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

What is Brain Heart Infusion (BHI) Agar?

  • Brain Heart Infusion (BHI) Agar is a specialized culture medium that was originally developed by Rosenow in 1919. It was designed specifically for cultivating streptococci, a group of bacteria commonly associated with various infections. Rosenow formulated a dextrose broth supplemented with brain tissue, which provided essential nutrients for the growth of streptococci.
  • Over time, the original formulation was modified by Hayden, who discovered that the addition of crushed marble facilitated the growth of dental pathogens. As a result, modern versions of BHI Agar have evolved to include infusion from calf brain instead of brain tissue. Furthermore, disodium phosphate has replaced calcium carbonate as an ingredient in the medium.
  • BHI Agar serves as a highly nutritious base that fulfills the growth requirements of various microorganisms, including bacteria, yeasts, and molds. It provides a rich environment for these organisms to thrive and reproduce. This makes BHI Agar a valuable tool in laboratory settings for the cultivation and study of different microorganisms.
  • One common application of BHI Agar is in the recovery of specific fungi. By supplementing BHI Agar with 5 to 10% defibrinated sheep blood, it becomes an ideal medium for the retrieval of dimorphic fungi like Histoplasma capsulatum, as well as other pathogenic fungi such as Coccidioides immitis. These fungi are known to cause serious infections in humans, and BHI Agar with blood serves as an effective medium for their growth and isolation in clinical and research settings.
  • Moreover, a selective variant of BHI Agar is available, which contains additional additives such as chloramphenicol and cycloheximide. This formulation allows for the recovery of pathogenic fungi while inhibiting the growth of a wide range of bacteria and saprophytic fungi. This selectivity is beneficial when trying to isolate and study specific fungal pathogens while minimizing interference from other microorganisms.
  • Overall, Brain Heart Infusion (BHI) Agar is a solid medium that is recommended for the cultivation of fastidious pathogenic bacteria, yeasts, and molds from both clinical and non-clinical samples. Its rich nutrient composition and versatility make it an essential tool in microbiology laboratories for studying various microorganisms and identifying pathogens.

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

The principle of Brain Heart Infusion (BHI) Agar revolves around its nutrient composition and its ability to support the growth of a wide range of microorganisms, including pathogens. BHI Agar has been widely used as a base medium and has served as the foundation for the development of new culture media when supplemented with blood or selective agents.


The nutrients in BHI Agar are derived from several components, including brain heart infusion, peptone, and glucose. Protease peptone and infusions present in the medium act as sources of carbon, nitrogen, vitamins, amino acids, and other essential growth factors necessary for microbial growth and reproduction.

Dextrose, a type of sugar, serves as the energy source in BHI Agar. It provides the microorganisms with the necessary fuel for metabolic processes and facilitates their growth.


Sodium chloride plays a crucial role in maintaining the osmotic equilibrium of the medium. It helps regulate the concentration of solutes within the agar, creating an environment conducive to microbial growth.

Disodium phosphate acts as a buffer in BHI Agar. It helps maintain the pH of the medium within a suitable range for the growth of microorganisms. By stabilizing the pH, disodium phosphate creates a favorable environment for the organisms to thrive.


In certain applications, BHI Agar is supplemented with defibrinated sheep blood. The addition of blood provides essential growth factors that are particularly important for the cultivation of more fastidious fungal organisms. These growth factors support the growth and development of these fungi, allowing for their isolation and study.

Overall, the principle of BHI Agar lies in its ability to provide a nutrient-rich environment through the combination of brain heart infusion, peptone, glucose, and other components. This composition supports the growth of various microorganisms and allows for the cultivation of both fastidious and non-fastidious pathogens.


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

The preparation and method of use for Brain Heart Infusion (BHI) Agar are as follows:

  1. Measure and suspend 52.0 grams of BHI Agar in 1000 ml of distilled water.
  2. Heat the suspension to boiling in order to completely dissolve the medium.
  3. Sterilize the medium by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
  4. Allow the medium to cool down to 45-50°C.
  5. Thoroughly mix the medium to ensure uniformity.
  6. Pour the sterilized BHI Agar into sterile Petri plates. Note: To create a selective medium for fungi, 20 units of Penicillin and 40 µg Streptomycin per ml of the medium may be added.

