Brain Heart Infusion (BHI) Agar- Composition, Principle, Preparation, Results, Uses

  • In 1919, Rosenow devised an excellent medium for culturing streptococci by using a dextrose broth supplemented with brain tissue.
  • Rosenow’s formulation was later modified by Hayden who found the addition of crushed marble resulted in favorable growth of dental pathogens.
  • Our current formulation contains infusion from calf brain in place of brain tissue and disodium phosphate has replaced calcium carbonate.
  • Brain Heart Infusion (BHI) Agar is a highly nutritious base that meets the growth requirements of many types of microorganisms including bacteria, yeasts, and molds.
  • BHI Agar supplemented with (5 to 10%) defibrinated sheep blood is used extensively for the recovery of dimorphic fungi such as Histoplasma capsulatum and other pathogenic fungi such as Coccidioides immitis.
  • A more selective formulation containing chloramphenicol and cycloheximide is also available that will allow the recovery of pathogenic fungi while inhibiting a wide range of bacteria and saprophytic fungi.
  • BHI Agar is thus a solid medium recommended for the cultivation of fastidious pathogenic bacteria, yeasts, and molds from clinical and non-clinical samples.
Brain Heart Infusion (BHI) Agar

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Composition of Brain Heart Infusion (BHI) Agar

HM infusion powder12.500
BHI powder5.000
Proteose peptone10.000
Dextrose (Glucose)2.000
Sodium chloride5.000
Disodium phosphate2.500

Final pH (at 25°C): 7.4±0.2

Principle of Brain Heart Infusion (BHI) Agar

  • Brain Heart Infusion has proven to be effective in the cultivation of a wide variety of microorganisms, including many types of pathogens. It has served as the base medium for new culture media formulations when supplemented with blood or with selective agents.
  • Brain Heart Infusion (BHI) Agar derives its nutrients from the brain heart infusion, peptone and glucose components.
  • Protease peptone and infusions used in the media serve as sources of carbon, nitrogen, vitamins, amino acids, along with essential growth factors.
  • Dextrose is the energy source.
  • Sodium chloride maintains the osmotic equilibrium of the medium while disodium phosphate buffers the medium.
  • Defibrinated sheep blood added to the basal medium provides essential growth factors for the more fastidious fungal organisms.

Preparation and Method of Use of Brain Heart Infusion (BHI) Agar

  1. Suspend 52.0 grams in 1000 ml distilled water.
  2. Heat to boiling to dissolve the medium completely.
  3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
  4. Cool to 45-50°C.
  5. Mix well and pour into sterile Petri plates.

Note: If desired, 20 units of Penicillin and 40 µg Streptomycin per ml of the medium may be added to make the medium selective for fungi.

  1. Streak the specimen as soon as possible after it is received in the laboratory using a sterile inoculating loop to obtain isolated colonies.
  2. For isolation of fungi from potentially contaminated specimens, a selective medium should be inoculated along with the nonselective medium.
  3. Incubate the plates at 25 to 30°C in an inverted position with increased humidity.
  4. For isolation of fungi causing systemic mycoses and the isolation of aerobic Actinomycetales, two sets of media should be inoculated, with one set incubated at 25 to 30°C and a duplicate set at 35 to 37°C.
  5. Depending on the clinical diagnosis and the agents suspected to cause the infection, other media should be included.
  6. All cultures should be examined at least weekly for growth and should be held for several weeks before being reported as negative.

Result Interpretation on Brain Heart Infusion (BHI) Agar

  • After sufficient incubation, the plates should show isolated colonies in streaked areas and confluent growth in areas of heavy inoculation.
  • Examine plates for fungal and/or bacterial colonies exhibiting typical color and morphology.
  • Biochemical tests and/or microscopic or serological procedures must be performed to confirm findings.
Candida albicansLuxuriant growth
Staphylococcus aureus subsp. aureus Luxuriant growth
Streptococcus pneumoniaeLuxuriant growth; grey-green colored colonies
Shigella flexneriLuxuriant growth
Escherichia coli Luxuriant growth
Listeria monocytogenesGrowth good to excellent
Trichophyton mentagrophytesGrowth good to excellent
Neisseria meningitidisGood growth; grey-brown colored colonies

Uses of Brain Heart Infusion (BHI) Agar

  • Brain Heart Infusion Agar is highly nutritious and can support a luxuriant growth of a wide variety of microorganisms.
  • It can be further enriched by the addition of blood or rendered selective by adding different antibiotics.
  • It is a general purpose medium used for primary isolation of aerobic bacteria from clinical specimens.
  • Addition of 50 mg/l chloramphenicol or 40mg/l streptomycin or a mixture of 50mg/l gentamicin and 50mg/l chloramphenicol along with 5-10% sterile defibrinated blood is often recommended for inhibition of bacteria and isolation of pathogenic systemic fungi.
  • Without supplementation, Brain Heart Infusion (BHI) Agar currently is recommended as a universal medium for aerobic bacteriology and for the primary recovery of fungi and Actinomycetales from clinical specimens and from nonclinical materials.

Limitations of BHI Agar

  • As organisms differ in their nutritional requirements, some fastidious organisms may be inhibited or may show poor growth.
  • Further biochemical tests must be carried out for complete identification.
  • Due to the non-selective nature of BD Brain Heart Infusion (BHI) Agar, specimens heavily contaminated with normal flora should also be streaked onto appropriate selective media to avoid overgrowth by the contaminating organisms.
  • If fastidious organisms known to require blood for growth are suspected, Brain Heart Infusion Agar with 10% Sheep Blood should be used.


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

About Author

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Sagar Aryal

Sagar Aryal is a microbiologist and a scientific blogger. He is doing his Ph.D. at the Central Department of Microbiology, Tribhuvan University, Kathmandu, Nepal. He was awarded the DAAD Research Grant to conduct part of his Ph.D. research work for two years (2019-2021) at Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Saarbrucken, Germany. Sagar is interested in research on actinobacteria, myxobacteria, and natural products. He is the Research Head of the Department of Natural Products, Kathmandu Research Institute for Biological Sciences (KRIBS), Lalitpur, Nepal. Sagar has more than ten years of experience in blogging, content writing, and SEO. Sagar was awarded the SfAM Communications Award 2015: Professional Communicator Category from the Society for Applied Microbiology (Now: Applied Microbiology International), Cambridge, United Kingdom (UK). Sagar is also the ASM Young Ambassador to Nepal for the American Society for Microbiology since 2023 onwards.

2 thoughts on “Brain Heart Infusion (BHI) Agar- Composition, Principle, Preparation, Results, Uses”

  1. It’s interesting to learn that brain-heart infusion broth can help support a variety of microorganisms to grow. I think if that was something that you needed to do, it would be smart to buy that from professionals that knew how to make it and what they were doing. That would help make sure that everything could be done correctly and you could get the best results.


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