BBL Mueller Hinton Chocolate Agar - BD

!
BBL Mueller Hinton Chocolate Agar
L007396 • Rev. 03 • January 2006
QUALITY CONTROL PROCEDURES
I INTRODUCTION
Mueller Hinton Chocolate Agar is an enriched medium for the isolation and cultivation of fastidious organisms, particularly
Haemophilus species.
II PERFORMANCE TEST PROCEDURE
1. Inoculate representative samples with the culture listed below.
a. Preparation of inoculum.
1) Prepare the Haemophilus culture by suspending the growth from a Chocolate II Agar plate in Mueller Hinton II Broth.
2) Standardize the culture to 0.1 absorbance at 625 nm.
b. Within 15 min after adjusting the turbidity of the inoculum, dip a sterile swab into the broth suspension. Rotate the swab
several times on the inside wall of the tube above the fluid level to remove excess inoculum from the swab.
c. Inoculate the surface of the plate by streaking the swab over the surface of the plate. Repeat this procedure two more
times, rotating the plate 60 degrees each time.
d. Incubate plates at 35 ± 2°C with 5–7% CO2 .
2. Examine plates after 16–18 h for growth.
3. Expected Results
Organism ATCC Recovery
*Haemophilus influenzae 10211 Growth
*Recommended organism strain for User Quality Control.
III ADDITIONAL QUALITY CONTROL
1. Examine plates as described under “Product Deterioration.”
2. Visually examine representative plates 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. Note the firmness of plates during the inoculation procedure.
5. Incubate uninoculated representative plates at 35 ± 2°C with 5–7% CO 2 for 72 h and examine for microbial contamination.
PRODUCT INFORMATION
IV INTENDED USE
Mueller Hinton Chocolate Agar is for use in qualitative procedures for the isolation and cultivation of fastidious organisms,
particularly Haemophilus species. It formerly was recommended by the Clinical and Laboratory Standards Institute (CLSI,
formerly NCCLS) for antimicrobial susceptibility testing of H. influenzae. However, it has been replaced in this procedure by
Haemophilus Test Medium (HTM) Agar.
Mueller Hinton Chocolate Agar is recommended by CLSI for antimicrobial susceptibility testing of bacteria isolated from animals. 1
V SUMMARY AND EXPLANATION
Mueller Hinton Agar was originally developed for the cultivation of pathogenic Neisseria. 2 In the 1960s, Bauer, Kirby and others
developed a standardized disc diffusion procedure for determining the susceptibility of bacteria to antibiotic and
chemotherapeutic agents in which Mueller Hinton Agar was selected as the test medium. 3
Because growth of fastidious organisms was poor, the use of Mueller Hinton Agar supplemented with 1% hemoglobin and a
defined supplement was adopted for testing of H. influenzae. 4 Known as Mueller Hinton Chocolate Agar, this formulation has
been replaced for this purpose by HTM Agar. 5
The medium is now recommended for routine cultivation of fastidious organisms.
VI PRINCIPLES OF THE PROCEDURE
The primary nutriments in Mueller Hinton Chocolate Agar are beef extract, which provides nitrogenous nutrients, vitamins and
minerals required for microbial growth, and acid hydrolysate of casein, which provides amino acids with the exception of
cystine, because casein contains little cystine, and tryptophan, which is destroyed by the acid treatment, as are vitamins. The
starch neutralizes toxic fatty acids that may be present in the agar. Hemoglobin provides X factor (hemin) for Haemophilus
species. IsoVitaleX Enrichment is a defined supplement that provides V factor (nicotinamide adenine dinucleotide, NAD) for
Haemophilus species and vitamins, amino acids, co-enzymes, dextrose, ferric ion and other factors for improved growth of
fastidious organisms; i.e., pathogenic Neisseria.
