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

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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.

Mueller-Hinton agar (MHA): Introduction, Composition, Principle, Preparation Requirements, Testing Procedure, Colony Characteristics, Uses, Keynotes, and MHA Footages
Fig. Freshly prepared Mueller-Hinton Agar (MHA) without inoculation of microbes

Composition of MHA

IngredientsGms / Litre
Beef infusion solids2.0
Starch1.5
Casein hydrolysate17.5
Agar17.0
Distilled water (D/W)1000 ml
Final pH at 25°C7.3 +/- 0.2
Table: Composition of Mueller-Hinton agar (MHA)

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)
Table:Control strains growth status

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)

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

Measuring Zone of Inhibition (ZOI) for antimicrobial disks

Measuring Zone of Inhibition (ZOI) for antimicrobial disks to say whether the organism is susceptible, intermediate, or resistant.
Fig. Measuring Zone of Inhibition (ZOI) for antimicrobial disks to say whether the organism is susceptible, intermediate, or resistant.

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

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

MIC of Vancomycin Determination by E-strip for Staphylococcus aureus

MIC of Vancomycin Determination by E-strip for Staphylococcus aureus
Fig. MIC of Vancomycin Determination by E-strip for Staphylococcus aureus

Pseudomonas aeruginosa Antimicrobial Sensitivity Testing (AST) Pattern

Pseudomonas aeruginosa Antimicrobial Sensitivity Testing (AST) Pattern
Fig. Pseudomonas aeruginosa Antimicrobial Sensitivity Testing (AST) Pattern

Enterobacteriaceae (Escherichia coli) AST pattern

Enterobacteriaceae (Escherichia coli) AST pattern-
Fig. Enterobacteriaceae (Escherichia coli) AST pattern

Staphylococcus Antimicrobial susceptibility Testing Pattern

Staphylococcus Antimicrobial susceptibility Testing Pattern
Fig. Staphylococcus Antimicrobial susceptibility Testing Pattern

Acinetobacter Antibiogram Pattern

Acinetobacter Antibiogram Pattern
Fig. Acinetobacter Antibiogram Pattern

Beta-hemolytic streptococci (BHS) Antimicrobial Susceptibility Testing Pattern

Beta-hemolytic streptococci (BHS) Antimicrobial Susceptibility Testing Pattern
Fig. Beta-hemolytic streptococci (BHS) Antimicrobial Susceptibility Testing Pattern

Streptococcus pneumoniae Antibiogram assaying pattern on modified MHA

Streptococcus pneumoniae Antibiogram assaying pattern on modified MHA
Fig. Streptococcus pneumoniae Antibiogram assaying pattern on modified MHA

Haemophilus Antibiogram on modified MHA

Haemophilus Antibiogram on modified MHA
Fig. 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

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  137. I do believe that a foreclosed can have a important effect on the applicant’s life. Real estate foreclosures can have a Six to decade negative influence on a debtor’s credit report. The borrower that has applied for home financing or any loans for that matter, knows that the worse credit rating is, the more tough it is to secure a decent bank loan. In addition, it could affect any borrower’s capability to find a really good place to lease or rent, if that gets to be the alternative real estate solution. Interesting blog post.

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  144. Thanks for your article. I would love to say that the very first thing you will need to conduct is check if you really need credit score improvement. To do that you will need to get your hands on a replica of your credit history. That should really not be difficult, because the government mandates that you are allowed to be issued one no cost copy of your actual credit report annually. You just have to inquire the right people. You can either look into the website with the Federal Trade Commission as well as contact one of the leading credit agencies directly.

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  146. You actually make it seem so easy along with your presentation however I to find this matter to be actually something that I believe I’d never understand. It sort of feels too complex and extremely wide for me. I’m taking a look ahead for your subsequent put up, I?ll try to get the hang of it!

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  147. The very crux of your writing while appearing agreeable in the beginning, did not really settle perfectly with me personally after some time. Somewhere within the sentences you actually managed to make me a believer but just for a very short while. I however have a problem with your leaps in assumptions and one would do nicely to fill in all those breaks. When you actually can accomplish that, I will undoubtedly end up being amazed.

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  150. The very heart of your writing while appearing agreeable at first, did not really settle very well with me personally after some time. Somewhere throughout the sentences you managed to make me a believer but just for a while. I still have got a problem with your jumps in assumptions and one would do nicely to fill in all those gaps. In the event that you actually can accomplish that, I could undoubtedly be impressed.

