BBL™ Mueller Hinton Broth

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8806691 • Rev. 02 • June 2012 U
QUALITY CONTROL PROCEDURES
I INTRODUCTION
Mueller Hinton Broth is a general-purpose medium that may be used in the cultivation of a wide variety of fastidious and
nonfastidious microorganisms.
II PERFORMANCE TEST PROCEDURE
1. Inoculate representative samples with the cultures listed below.
a. Inoculate 1 µL (0.001 mL) from a 4 – 5 h culture of Trypticase™ Soy Broth diluted to yield 106-107 CFU/mL.
b. Incubate at 36 ± 1°C under appropriate atmospheric conditions.
2. Examine tubes after 18 – 24 h for growth.
3. Expected Results
Organisms ATCC™ Recovery
*Escherichia coli 25922 Moderate to heavy growth
*Enterococcus faecalis 29212 Moderate to heavy growth
*Staphylococcus aureus 25923 Moderate to heavy growth
*Recommended organism strain for User Quality Control.

III ADDITIONAL QUALITY CONTROL

1. Examine tubes as described under "Product Deterioration."
2. Visually examine representative tubes to assure that any existing physical defects will not interfere with use.
3. Determine the pH potentiometrically at room temperature for adherence to the specification of 7.3 ± 0.2.
4. Incubate uninoculated representative tubes at 33 – 37°C and 20 – 25°C for 72 h and examine for microbial contamination.

PRODUCT INFORMATION
IV INTENDED USE
Mueller Hinton Broth is a general-purpose medium that may be used in the cultivation of a wide variety of fastidious and
nonfastidious microorganisms. This formulation has not had its calcium and magnesium ion concentrations adjusted to make it
suitable for use in quantitative procedures for antimicrobial susceptibility testing.
V SUMMARY AND EXPLANATION
The Mueller Hinton formulation was originally developed as a simple, transparent agar medium for the cultivation of
pathogenic Neisseria.1 Other media were developed that replaced the use of Mueller Hinton Agar for the cultivation of
pathogenic Neisseria, but it became widely used in the determination of sulfonamide resistance of gonococci and other
organisms. It now is recommended as the test medium for use in antimicrobial susceptibility testing.2-3
Mueller Hinton Broth, unadjusted, has a formula similar to that of the solid medium, but without agar, for use when fluid
medium is preferred. It may be used for the general cultivation of bacteria.
VI PRINCIPLES OF THE PROCEDURE
Acid hydrolysate of casein and beef extract supply amino acids and other nitrogenous substances, minerals, some vitamins
and other nutrients to support the growth of microorganisms. Starch acts as a protective colloid against toxic substances
that may be present in the medium. Hydrolysis of the starch during autoclaving provides a small amount of dextrose, which
is a source of energy.
VII REAGENTS
Mueller Hinton Broth
Approximate Formula* Per Liter Purified Water
Acid Hydrolysate of Casein..................................................................... 17.5 g
Beef Extract................................................................................................ 3.0 g
Starch.......................................................................................................... 1.5 g
*Adjusted and/or supplemented as required to meet performance criteria.
Warnings and Precautions: For in vitro Diagnostic Use.
Tubes and bottles with tight caps should be opened carefully to avoid injury due to breakage of glass.
Observe aseptic techniques and established precautions against microbiological hazards throughout all procedures. After use,
prepared tubes, specimen containers and other contaminated materials must be sterilized by autoclaving before discarding.
Storage Instructions: On receipt, store tubes and bottles in the dark at 2 – 25°C. Avoid freezing and overheating. Do not
open until ready to use. Minimize exposure to light. Tubed and bottled media stored as labeled until just prior to use may be
inoculated up to the expiration date and incubated for the recommended incubation times. Allow the medium to warm to
room temperature before inoculation.
Product Deterioration: Do not use tubes or bottles if they show evidence of microbial contamination, evaporation,
precipitation or other signs of deterioration.
VIII SPECIMEN COLLECTION AND HANDLING
This medium is not suitable for use directly with specimens or other materials containing mixed microbial flora except as a
“backup” enrichment broth in addition to primary plating media. Consult appropriate references for further information.4-6

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IX PROCEDURE
Material Provided: Mueller Hinton Broth
Materials Required But Not Provided: Ancillary culture media, reagents, quality control organisms and laboratory equipment as
required.
Test Procedure: Observe aseptic techniques and established precautions against microbiological hazards throughout all
procedures.
Organisms to be subcultured must first be isolated in pure culture on an appropriate solid medium. Transfer growth from the
isolation medium to Mueller Hinton Broth using standard bacteriologic techniques.5-7
For enrichment purposes, inoculate the specimen onto primary media and then into the broth according to recommended
procedures.
Incubate tubes and bottles at 35ºC under conditions appropriate for the organism being cultured.
User Quality Control: See "Quality Control Procedures."
Quality control requirements must be performed in accordance with applicable local, state and/or federal regulations or
accreditation requirements and your laboratory’s standard Quality Control procedures. It is recommended that the user refer to
pertinent CLSI guidance and CLIA regulations for appropriate Quality Control practices.
X RESULTS
Growth in broth media is indicated by the presence of turbidity compared with an uninoculated control.
XI LIMITATIONS OF THE PROCEDURE
Enrichment broths should not be used as the sole isolation medium. They are to be used in conjunction with selective and
nonselective plating media to increase the probability of isolating pathogens, especially when they may be present in small
numbers.
For identification, organisms must be in pure culture. Morphological, biochemical and/or serological tests should be performed
for final identification. Consult appropriate texts for further information.4-7
XII PERFORMANCE CHARACTERISTICS
Prior to release, all lots of Mueller Hinton Broth are tested for performance characteristics. Representative samples of the lot
are tested with cell suspensions of Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922 and Staphylococcus aureus
ATCC 25923, diluted in normal saline to yield 103 to 104 CFU per tube of broth. Tubes are incubated with loose caps at 35 ± 2°C
for one day in an aerobic atmosphere. Growth is observed with all organisms.
XIII AVAILABILITY
Cat. No. Description
296164 BBL™ Mueller Hinton Broth, 2 mL, Ctn. of 100 size K tubes
XIV REFERENCES
1. Mueller, J.H., and J. Hinton. 1941. A protein-free medium for primary isolation of he gonococcus and meningooccus. Proc.
Soc. Exp. Biol. Med. 48:330-333.
2. Clinical and Laboratory Standards Institute. 2009. Approved standard M7-A8. Methods for dilution antimicrobial susceptibility
tests for bacteria that grow aerobically, 8th ed. CLSI, Wayne, Pa.
3. Clinical and Laboratory Standards Institute. 2009. Approved standard M2-A10. Performance standards for
antimicrobial disk susceptibility tests, 10th ed. CLSI, Wayne, Pa.
4. Washington, J.A. (ed.). 1985. Laboratory procedures in clinical microbiology, 2nd ed. Springer-Verlag, New York.
5. Forbes, B.A., D.F. Sahm, and A.S. Weissfeld. 1998. Bailey & Scott’s diagnostic microbiology, 10th ed. Mosby, Inc., St. Louis.
6. Murray, P.R., E.J. Baron, M.A. Pfaller, F.C. Tenover, and R.H. Yolken (ed.). 1995. Manual of clinical microbiology, 6th ed.
American Society for Microbiology, Washington, D.C.
7. Holt, J.G., N.R. Krieg, P.H.A. Sneath, J.T. Staley, and S.T. Williams (ed.). 1994. Bergey’s Manual™ of determinative bacteriology,
9th ed. Williams & Wilkins, Baltimore.

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