VII REAGENTS
Mueller Hinton Chocolate Agar
Approximate Formula* Per Liter Purified Water
Beef Extract ..................................................................2.0 g
Acid Hydrolysate of Casein........................................17.5 g
Starch ............................................................................1.5 g
Agar ............................................................................17.0 g
Hemoglobin ................................................................10.0 g
IsoVitaleX Enrichment** ..........................................10.0 mL
*Adjusted and/or supplemented as required to meet performance criteria.
L007396 1 of 3

**IsoVitaleX Enrichment
Approximate Formula* Per Liter Purified Water
Vitamin B12 ..................................................................0.01 g
L-Glutamine ................................................................10.0 g
Adenine ........................................................................1.0 g
Guanine Hydrochloride................................................0.03 g
ρ-Aminobenzoic Acid ..................................................0.013 g
Nicotinamide Adenine Dinucleotide (NAD) ..............0.25 g
Thiamine Pyrophosphate ............................................0.1 g
Ferric Nitrate ................................................................0.02 g
Thiamine Hydrochloride ..............................................0.003 g
Cysteine Hydrochloride..............................................25.9 g
L-Cystine........................................................................1.1 g
Dextrose ....................................................................100.0 g
*Adjusted and/or supplemented as required to meet performance criteria.
Warnings and Precautions: For in vitro Diagnostic Use.
If excessive moisture is observed, invert the bottom over an off-set lid and allow to air dry in order to prevent formation of a
seal between the top and bottom of the plate during incubation.
Pathogenic microorganisms, including hepatitis viruses and Human Immunodeficiency Virus, may be present in clinical
specimens. "Standard Precautions" 6-9 and institutional guidelines should be followed in handling all items contaminated with
blood and other body fluids. After use, prepared plates, specimen containers and other contaminated materials must be
sterilized by autoclaving before discarding.
Storage Instructions: On receipt, store plates in the dark at 2–8°C. Avoid freezing and overheating. Do not open until ready to
use. Minimize exposure to light. Prepared plates stored in their original sleeve wrapping at 2–8°C until just prior to use may be
inoculated up to the expiration date and incubated for recommended incubation times. Allow the medium to warm to room
temperature before inoculation.
Product Deterioration: Do not use plates if they show evidence of microbial contamination, discoloration, drying, cracking or
other signs of deterioration.
VIII SPECIMEN COLLECTION AND HANDLING
Specimens suitable for culture may be handled using various techniques. For detailed information, consult appropriate texts. 10,11
Specimens should be obtained before antimicrobial therapy has been administered. Provision must be made for prompt delivery
to the laboratory.
IX PROCEDURE
Material Provided: Mueller Hinton Chocolate Agar
Materials Required But Not Provided: Ancillary culture media, reagents, quality control organisms and laboratory equipment as
required.
Test Procedure: Observe aseptic techniques.
The agar surface should be smooth and moist, but without excessive moisture.
Streak the specimen as soon as possible after it is received in the laboratory. The streak plate is used primarily to isolate pure
cultures from specimens containing mixed flora.
Alternatively, if material is being cultured directly from a swab, roll the swab over a small area of the surface at the edge; then
streak from this inoculated area.
Incubate plates at 35 ± 2°C for 18–24 h and up to 72 h, if necessary, in an aerobic atmosphere enriched with 5–7% carbon dioxide. 12
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 (formerly NCCLS) guidance and CLIA regulations for appropriate Quality Control practices.
X RESULTS
After a minimum of 18 h of incubation, the plates should show isolated colonies in streaked areas and confluent growth in
areas of heavy inoculation.
The growth of Haemophilus appears as small (1 mm), moist, pearly colonies with a characteristic “mousy” odor.
