BRAIN HEART INFUSION (BHI) AGAR

Cat. no. L35 BHI Agar, 20x125mm Tube, 10ml Slant 20 tubes/box
Cat. no. L36 BHI Agar, 16x100mm Tube, 5.5ml Slant 20 tubes/box
Cat. no. Q16 BHI Agar, 20x150mm Tube, 18ml Deep 20 tubes/box
Cat. no. W15 BHI Agar, 15x100mm Plate, 26ml 10 plates/bag
Cat. no. W163 BHI Agar, 25x100mm Plate, 60ml 5 plates/bag
Cat. no. X10 BHI Agar, 50ml HardyFlask™, 12ml Slant 20 flasks/box
Cat. no. A20 BHI Agar with Blood, 15x100mm Plate, 18ml 10 plates/bag
Cat. no. W185BX BHI Agar with Blood, 25x100mm Plate, 37ml 50 plates/box
Cat. no. X13 BHI Agar with Blood, 50ml HardyFlask™, 12ml 20 flasks/box
Cat. no. L31 BHI Agar with Blood, 20x125mm Tube, 10ml Slant 20 tubes/box
Cat. no. X12 BHI Agar with Blood and Gentamicin, 50ml HardyFlask™, 12ml 20 flasks/box
Cat. no. L114 BHI Agar with Blood, Choramphenicol and Gentamicin, 20x125mm Tube, 10ml Slant 20 tubes/box
Cat. no. W65 BHI Agar with Blood, Chloramphenicol and Gentamicin, 15x100mm Plate, 26ml 10 plates/bag
Cat. no. X14 BHI Agar with Blood, Chloramphenicol and Gentamicin, 50ml HardyFlask™, 12ml 20 flasks/box
Cat. no. X11 BHI Agar with Chloramphenicol and Cycloheximide, 50ml HardyFlask™, 12ml 20 flasks/box

INTENDED USE

Hardy Diagnostics Brain Heart Infusion (BHI) Agar is a general purpose nutrient medium recommended for the cultivation and isolation of a variety of microorganisms, including bacteria, yeasts, and molds. The addition of defibrinated sheep blood and antimicrobics produces a selective medium used for the isolation of pathogenic fungi from specimens heavily contaminated with bacteria and saprophytic fungi.(2)

SUMMARY

Rosenow, by adding brain tissue to dextrose broth, discovered a medium useful in the cultivation of streptococci.(8) Formula modifications were made by various researchers who found the medium effective in the recovery of dental pathogens.(4,5,8) The medium was further modified by the addition of agar and a variety of supplements and enrichments which further enhanced the recovery of microorganisms.

The brain heart infusion, peptone and dextrose components of the medium provide the nutrients to BHI Agar. Organic nitrogen, carbon, sulfur, vitamins and trace substances are provided by the peptones and infusion. Dextrose provides the carbohydrate source for fermentative microorganisms. Disodium phosphate is added to the medium in order to maintain an optimal pH. Additionally, a variety of supplements has been added to further enhance the recovery of fastidious microorganisms.

BHI Agar with Blood is used for the recovery of fungal species.(13) Blood provides essential growth factors for the more fastidious fungal organisms.

BHI Agar with Blood, Gentamicin and Chloramphenicol is used for the selective isolation of fungi from specimens heavily contaminated with bacteria.(2) Gentamicin inhibits the growth of gram-negative and some gram-positive bacteria. A wide range of gram-positive and gram-negative bacteria are inhibited by chloramphenicol, a broad-spectrum antimicrobic.

