to isolate and maintain Neisseria and Moraxella species. Use of media other than Mueller-Hinton agar may result in erroneous results. Now is recommended by various institutions like the American Society of Microbiology (ASM), Clinical, Laboratory Standards Institute (CLSI), and  FDA, World Health Organization (WHO)  for antibiotic susceptibility testing of bacteria.

Composition of MHA

Ingredients	Gms / Litre
Beef infusion solids	2.0
Starch	1.5
Casein hydrolysate	17.5
Agar	17.0
Distilled water (D/W)	1000 ml
Final pH at 25°C	7.3 +/- 0.2

Principle of MHA

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

Properties of  MHA

Due to the following properties, Muller- Hinton agar uses for routine antibiotic sensitivity testing (AST)-

1. Defined medium concentration.
2. Identical from batch to batch.
3. No enrichment or selective in nature.
4. It supports the growth of nearly all types of pathogens.
5. Constituents are not antagonistic to any drugs.
6. Well-adjusted pH(7.3) for all types of antibiotic susceptibility tests.
7. The Agar-broth version has the same formula.
8. Isotonic
9. Appropriate for adding blood and serum.
10. Adjusted mineral salts.
11. It contains starch and it is known to absorb toxins released from bacteria so that they cannot interfere with antibiotics.

Note: Mueller- Hinton agar (MHA) is available in the market by commercial suppliers both ready-made form and dehydrated medium. Be sure to prepare the media according to the manufacturer’s directions.

Requirements for Preparation of Muller- Hinton Agar

• Dehydrated Mueller-Hinton agar powder
• Electronic balance
• Distilled water
• Autoclave
• Measuring cylinder
• Conical flask
• Hot plate
• Petri plates or dish
• Sheep blood ( optional  for Mueller Hinton blood agar and Mueller Hinton chocolate agar)
• Control strains for testing the quality of prepared media

Preparation of  Mueller-Hinton agar (MHA)

1. Suspend 38 g of medium in 1 liter of distilled water and mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the components.
3. Autoclave at 121°C for 15 minutes.
4. Cool to 45°C
5. Pour cooled Mueller Hinton Agar into sterile Petri dishes on a level, horizontal surface to give uniform depth.
6. Check the prepared Mueller Hinton agar to ensure the final pH is 7.3 ±1 at 25°C.
7. Prepared media can be stored at 4 – 8°C. Mueller-Hinton agar is stable for approximately 70 days from the date of preparation but should be protected from direct light.

Storage and Shelf life of MHA

• Store at 2-8ºC  and away from direct light.
• Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), or contamination.
• The product is light and temperature sensitive; protects from light, excessive heat, moisture, and freezing.

Test Requirements for Mueller-Hinton agar (MHA)

• Pure cultures of the organism isolated from the clinical specimen
• Muller Hinton Agar
• Antibiotic Disks
• Turbidity Standard
• Swabs

Test procedure (specimen/organism inoculation)

1. Mostly Muller Hinton agar( MHA) is used in this antibiotic susceptibility test.
2. Emulsify 2-3 colonies in sterile saline matching the turbidity that standard (0.5 McFarland).
3. Place a sterile cotton swab in the bacterial suspension and remove the excess fluid by pressing and rotating the cotton against the inside of the tube above the fluid level.
4. The swab is streaked in three directions over the surface of the MHA  to obtain uniform growth.
5. Allow the plates to dry for 10-15 minutes.
6. Using sterile forceps or a suitable disk dispenser, place paper disks impregnated with a fixed concentration of an antibiotic, on the surface agar plates having a distance disc to disc 25 mm while the plate border to disk 15 mm.
7. Incubate the plates at 37°C for 24 hours.
8. Following overnight incubation, measure the diameter of the zone of inhibition in millimeters (mm) around each disc.

Result -Interpretation

• Using a standard table of antibiotic susceptibilities, determine if the strain is resistant, intermediate, or susceptible to the antibiotics tested.

Modifications of Mueller-Hinton agar (MHA)

• Mueller Hinton agar medium supplemented with 5% sheep blood and nicotinamide adenine dinucleotide (NAD) is recommended for determining the antimicrobial susceptibility of Streptococcus species, Neisseria, and 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 the following ingredients: 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 interfere with the activity of aminoglycoside antibiotics. To overcome this problem MHA No. 2 is manufactured to contain low levels of thymine, thymidine, and controlled levels of calcium and magnesium.

Quality Control

Prepared Appearance: Light yellow to amber-colored clear to very slightly opalescent gel forms in Petri plates.Cultural Response: Cultural characteristics were observed after incubation at 35°C-37°C for 18-24 hours.

