Mueller Hinton Agar
Intended Use:
Recommended for determination of susceptibility of microorganisms to antimicrobial agents isolated from clinical samples.
Composition**
Ingredients Gms / Litre
HM infusion B from # 300.000
Acicase ## 17.500
Starch 1.500
Agar 17.000
Final pH ( at 25°C) 7.3±0.1
Directions
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 (1). 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
(2). 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
reasons:
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 (4).
Kirby-Bauer et al recommended this medium for performing antibiotic susceptibility tests using a single disc of high concentration (5). WHO Committee on Standardization of Susceptibility Testing has accepted Mueller Hinton Agar for determining the susceptibility of microorganisms because of its reproducibility (6). 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 (7,1,2). 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 (4). 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 (4,8).
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 (9).
Type of specimen
Clinical samples : Isolated microorganisms from urine , stool etc.
Specimen Collection and Handling
For clinical samples follow appropriate techniques for handling specimens as per established guidelines (3,5).
After use, contaminated materials must be sterilized by autoclaving before discarding.
Warning and Precautions
In Vitro diagnostic use only. For professional 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.
Limitations
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.
Quality Control
Appearance
Cream to yellow homogeneous free flowing powder.
Gelling
Firm, comparable with 1.7% agar gel.
Colour and Clarity of prepared medium
Light amber coloured clear to slight opalscent gel froms in Petri plates.
Reaction
Reaction of 3.8% w/v aqueous solution at 25°C. pH : 7.3±0.1
pH: 7.20-7.40
Cultural Response
Cultural characteristics observed after incubation at 30-35°C for 18 -24 hours for bacterial cultures.
For testing S. pneumoniae : The medium was supplemented with 5% Sheep blood and incubated at 35°C for 16-18 hours at
5% CO2 .
For testing H. influenaze : The medium was supplemented with 5g/l of Yeast extract & 2 vials /l of Haemophilus Growth
Supplement (FD117 containing 15 mg/l of Haematin + 15 mg/l of NAD) and incubated at 35°C for 20-24 hours at 5% CO2.
Antibiotic Sensitivity test
Various discs were tested for standard ATCC strains and zone of inhibition were measured after an incubation 30-35°C for
18 hours. (As per the latest CLSI Protocol M6 & Standards as per the current CLSI M100).
Thymine/Thymidine Content
# The zones for these discs are indicative of the Thymine/Thymidine content of the medium.
Divalent Cation Content
$ The zones for these discs are indicative of the Divalent Cation content of the medium.
Storage and Shelf Life
Store between 10-30°C in a tightly closed container and the prepared medium at 20-30°C. Use before expiry date on
the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition Seal the container tightly after use.
Product performance is best if used within stated expiry period.
Disposal
User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow
established laboratory procedures in disposing of infectious materials and material that comes into contact with clinical
sample must be decontaminated and disposed of in accordance with current laboratory techniques.