BHI Agar with Chloramphenicol and Cycloheximide is used for the selective isolation of pathogenic fungi. Cycloheximide inhibits saprophytic mold while Chloramphenicol acts as an antibacterial agent.(2)

FORMULA

Ingredients per liter of deionized water:*

BHI Agar with Blood, Chloramphenicol and Gentamicin**:
BHI Agar:
Pancreatic Digest of Casein 16.0gm
Brain Heart Infusion from Solids 10.0gm
Peptic Digest of Animal Tissue 5.0gm
Sodium Chloride 3.0gm
Disodium Phosphate 2.5gm
Dextrose 2.0gm
Agar 13.5gm
BHI Agar with Blood:
In addition to the above formulation, contains:
Sheep Blood 50.0ml
BHI Agar with Blood and Gentamicin:
In addition to the above formulation, contains:
Gentamicin 5.0mg
Sheep Blood 50.0ml
In addition to the above formulation, contains:
Chloramphenicol 50.0mg
Gentamicin 5.0mg
Sheep Blood 50.0ml
BHI Agar with Chloramphenicol and Cycloheximide**:
Pancreatic Digest of Casein 16.0gm
Brain Heart Infusion 8.0gm
Peptic Digest of Animal Tissue 5.0gm
Sodium Chloride 5.0gm
Disodium Phosphate 2.5gm
Dextrose 2.0gm
Cycloheximide 0.5gm
Chloramphenicol 0.05gm
Agar 13.5gm

Final pH 7.4 +/- 0.3 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

** This media contains chloramphenicol and/or cycloheximide which are ingredients considered potentially hazardous.

STORAGE AND SHELF LIFE

Storage: Upon receipt store all BHI Agar products* at 2-8ºC (*except Cat. nos. L35, X10 and X11). Cat. nos. L35, X10 and X11 may be stored at 2-30ºC. Products should not be used if there are any signs of contamination, deterioration (shrinking, cracking, or discoloration), hemolysis, contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: Consult listed references for information on specimen collection.(1-3,6) Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport medium and refrigerated until inoculation.

Method of Use for Cultivation and Isolation of Bacteria: Consult listed references for the appropriate cultivation techniques using this medium.(1-3,6) Medium should be brought to room temperature and agar surface should be dry prior to inoculation. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface. Streak for isolation with a sterile loop. Incubate plates aerobically at 35 +/- 2.0ºC for 24 to 48 hours. Protect from light. Examine plates for colonial morphology.

Method of Use for Cultivation and Isolation of Fungi: A selective and non-selective medium should be inoculated for isolation of fungi from potentially contaminated specimens. Media should be incubated at room temperature (15-30ºC) with increased humidity. Two sets of media should be inoculated for isolation of fungi causing systemic mycoses; one set incubated at 15-30ºC and one set at 35 +/- 2.0ºC. Cultures should be examined weekly for fungal growth. Media should be held for four to six weeks before being reported as negative.

BHI Agar slants (without supplements or antimicrobics) are used primarily for the cultivation and maintenance of pure cultures of microorganisms.

INTERPRETATION OF RESULTS

Media should be examined for typical bacterial and fungal colonial morphology. Consult listed references for the interpretation of growth and other identification tests to identify growth of organism(s) on this media.(1-3,6)

LIMITATIONS

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
BHI Agar:
Escherichia coli
ATCC ® 25922
A 24-48hr 35°C Aerobic Growth
Staphylococcus aureus
ATCC ® 25923
A 24-48hr 35°C Aerobic Growth
Staphylococcus epidermidis
ATCC ® 12228
A 18-24hr 35°C Aerobic Growth
BHI Agar with Blood:
Streptococcus pyogenes
ATCC ® 19615
A 18-24hr 35°C Aerobic Growth; beta-hemolysis
Streptococcus pneumoniae
ATCC ® 6305**
A 18-24hr 35°C Aerobic Growth; alpha-hemolysis
Aspergillus brasiliensis
ATCC ® 16404
G 5-7 days 30°C Aerobic Growth
Staphylococcus aureus
ATCC ® 25923
A 18-24hr 35°C Aerobic Growth
Escherichia coli
ATCC ® 25922
A 18-24hr 35°C Aerobic Growth
BHI Agar with Blood and Gentamicin / BHI Agar with Blood, Chloramphenicol, and Gentamicin:
Candida albicans
ATCC ® 10231**
A 18-24hr 35°C Aerobic Growth
Trichophyton mentagrophytes
ATCC ® 9533
G 7 days 15-30°C Aerobic Growth
Escherichia coli
ATCC ® 25922
B 18-24hr 35°C Aerobic Partial to complete inhibition
Pseudomonas aeruginosa
ATCC ® 27853
B 24hr 35°C Aerobic Partial to complete inhibition
BHI Agar with Chloramphenicol and Cycloheximide:
Candida albicans
ATCC ® 10231**
A 18-24hr 35°C Aerobic Growth
Trichophyton mentagrophytes
ATCC ® 9533**
G 7 days 15-30°C Aerobic Growth
Escherichia coli
ATCC ® 25922**
B 18-24hr 35°C Aerobic Partial to complete inhibition
Aspergillus brasiliensis
ATCC ® 16404**
G 7 days 15-30°C Aerobic Partial to complete inhibition

