Müller-Hinton agar principle and antimicrobial susceptibility testing

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Müller-Hinton agar formulation was originally developed as a simple, transparent agar medium for the growth of pathogenic Neisseria species by John Howard Mueller and Jane Hinton at Harvard.It is now used as a test medium for antimicrobial susceptibility testing .It is recommended for the diffusion of antimicrobial agents impregnated on paper disc through the agar gel . Mueller Hinton supports the growth of most non-fastidious bacterial pathogen. Kirby-Bauer et al also recommended this medium for performing antibiotic susceptibility tests using a single disc of high concentration.

Müller-Hinton agar is not appropriate for assay by disc diffusion method of  antibiotic susceptibility tests with slow growing microorganisms, anaerobes and capnophiles because with slow growing organisms, increased incubation may cause deterioration of diffusing antibiotic and produce unprecise readings

Muller Hinton has a few properties that makes it  excellent for antibiotic use. First of all

  • it is a nonselective, nondifferential medium. This means that almost all organisms plated on it will grow.
  • Muller Hinton  contains starch. Starch is known to absorb toxins released from bacteria, so that they cannot interfere with the antibiotics.
  • it is a loose agar. This allows for better diffusion of the antibiotics than most other plates. A better diffusion leads to a truer zone of inhibition.

Mueller Hinton Agar has also been selected by the Clinical and Laboratory Standards Institute (CLSI) for several reasons which are

  • It demonstrates good batch-to-batch reproducibility for susceptibility testing
  • It is low in sulfonamide, trimethoprim and tetracycline inhibitors
  • It supports the growth of most non-fastidious bacterial pathogens
  • Many data and much experience regarding its performance have been recorded

Principle of Mueller Hinton Agar

  • The HM infusion B from and acicase provide nitrogenous compounds, sulphur,carbon, and other essential nutrients needed by microorganism.
  • Starch in the media acts as a protective colloid against toxic substances present in the medium and the  hydrolysis of starch yields dextrose, which serves as a source of energy for the organism.
  • These ingredients are selected for low thymine and thymidine content as determined by Minimum Inhibitory Concentration (MIC)  values for Enterococcus faecalis with sulfamethoxazole trimethoprim (SXT) antibiotics.
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Composition of Mueller Hinton Agar

HM infusion B from 300.000
Starch 1.500
Acicase 17.500
Final pH ( at 25°C)7.4±0.1

How to Prepare Mueller Hinton Agar

The preparation of Mueller Hinton Agar depends on each laboratory workload or the numbers of culture plates available. Anyway, note that a standard Petri dish is equivalent to 20ml when poured completely.

  • Suspend 38.0 grams of Mueller Hinton powder in 1000 ml distilled water.
  • Heat to boiling to dissolve the medium completely
  • Now sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes
  • Cool the media to 45-50°C.
  • Mix well and pour into sterile Petri plates.

NB: It is also advisable to incubate a petri dish for 24 hour for quality control in order to avoid wastage of resources and ease early detection of contamination after the preparation

Appearance of  Mueller Hinton Agar

The media is Light amber coloured clear to slightly opalescent gel forms in Petri plates

Type of Inoculums

Clinical samples such as pure cultures isolated from

  • Urine
  • Stool
  • blood
  • Water etc

Culture Procedure of Mueller Hinton Agar

  • Pick isolated colonies with an inoculating needle and completely streak the entire surface of Mueller hinton agar plate
  • incubated at 30-35ºC. for 18-24 hours

Cultural Response after Incubation

Cultural characteristics observed after incubation at 30-35°C for 18 -24 hours for bacterial cultures.

  • For testing Streptococcus pneumoniae : The medium was supplemented with 5% Sheep blood and incubated at 35°C for 16-18 hours at 5% CO2
  • Haemophilus influenzae : 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 inhibitory zones

