Mueller Hinton Agar is used for cultivation of Neisseria and for determination of susceptibility of microorganisms to antimicrobial agents.
Ingredients Gms / Litre
HM infusion B from # 300.000
Acicase ## 17.500
Final pH ( at 25°C) 7.3±0.1
**Formula adjusted, standardized to suit performance parameters
# – Equivalent to Beef infusion from
## – Equivalent to Casein acid hydrolysate
Suspend 38.0 grams in 1000 ml purified/ distilled water. Heat to boiling to dissolve the medium completely.
Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool to 45-50°C. Mix well and pour into sterile
Petri plates. Note: The performance of this batch has been tested and standardised as per the current CLSI
(formerly, NCCLS) document M6-protocols for Evaluating Dehydrated Mueller Hinton Agar.
Principle And Interpretation
The Mueller Hinton formulation was originally developed as a simple, transparent agar medium for the cultivation of
pathogenic Neisseria species (6). Other media were subsequently developed that replaced the use of Mueller Hinton Agar for
the cultivation of pathogenic Neisseria species, but it became widely used in the determination of sulfonamide resistance of
gonococci and other organisms. Mueller Hinton Agar is now used as a test medium for antimicrobial susceptibility testing
(9). Mueller Hinton Agar is recommended for the diffusion of antimicrobial agents impregnated on paper disc through an
agar gel as described in CLSI Approved Standard (3). Mueller Hinton Agar has been selected by the CLSI for several
i. It demonstrates good batch-to-batch reproducibility for susceptible testing.
ii. It is low in sulfonamide, trimethoprim and tetracycline inhibitors.
iii. It supports the growth of most non-fastidious bacterial pathogens and
iv. Many data and much experience regarding its performance have been recorded (7).
Kirby-Bauer et al recommended this medium for performing antibiotic susceptibility tests using a single disc of high
concentration (4). WHO Committee on Standardization of Susceptibility Testing has accepted Mueller Hinton Agar for
determining the susceptibility of microorganisms because of its reproducibility (11). Mueller Hinton Agar with 5% sheep
blood and Mueller Hinton Agar with Hemoglobin have been recommended for antimicrobial susceptibility testing of
Streptococcus pneumoniae and Haemophilus influenzae.
HM infusion B from and acicase provide nitrogenous compounds, carbon, sulphur and other essential nutrients. Starch acts
as a protective colloid against toxic substances present in the medium. Starch hydrolysis yields dextrose, which serves as a
source of energy. These ingredients are selected for low thymine and thymidine content as determined by MIC values for
Enterococcus faecalis with sulfamethoxazole trimethoprim (SXT).
The Kirby-Bauer procedure is based on agar diffusion of antimicrobial substances impregnated on paper discs. This method
employs disc with a single concentration of antimicrobial agent and the zone diameters observed are correlated with minimum
inhibitory concentration (MIC) values (2,6,9). A standardized suspension of the organism is swabbed over the entire surface
of the medium. Paper discs impregnated with specific amounts of antimicrobial agents are then placed on the surface of the
medium, incubated and zones of inhibition around each disc are measured. The susceptibility is determined by comparing
with CLSI standards (7). The various factors, which influence disc diffusion susceptibility tests, are agar depth, disc potency,
inoculum concentration, pH of the medium and beta-lactamase production by test organisms (7,8).
Mueller Hinton Agar is not appropriate for assay by disc diffusion method with slow growing organisms, anaerobes and
capnophiles. With slow growing organisms, increased incubation may cause deterioration of diffusing antibiotic and produce
unprecise readings (5).
Type of specimen
Clinical samples : Pure cultures isolated from urine , stool, blood etc.
Specimen Collection and Handling
For clinical samples follow appropriate techniques for handling specimens as per established guidelines (3,4).
Warning and Precautions
In Vitro diagnostic use only. Read the label before opening the container. Wear protective gloves/protective clothing/
eye protection/face protection. Follow good microbiological lab practices while handling specimens and culture. Standard
precautions as per established guidelines should be followed while handling clinical specimens. Safety guidelines may be
referred in individual safety data sheets.
1. This medium is recommended for susceptibility testing of pure cultures only.
2. Inoculum density may affect the zone size. Heavy inoculum may result in smaller zones or too less inoculum may result
in bigger zones.
3. Fastidious organisms may not grow on this medium and may require supplementation of blood.
4. Fastidious anaerobes may not grow on this medium.
5. As antimicrobial susceptibility is carried with antibiotic disc, proper storage of the disc is desired which may affect the
potency of the disc.
6. Under certain circumstances, the in vitro results of antibiotic susceptibility may not show the same in vivo.
Performance and Evaluation
Performance of the medium is expected when used as per the direction on the label within the expiry period when stored
at recommended temperature.