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  178. I have really learned some new things from the blog post. One more thing to I have observed is that in most cases, FSBO sellers may reject people. Remember, they will prefer not to use your services. But if an individual maintain a steady, professional relationship, offering help and being in contact for about four to five weeks, you will usually be capable of win a conversation. From there, a house listing follows. Many thanks

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  179. Thanks for enabling me to achieve new tips about computer systems. I also have the belief that certain of the best ways to keep your notebook computer in best condition is by using a hard plastic-type material case, as well as shell, that suits over the top of the computer. Most of these protective gear are usually model specific since they are made to fit perfectly over the natural outer shell. You can buy all of them directly from the seller, or from third party sources if they are available for your laptop, however don’t assume all laptop could have a spend on the market. Once more, thanks for your ideas.

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  180. I do like the manner in which you have presented this particular challenge plus it does give me some fodder for thought. However, coming from what I have personally seen, I only hope when the commentary pile on that people today keep on point and in no way embark on a soap box associated with the news du jour. Yet, thank you for this fantastic point and whilst I can not really concur with it in totality, I value the viewpoint.

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  194. A powerful share, I just given this onto a colleague who was doing a bit evaluation on this. And he in fact bought me breakfast because I found it for him.. smile. So let me reword that: Thnx for the treat! However yeah Thnkx for spending the time to discuss this, I really feel strongly about it and love reading extra on this topic. If potential, as you turn into experience, would you thoughts updating your weblog with more particulars? It’s highly useful for me. Large thumb up for this blog submit!

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  196. The crux of your writing while sounding reasonable initially, did not settle very well with me personally after some time. Somewhere throughout the sentences you actually were able to make me a believer but only for a short while. I still have got a problem with your jumps in logic and you might do nicely to fill in all those gaps. In the event you can accomplish that, I will definitely be amazed.

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  199. One important issue is that when you find yourself searching for a education loan you may find that you’ll want a cosigner. There are many conditions where this is true because you might discover that you do not have a past credit rating so the mortgage lender will require you have someone cosign the money for you. Interesting post.

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  215. An additional issue is really that video gaming became one of the all-time most important forms of entertainment for people of all ages. Kids enjoy video games, plus adults do, too. The actual XBox 360 is just about the favorite gaming systems for many who love to have a huge variety of video games available to them, along with who like to learn live with other people all over the world. Thank you for sharing your ideas.

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  221. In my opinion that a foreclosures can have a major effect on the borrower’s life. Property foreclosures can have a Several to few years negative affect on a applicant’s credit report. The borrower who may have applied for home financing or almost any loans for that matter, knows that the worse credit rating can be, the more challenging it is to acquire a decent mortgage loan. In addition, it may affect any borrower’s capacity to find a respectable place to lease or rent, if that results in being the alternative homes solution. Thanks for your blog post.

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  230. You really make it seem so easy with your presentation but I find this topic to be really something that I think I would never understand. It seems too complicated and extremely broad for me. I am looking forward for your next post, I?ll try to get the hang of it!

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  250. I have discovered that charges for internet degree specialists tend to be an awesome value. For example a full Bachelor’s Degree in Communication in the University of Phoenix Online consists of Sixty credits with $515/credit or $30,900. Also American Intercontinental University Online makes available Bachelors of Business Administration with a full program feature of 180 units and a tariff of $30,560. Online studying has made taking your college degree been so cool because you could earn your current degree through the comfort of your dwelling place and when you finish working. Thanks for other tips I have really learned through the website.

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  273. Thanks for your handy post. Through the years, I have come to understand that the actual symptoms of mesothelioma are caused by the actual build up connected fluid between lining of the lung and the chest muscles cavity. The disease may start in the chest region and distribute to other body parts. Other symptoms of pleural mesothelioma include weight reduction, severe breathing trouble, vomiting, difficulty eating, and puffiness of the face and neck areas. It should be noted that some people living with the disease will not experience any serious symptoms at all.

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  288. An added important issue is that if you are a mature person, travel insurance for pensioners is something you need to really consider. The older you are, the more at risk you’re for having something bad happen to you while overseas. If you are not covered by quite a few comprehensive insurance plan, you could have several serious challenges. Thanks for expressing your guidelines on this blog.

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  385. Thanks for your intriguing article. One other problem is that mesothelioma is generally the result of the breathing of materials from asbestos, which is a positivelly dangerous material. It truly is commonly noticed among laborers in the construction industry who may have long experience of asbestos. It is also caused by living in asbestos covered buildings for a long period of time, Genetic makeup plays a crucial role, and some individuals are more vulnerable to the risk when compared with others.

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