OrganisimsAntibioticssStandard Zone of Inhibtion
Escherichia coli ATCC 25922 (00013*)Cephalothin CEP 30mcg29-37 mm
Chloramphenicol C 30 mcg21-27 mm
Co-Trimoxazole COT 25 23-29 mm
Cefotaxime CTX 30 mcg29-35 mm
Gentamicin GEN 10 mcg19-26 mm
Sulphafurazole SF 300 mcg15-23 mm
Staphylococcus aureus subsp. aureus ATCC 25923 (00034*)Co-Trimoxazole COT 25# 20 mm (Clear zone) >=20 mm
Erythromycin E 15 mcg22-30 mm
Linezolid LZ 30 mcg25-32 mm
Oxacillin OX 1mcg18-24 mm
Pristinomycin RP 15 mcg21-28 mm
Tetracycline TE 30 mcg $18-25 mm
Ciprofloxacin CIP 5mcg22-30 mm
Cefoxitin CX 30 mcg23-29 mm
Pseudomonas aeruginosa ATCC 27853 (00025*)Ceftazidime CAZ 30 mcg22-29 mm
Ciprofloxacin CIP 5mcg 30-40 mm
Tobramycin TOB 10 mcg 19-25 mm
Amikacin AK 30 mcg 18-26 mm
Aztreonam AT 3mcg23-29 mm
Cephotaxime CTX 30 mcg18-22 mm
Gentamicin GEN 10 mcg16-21 mm
Imipenem IPM 10 mcg 20-28 mm
Piperacillin PI 100 mcg12-18 mm
Escherichia coli ATCC 35218Amoxyclav AMC 30 mcg18-24 mm
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Piperacillin/Tazobactam PIT 100/10 mcg24-30 mm
Ticarcillin TI 75 mcg6 mm
Ticarcillin/Clavulanic acid TCC 75/10mcg20-28 mm
Ampicillin AMP 10 mcg16-22 mm
Ampicillin/Sulbactam A/S 10/10 mcg29-37 mm
Enterococcus faecalis ATCC 29212 (00087*)Trimethoprim TR 5 mcg20 mm
Vancomycin VA 30 mcg17-21 mm
Staphylococcus aureus subsp. aureusATCC 43300 (MRSA) (00211*) Oxacillin OX 1 mcgVery Hazy to No Zone

Limitations of Mueller Hinton

Mueller Hinton  is recommended for susceptibility testing of pure cultures only

  • Inoculum density (Quantity or size of inoculum) may affect the zone size therefore heavy inoculum may result in smaller zones or too less inoculum may result in bigger zones.
  • Fastidious organisms may not grow on this medium and may require supplementation of blood for enrichment.
  • Fastidious anaerobes may not grow on this medium.
  • As antimicrobial susceptibility is carried with antibiotic disc, proper storage of the disc is desired which may affect the potency of the disc.

Note that Under certain circumstances, the in vitro results of antibiotic susceptibility may not show the same in vivo

Limitations of Mueller Hinton

Mueller Hinton  is recommended for susceptibility testing of pure cultures only

Note that Under certain circumstances, the in vitro results of antibiotic susceptibility may not show the same in vivo


  • Mueller J. H. and Hinton J., 1941, Proc. Soc. Exp. Biol. Med., 48:330.
  • National Committee for Clinical Laboratory Standards, 2000, Approved Standard: M7-A5. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that grow aerobically, 5th Ed., NCCLS, Wayne, Pa.
  • NCCLS Approved Standard: ASM-2, 1979, Performance Standards for Antimicrobic disc Susceptibility Tests, 2nd Ed., National Committee for Clin. Lab. Standards.
  • Bauer A. W., Kirby W. M., Sherris J. L. and Turck M., 1966, Am. J. Clin. Pathol., 45:493.
  • Present Status and Future Work, WHO Sponsored collaborative study, Chicago, Oct. 1967.
  • Ericsson H. M. and Sherris J. L., 1971, Acta Pathol. Microbiol., Scand. Sect B Suppl., 217:1.
  • National Committee for Clinical Laboratory Standards, 1986, Proposed Standards, M6-P, NCCLS, Villanova, Pa. 8. MacFaddin J. F., 1985, Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, Vol. 1, Williams and Wilkins, Baltimore
  • Murray P. R., Baron J. H., Pfaller M. A., Jorgensen J. H. and Yolken R. H., (Ed.), 2003, Manual of Clinical Microbiology, 8th Ed., American Society for Microbiology, Washington, D.C. ,,
  • Isenberg, H.D. Clinical Microbiology Procedures Handb0ook. 2 nd Edition.
  • Jorgensen,J.H., Pfaller , M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.
Also read  Pseudomonas aeruginosa : Morphology,Diagnosis and Cultural characteristics

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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.

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