PREPARATION OF MUELLER HINTON AGAR MEDIA (MHA) IN LABORATORY

The well-known solid media for the Antibiotic Sensitivity Test a.k.a. Antibiotic Susceptibility Test was developed by John Howard Mueller and Jane Hinton in 1941 and named as Mueller Hinton Agar. This Mueller Hinton agar (MHA) was a protein-free medium for isolating the pathogenic strains of Neisseria.

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).

Later, A. L. Barry & G. D. Fay investigated the effects of altering the depth of plated Mueller Hinton Agar medium on disk diffusion testing, and determined a standardized depth of the medium in the plate to be approximately 4 millimeters is sufficient to avoid false results.

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 OF MUELLER HINTON AGAR  (MHA) MEDIUM

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. The WHO Committee on Standardization of Susceptibility Testing has accepted Mueller Hinton Agar for determining the susceptibility of microorganisms because of its reproducibility.

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.

Meat infusion or Beef extract and Casein acid hydrolysate present in the MHA medium provides nitrogenous compounds, carbon, sulfur and other essential nutrients to the bacterium.

Starch, present in MHA medium acts as a protective colloid against the toxic substances present in the medium. Also, the hydrolysis of Starch yields the dextrose, which serves as the source of energy.

Agar-Agar – It is often called as Agar, is a complex polysaccharide, a carbohydrate consisting of 3, 6-anhydro-L-galactose and D-galactopyranose, free of nitrogen, produced from various red-purple algae belonging to Gelidium, Gracilaria, Gigastina etc. It liquefies on heating to 96 °C and hardens into a jelly on cooling 40-45 °C.

These ingredients are selected for low thymine and thymidine content as determined by MIC values for Enterococcus faecalis with sulfamethoxazole-trimethoprim (SXT).

The methodology explained in this article is based on HiMedia Labs Mueller Hinton Agar Medium which you can check here

MATERIALS REQUIRED FOR THE PREPARATION OF MUELLER HINTON AGAR (MHA) MEDIUM

• 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
• Butter Paper

PROCEDURE FOR THE PREPARATION OF MUELLER HINTON AGAR (MHA) MEDIUM

⇒ Weigh the quantity of Beef Extract, Casein acid hydrolysate, Agar and Starch using the weighing scale for 1000 ml of Mueller Hinton Agar Medium as follows:

COMPONENTS	QUANTITY (in grams)
Beef Extract	2
Casein acid hydrolysate	17.5
Starch	1.5
Agar-Agar	17

⇒ Put the butter paper on the weighing scale and transfer the required quantity of Beef Extract on to the paper using the spatula. Repeat the step to obtain the required quantity of Casein Acid Hydrolysate, starch and Agar-Agar powder.

Note: Use the separate piece of butter paper to avoid the errors in measurements.

Alternatively, You can also use the commercially available MHA media powders and simply weigh the mixture of content as prescribed on the carton or box of the manufacturer. As per HiMedia, its 38 grams for 1000 ml medium.

⇒ Take a clean and dry Conical Flask/ Erlenmeyer flask.

Note: The size of the flask should be at least 1.5 times larger than the quantity of media you are preparing, for e.g. use 1500 or 2000 ml flask to prepare 1000ml of broth.

⇒ 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.

⇒ Now add the weighed quantity of Agar-Agar to the above solution.

Note: If you are using Dehydrated MHA Medium, directly add the 38 gram of powder to the Distilled water and mix thoroughly.

⇒ Mix well the content, Heat it with continuous agitation and boil it for 1-2 minutes to dissolve the constituents.

⇒ Now add more distilled water to the medium and make the volume 1000 ml and mix thoroughly to make the solution homogeneous.

⇒ 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.

Note: 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.

⇒ Mix well the content and apply the Non-absorbent cotton plug to the flask.

⇒ Autoclave the content at 121 °C and 15 psi pressure for 15 minutes.

⇒ 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.

Note: Pour the MHA media to a depth of 4 mm to avoid any errors in results (For this, approximately 25 ml of liquid MHA medium is required for 100-mm Petri plates and 60 ml of liquid MHA medium for 150-mm Petri plates). This step is crucial as the Plates that are too shallow will produce false positive susceptible results because the antimicrobial compound will diffuse further than it should have to which may create larger zones of inhibition.  Conversely, the plates poured to a depth greater than 4 mm will result in the false resistant results of the test.

⇒ The prepared MHA medium can be stored at 4 to 8°C and is stable for approximately 70 days from the date of preparation.

⇒ Use the prepared media plates to inoculate the specimen to be cultured and then place in the incubator at optimum temperature.

PRECAUTIONS TO BE TAKEN WHILE PREPARING MHA MEDIUM

⇒ Carefully measure the quantity of beef extract, Casein acid Hydrolysate, Starch and Agar-Agar as per the quantity of medium to be prepared.

⇒ Maintain the pH of the medium carefully. A single extra drop can change the pH of the solution.

⇒ Store the Medium at low temperature in dust and contamination free environment for later use.

⇒ Acid and Alkali are corrosive to the skin, handle with care.

⇒ Carefully pour the liquid MHA medium in the Petri plates so that its depth should not be less or more than 4 mm to get the error free results.

Check Out the Preparation of Nutrient Agar Medium in Laboratory