For the method of use of BHI Agar:

  1. As soon as a specimen is received in the laboratory, streak it onto BHI Agar using a sterile inoculating loop to obtain isolated colonies.
  2. If the specimen is potentially contaminated, it is recommended to inoculate both a nonselective medium and a selective medium for the isolation of fungi.
  3. Incubate the inoculated plates at a temperature range of 25 to 30°C. Place the plates in an inverted position to prevent condensation from falling onto the growing colonies.
  4. For the isolation of fungi causing systemic mycoses and aerobic Actinomycetales, two sets of media should be inoculated. One set should be incubated at 25 to 30°C, while the duplicate set should be incubated at 35 to 37°C.
  5. Depending on the suspected agents causing the infection and the clinical diagnosis, other media may need to be included.
  6. Regularly examine the cultures at least weekly for any signs of growth.
  7. Negative cultures should be held for several weeks before being reported as negative to allow sufficient incubation time for slow-growing organisms.

By following these preparation and method of use guidelines, BHI Agar can be effectively utilized for the cultivation and isolation of various microorganisms, particularly fungi, in clinical and research settings.

Result Interpretation on Brain Heart Infusion (BHI) Agar

The interpretation of results on Brain Heart Infusion (BHI) Agar involves the examination of the colonies that have grown on the plates after sufficient incubation. Here are the key points to consider:

  1. Isolated colonies: In areas where the specimen was streaked onto the agar, there should be individual, discrete colonies. These isolated colonies indicate the presence of microorganisms and suggest successful growth of the organisms in the sample.
  2. Confluent growth: In regions of the plate where the inoculation was heavy or where there was a high concentration of microorganisms, the growth may appear as a confluent mass, covering a larger area. This indicates abundant microbial growth and suggests that the microorganisms were able to proliferate in those areas.
  3. Colony characteristics: Examine the colonies for their color, shape, and overall morphology. Different microorganisms may exhibit characteristic appearances on BHI Agar. This visual examination can provide initial clues about the types of microorganisms present in the sample.
  4. Confirmatory tests: It is important to note that further tests are necessary to confirm the identity of the microorganisms. Biochemical tests, microscopic examinations, or serological procedures may be required to accurately identify the specific species of microorganisms present. These additional tests provide more detailed information about the microorganisms’ characteristics, enabling accurate diagnosis and appropriate treatment if needed.

Interpreting the results on BHI Agar requires a combination of visual observations and confirmatory tests to ensure accurate identification of the microorganisms. By carefully examining the isolated and confluent colonies and performing additional tests, researchers and healthcare professionals can gain insights into the types of microorganisms present in the sample and make informed decisions regarding diagnosis and treatment.

Candida albicansLuxuriant growth
Staphylococcus aureus subsp. aureusLuxuriant growth
Streptococcus pneumoniaeLuxuriant growth; grey-green colored colonies
Shigella flexneriLuxuriant growth
Escherichia coliLuxuriant growth
Listeria monocytogenesGrowth good to excellent
Trichophyton mentagrophytesGrowth good to excellent
Neisseria meningitidisGood growth; grey-brown colored colonies

Quality Control Organisms

Organism TestedResultsTime
Bacteroides fragilisGrowth24 hrs
Prevotella melaninogenicaGrowth24 – 48 hrs
Fusobacterium mortiferumGrowth24 – 48 hrs
Fusobacterium necrophorumGrowth24 – 48 hrs
Clostridium perfringensGrowth24 hrs
Clostridium sporogenesGrowth24 hrs
Peptostreptococcus anaerobiusGrowth24 hrs
Proteus mirabilisGrowth24 hrs
Propionibacterium acnes orClostridium difficileGrowth24 – 48 hrs24 hrs
Staphylococcus aureus orEnterococcus faecalisGrowth24 hrs24 hrs
Escherichia coliGrowth24 hrs

Uses of Brain Heart Infusion (BHI) Agar

Brain Heart Infusion (BHI) Agar has a range of uses due to its highly nutritious composition and versatility. Here are the key applications of BHI Agar:

  1. General purpose medium: BHI Agar is a highly nutritious medium that can support the growth of a wide variety of microorganisms. It is commonly used as a general purpose medium for the primary isolation of aerobic bacteria from clinical specimens. Its rich nutrient content provides an optimal environment for bacterial growth.
  2. Enrichment: BHI Agar can be further enriched by the addition of blood, typically in the form of 5-10% sterile defibrinated blood. The addition of blood enhances the medium by providing additional growth factors, particularly for fastidious organisms. This enrichment is particularly useful for the isolation and cultivation of pathogenic systemic fungi.
  3. Selective medium: BHI Agar can be modified to become a selective medium by the addition of different antibiotics. For example, the inclusion of 50 mg/l chloramphenicol, 40 mg/l streptomycin, or a combination of 50 mg/l gentamicin and 50 mg/l chloramphenicol can inhibit the growth of certain bacteria while facilitating the isolation of pathogenic systemic fungi. These selective additions help create an environment that promotes the growth of specific microorganisms while suppressing others.
  4. Universal medium: In its standard formulation without supplementation, BHI Agar is recommended as a universal medium for aerobic bacteriology. It can support the growth of a broad range of bacteria and is suitable for primary recovery of fungi and Actinomycetales from clinical specimens as well as nonclinical materials. Its versatility and nutrient richness make it a widely used medium in microbiology laboratories.

