Mueller-hintot (MH) agar plate preparation assay

Summary

Mueller-hinton (MH) agar can be applied to (1) antibiotic susceptibility testing; (2) isolation and maintenance of Neisseria and Moraxella catarrhalis. MH agar plate, special for drug susceptibility test, this experiment is from Mudanjiang Medical College Undergraduate 5-year laboratory guide for testing majors

Operation method

Dehydrated media preparation method

Principle

Mueller-Hinton agar was developed by John Howard Mueller and Jane Hinton of Harvard University as a culture of gonococci and meningococci, and the method was published in 1941. It usually contains: beef extract: 2.0 grams; acid hydrolysis product of casein: 17.5 grams; starch: 1.5 grams; agar: 17.0 grams.

Materials and Instruments

Beef extract
Acid hydrolysis products of casein Starch Agar Purified water
Petri dishes

Move

1. Suspend 38 grams of medium (or components listed above) in 1 liter of purified water.

2. Mix thoroughly.

3. Heat and boil with frequent stirring for 1 minute to completely dissolve the components.

4. Autoclave at 121°C for 15 minutes.

5. Cool to 45°C

6. Pour cooled Mueller Hinton Agar into a sterile petri dish on a horizontal level surface to obtain a uniform depth.

(Note: Plates must be poured to a depth of 4 mm (for 100 mm plates, approximately 25 ml of liquid agar, for 150 mm plates, approximately 60 ml of liquid agar, but in any case, measure to a depth of 4 mm). Plates that are too shallow will produce falsely sensitive results because the anti-microbial compound will diffuse further than it should, producing a larger zone of inhibition. Conversely, casting the plate to a depth of > 4 mm will lead to anti-false results).

7. Cure at room temperature.

8. Check the prepared Mueller Hinton agar to ensure that the final pH is 7.3 ± 1 at 25°C.

(Note: If pH < 7.2, some drugs appear to lose potency (aminoglycosides, quinolones, macrolides), while others appear to have excess activity (tetracyclines). The opposite may occur if pH > 7.4).

9.Prepared media may be stored at temperatures from 4 to 8°C. Mueller-Hinton agar is stable for approximately 70 days from the date of preparation.

Caveat

1.Mueller Hinton Agar should be tested at least weekly with known strains of organisms to verify that the medium and culture are working as intended.

2. Mueller Hinton agar medium with 5% sheep blood can be used to determine antimicrobial drug susceptibility (e.g., Streptococcus pneumoniae, Neisseria meningitidis).

3. 5% sheep's blood and nicotinamide adenine dinucleotide can also be added when performing drug susceptibility testing on Streptococcus spp. species. This type is also commonly used for drug sensitivity testing of Campylobacter.

Common Problems

Mueller-hinton (MH) agar is well suited for antibiotic use. First, it is a non-selective, non-discriminatory medium. This means that almost any organism plated on it will grow. In addition, it contains starch. Starch is known to absorb toxins released by bacteria, so they do not interfere with antibiotics. Secondly, it is a loose agar. This allows for better diffusion of the antibiotic compared to most other plates. Better diffusion leads to truer zones of inhibition.

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