** Recommended QC strains for User Quality Control according to the CLSI document M22 when applicable.

User Quality Control

PHYSICAL APPEARANCE

BHI Agar and BHI Agar with Chloramphenicol and Cycloheximide should appear slightly opalescent, and light amber in color.

BHI Agar with Blood; BHI Agar with Blood and Gentamicin; and BHI Agar with Blood, Chloramphenicol, and Gentamicin should appear opaque, and cherry to brick-red in color.

BHI Agar

Sterile plate of BHI Agar (Cat. no. W15).

E. coli growing on BHI Agar

Escherichia coli (ATCC ® 25922) colonies growing on BHI Agar (Cat. no. W15). Incubated aerobically for 24 hours at 35ºC.

BHI Agar with Blood

Uninoculated plate of BHI Agar with Blood (Cat. no. A20).

C. albicans growing on BHI Agar with Blood, Chloramphenicol and Gentamicin

Candida albicans (ATCC ® 10231) colonies growing on BHI Agar with Blood, Chloramphenicol and Gentamicin (Cat. no. W65). Incubated aerobically for 24 hours at 35ºC.

P. pneumoniae growing on BHI Agar with Blood

Streptococcus pneumoniae (ATCC ® 6305) colonies growing on BHI Agar with Blood (Cat. no. A20). Incubated aerobically for 24 hours at 35ºC.

E. coli inhibited on BHI Agar with Blood, Chloramphenicol and Gentamicin

Escherichia coli (ATCC ® 25922) growth inhibited on BHI Agar with Blood, Chloramphenicol and Gentamicin (Cat. no. W65). Incubated aerobically for 24 hours at 35ºC.

T. mentagrophytes growing on BHI Agar with Blood, Chloramphenicol and Gentamicin

Trichophyton mentagrophytes (ATCC ® 9533) colonies growing on BHI Agar with Blood, Chloramphenicol and Gentamicin (Cat. no. W65). Incubated aerobically for 96 hours at 30ºC.


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REFERENCES

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Versalovic, J., et al. Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C.

3. Tille, P.M., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

4. Falk, C.R., et al. 1939. J. Bacteriol.; 37:121.

5. Haden, R.L. 1932. Arch. Internal Med.; 32:828.

6. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

7. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD.

8. Hitchens, A.P. 1921. J. Infect. Disease; 29:390.

9. Rosenow, E.C. 1919. J. Dental Research; 1:205.

10. Quality Assurance for Commercially Prepared Microbiological Culture Media , M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

11. Fildes, P. 1920. Br. J. Exp. Pathol.; 1:129.

12. Fildes, P. 1921. Br. J. Exp. Pathol.; 2:16.

13. Creitz and Puckett. 1954. J. Clin. Pathol.; 24:1318.

14. J. Chron. Dis.; 5:545, 1957.

15. J. Lab and Clin. Med.; 55:116, 1960.


ATCC is a registered trademark of the American Type Culture Collection.

121415hh