Overall, BHI Agar’s uses range from general bacterial isolation to selective cultivation of specific microorganisms, including fungi. Its adaptability through the addition of blood or antibiotics makes it a valuable tool in various laboratory settings for studying and identifying different types of microorganisms.

Limitations of Brain Heart Infusion (BHI) Agar

BHI Agar, despite its versatility, has certain limitations that should be considered. Here are the key limitations of BHI Agar:

  1. Growth inhibition of fastidious organisms: BHI Agar may not support the optimal growth of certain fastidious organisms. As microorganisms vary in their nutritional requirements, some fastidious organisms may be inhibited or exhibit poor growth on BHI Agar. Additional specialized media or supplements may be required to meet the specific nutritional needs of these organisms.
  2. Further tests for identification: While BHI Agar provides a suitable environment for the growth of various microorganisms, it does not provide definitive identification. Additional biochemical tests, microscopic examinations, or serological procedures are necessary for accurate identification and characterization of the microorganisms present on the agar. These tests provide detailed information about the physiological and biochemical characteristics of the organisms.
  3. Non-selective nature: BHI Agar is a non-selective medium, meaning it supports the growth of a wide range of microorganisms, including both desired pathogens and normal flora. In cases where specimens are heavily contaminated with normal flora, overgrowth by contaminating organisms may occur, making it difficult to isolate and identify the target pathogens. To address this, appropriate selective media should be used alongside BHI Agar to prevent overgrowth by contaminating organisms.
  4. Use of blood for growth: Some fastidious organisms require blood as a growth factor. In situations where these organisms are suspected, BHI Agar supplemented with 10% sheep blood should be used to provide the necessary growth factors for their optimal growth and recovery.

Considering these limitations, it is important to select the appropriate media and supplements based on the specific requirements of the microorganisms being studied or isolated. BHI Agar can be a valuable component in the laboratory workflow, but its limitations should be recognized and addressed through additional testing and appropriate selective media when necessary.


What is Brain Heart Infusion (BHI) Agar?

BHI Agar is a highly nutritious culture medium used for the cultivation of various microorganisms, including bacteria, yeasts, and molds.

What are the main components of BHI Agar?

BHI Agar contains brain heart infusion, peptone, glucose, sodium chloride, and disodium phosphate as its main components.

What is the purpose of using BHI Agar?

BHI Agar serves as a general purpose medium for the isolation and cultivation of aerobic bacteria from clinical specimens. It can also be used for the primary recovery of fungi and Actinomycetales.

Can BHI Agar support the growth of fastidious organisms?

While BHI Agar is highly nutritious, it may not support the optimal growth of some fastidious organisms. Additional supplements or specialized media may be required for their cultivation.

Can BHI Agar be used for the selective isolation of specific microorganisms?

BHI Agar can be modified to become a selective medium by adding antibiotics or other selective agents. This allows for the growth and isolation of specific microorganisms while inhibiting others.

How is BHI Agar prepared?

BHI Agar is prepared by suspending the appropriate amount of BHI Agar powder in distilled water, boiling to dissolve the medium, sterilizing by autoclaving, and then pouring it into sterile Petri plates.

How should BHI Agar plates be incubated?

BHI Agar plates should be incubated at a temperature range of 25 to 30°C for the cultivation of bacteria and fungi. For specific organisms, different incubation temperatures may be required.

How can BHI Agar be used for the recovery of fungi?

BHI Agar can be supplemented with 5-10% defibrinated sheep blood to enhance the growth of fungi. This enriched medium provides essential growth factors for the recovery of fastidious fungal organisms.

What are the limitations of BHI Agar?

BHI Agar may inhibit the growth of some fastidious organisms, and additional tests are required for complete identification. It is also non-selective, so heavily contaminated specimens may require additional selective media.

Is BHI Agar suitable for the isolation of fastidious organisms requiring blood?

Yes, BHI Agar supplemented with 10% sheep blood can be used for the isolation of fastidious organisms that require blood as a growth factor.



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