XI LIMITATION OF THE PROCEDURE
For identification, organisms must be in pure culture. Morphological, biochemical, and/or serological tests should be performed
for final identification. Consult appropriate texts for detailed information and recommended procedures. 10,11,13-16
XII AVAILABILITY
Cat. No. Description
221860 BBL Mueller Hinton Chocolate Agar, Pkg. of 20 plates
221869 BBL Mueller Hinton Chocolate Agar, Pkg. of 8, 150 mm–style plates
221802
XIII REFERENCES
BBL Mueller Hinton Chocolate Agar, Ctn. of 24, 150 mm–style plates
1. National Committee for Clinical Laboratory Standards. 2002. Approved Standard M31-A2. Performance standards for antimicrobial disk and
dilution susceptibility tests for bacteria isolated from animals, 2nd ed. NCCLS, Wayne, PA.
2. Mueller, J.H., and J. Hinton. 1941. A protein-free medium for primary isolation of the gonococcus and meningococcus. Proc. Soc. Exp. Biol.
Med. 48:330-333.
3. Bauer, A.W., W.M.M. Kirby, J.C. Sherris, and M. Turck. 1966. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin.
Pathol. 45:493-496.
L007396 2 of 3

4. National Committee for Clinical Laboratory Standards. 1984. Approved standard: M2-A3. Performance standards for antimicrobial disk
susceptibility tests, 3rd ed. National Committee for Clinical Laboratory Standards, Villanova, Pa.
5. National Committee for Clinical Laboratory Standards. 2003. Approved standard: M2-A8. Performance standards for antimicrobial disk
susceptibility tests, 8th ed. National Committee for Clinical Laboratory Standards, Wayne, Pa.
6. National Committee for Clinical Laboratory Standards. 2001. Approved Guideline M29-A2. Protection of laboratory workers from
occupationally acquired infections, 2nd ed. NCCLS, Wayne, PA.
7. Garner, J.S. 1996. Hospital Infection Control Practices Advisory Committee, U.S. Department of Health and Human Services, Centers for
Disease Control and Prevention. Guideline for isolation precautions in hospitals. Infect. Control Hospital Epidemiol. 17:53-80.
8. U.S. Department of Health and Human Services. 1999. Biosafety in microbiological and biomedical laboratories, HHS Publication (CDC), 4th
ed. U.S. Government Printing Office, Washington, D.C.
9. Directive 2000/54/EC of the European Parliament and of the Council of 18 September 2000 on the protection of workers from risks related to
exposure to biological agents at work (seventh individual directive within the meaning of Article 16(1) of Directive 89/391/EEC). Official
Journal L262, 17/10/2000, p. 0021-0045.
10. Murray, P.R., E.J. Baron, J.H. Jorgensen, M.A. Pfaller, and R. H. Yolken (ed.). 2003. Manual of clinical microbiology, 8th ed. American Society
for Microbiology, Washington, D.C
11. Forbes, B.A., D.F. Sahm, and A.S. Weissfeld. 2002. Bailey and Scott's diagnostic microbiology, 11th ed. Mosby, Inc., St. Louis.
12. Campos, J.M. 1999. Haemophilus, p. 604-613. In P.R. Murray, E.J. Baron, M.A. Pfaller, F.C. Tenover, and R.H. Yolken (ed.), Manual of clinical
microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
13. 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.
14. MacFaddin, J.F. 2000. Biochemical tests for identification of medical bacteria, 3rd ed. Lippincott Williams & Wilkins, Baltimore.
15. Koneman, E.W., S.D. Allen, W.M. Janda, P.C. Schreckenberger, and W.C. Winn, Jr. 1997. Color atlas and textbook of diagnostic microbiology,
5th ed. Lippincott-Raven, Philadelphia.
16. Isenberg, H.D. (ed.). 2004. Clinical microbiology procedures handbook, vol. 1, 2 and 3, 2nd ed. American Society for Microbiology,
Washington, D.C.
Becton, Dickinson and Company
7 Loveton Circle
Sparks, Maryland 21152 USA
800-638-8663
ATCC is a trademark of the American Type Culture Collection.
BD, BD Logo, BBL and IsoVitaleX are trademarks of Becton, Dickinson and Company. ©2006 BD.
L007396 3 of 3