Organism	Growth
Escherichia coli (ATCC 25922)	Good
H. influenzae (ATCC 49247)	Good (on Mueller Hinton chocolate agar)
Neisseria gonorrhoeae (ATCC 49226)	Good
Pseudomonas aeruginosa (ATCC 27853)	Good
Staphylococcus aureus (ATCC 25923)	Good
Enterococcus faecalis (ATCC 29212)	Good
S.  pneumoniae (ATCC 6305)	Good (on Mueller Hinton blood agar)

Other Media Uses for AST

1. Bacteria: Aerobic and facultative anaerobes -Sensitest agar ( most common use outside of USA), Diagnostic sensitivity agar.
2. Wilkins-Chalgren agar is recommended for testing anaerobic bacteria.
3. For antiviral susceptibility testing, RPMI 1640 medium is used.
4. Fungal susceptibility testing media are  RPMI 1640 and Muller Hinton Agar + 2% Glucose and 0.5 mg/ml Methylene Blue Dye.
5. Mycobacterium tuberculosis sensitivity testing media are Middle brook 7H- 9 ( broth + glycerol), 10 and 11 agar form.

Applications of  MHA

1. MHA is the common medium to use for routine antibiotics susceptibility testing for non-fastidious bacteria (both aerobes and facultative anaerobes).
2. It is the standard medium for AST by the Bauer-Kirby method and its performance is specified by the CLSI.
3. It is also an 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 a useful medium for the 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. pneumoniae) and 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.

Limitations of  MHA

• It is recommended medium for susceptibility testing of pure cultures only.
• Inoculum density may affect the size of the 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 the supplementation of blood.
• Fastidious anaerobes may not grow on this medium.
• As antimicrobial susceptibility testing is carried out with the antibiotic disc, proper storage of the disc is desired which may affect its potency of the disc.

Some keynotes on MHA

1. The plates must be poured to a depth of 4 mm (approximately 25 ml of liquid agar for 100-mm plates and 60 ml of liquid agar for 150-mm plates, but in any case to a measured depth of 4 mm).  Plates that are too shallow will produce false susceptible results as the antimicrobial compound will diffuse further than it should, creating larger zones of inhibition.  Conversely, plates poured to a depth >4 mm will result in false resistant results.
2. If the pH is <7.2 certain drugs will appear to lose potency (aminoglycosides, quinolones, macrolides), while other agents may appear to have excessive activity (tetracycline). If the pH is >7.4, the opposite results may occur.
3. Mueller Hinton agar should be tested with known strains of the organism at least weekly in order to verify that the media and disks are working as expected.
4. It is also applied in  E-test for MIC determination.
5. It also uses antibiotic sensitivity testing by Stoke’s method in which both test and control organisms are inoculated on the same plates.
6. WHO recommended Kirby-Bauer method of AST has the following features-

• Test and control are placed separately.
• Discs are placed 15 mm from the edge of the plate and 25 mm from the disc to the disc distance.
• The depth of MHA  should be  4 mm.
• The diameter of the antibiotic disc should be 6 mm.
• Therefore a number of discs are applied that fully depend on the size of the Petri plate or dish.
• In a 9 cm agar plate, 6 discs are applied whereas 80 mm plate only 5 discs used.

MHA Footages

Applying antimicrobial disks on Muller-Hinton agar (MHA) for antimicrobial susceptibility testing (AST)

Measuring Zone of Inhibition (ZOI) for antimicrobial disks

Apply E-Test strip on Muller-Hinton agar (MHA) to determine the MIC

MIC of Vancomycin Determination by E-strip for Staphylococcus aureus

Pseudomonas aeruginosa Antimicrobial Sensitivity Testing (AST) Pattern

Enterobacteriaceae (Escherichia coli) AST pattern

Staphylococcus Antimicrobial susceptibility Testing Pattern

Acinetobacter Antibiogram Pattern

Beta-hemolytic streptococci (BHS) Antimicrobial Susceptibility Testing Pattern

Streptococcus pneumoniae Antibiogram assaying pattern on modified MHA

Haemophilus Antibiogram on modified MHA

Further Readings

1. https://www.thermofisher.com/order/catalog/product/R454082#/R454082
2. http://himedialabs.com/TD/M173.pdf
3. https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/Datasheet/1/70191dat.pdf
4. Murray P. R., Baron J. H., Pfaller M. A., Jorgensen J. H. and Yolken R. H., (Ed.), 2007, Manual of Clinical
5. Microbiology, 9th Ed., American Society for Microbiology, Washington, D.C.
6. Mueller J. H. and Hinton J., 1941, Proc. Soc. Exp. Biol. Med.,48:330
7. http://www.tulipgroup.com/MicroExpress/Accumix/PackInsert/Dehydrated%
8. https://www.researchgate.net/post/Why_muellerhinton_agar_is_used_in_routine_antibiotic_susceptibility_testing
9. http://www.himedialabs.com/HML/images/literature/pdf/100000027/68.pdf
10. https://en.wikipedia.org/wiki/Mueller-Hinton_agar