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

Brain Heart Infusion (BHI) Agar is a widely used culture medium for the isolation and cultivation of a variety of microorganisms, especially those that are pathogenic or fastidious. It is based on the infusion of brain and heart tissues from animals, which provide a rich source of nutrients and growth factors for the microbes. BHI Agar can support the growth of bacteria, fungi, yeasts, molds and actinomycetes. It can also be modified by adding blood or antibiotics to enhance or inhibit the growth of certain organisms.

BHI Agar was first developed by Rosenow in 1916 as a medium for the cultivation of pneumococci and streptococci. Later, it was found to be useful for other organisms as well. BHI Agar is now one of the most commonly used media in clinical microbiology and research laboratories. It is also recommended by various standard methods and guidelines for the isolation and identification of microorganisms from different sources, such as blood, cerebrospinal fluid, respiratory specimens, urine, feces, wounds and environmental samples.

BHI Agar is a versatile medium that can be used for different purposes, such as:

• Primary isolation of aerobic bacteria from clinical specimens
• Isolation of pathogenic systemic fungi and actinomycetes
• Subculture and maintenance of bacterial and fungal strains
• Preparation of inocula for antimicrobial susceptibility testing
• Production of bacterial toxins and antigens
• Induction of sporulation in some bacteria
• Detection of hemolysis and other biochemical reactions

BHI Agar is a complex medium that contains various ingredients that provide nutrients and growth factors for a wide range of microorganisms. The main components of BHI Agar are:

• Brain heart infusion: This is a mixture of brain and heart tissue from bovine or porcine sources that is rich in proteins, peptides, amino acids, vitamins, minerals and other essential substances for microbial growth. Brain heart infusion also provides some buffering capacity to the medium.
• Proteose peptone: This is a partially digested protein that contains short chains of amino acids and peptides. Proteose peptone serves as an additional source of nitrogen, carbon and energy for the microorganisms.
• Dextrose: This is a simple sugar that serves as the main source of energy and carbon for the microorganisms. Dextrose also helps to maintain the osmotic balance of the medium.
• Sodium chloride: This is a common salt that helps to maintain the osmotic balance and the electrolyte concentration of the medium. Sodium chloride also enhances the growth of some halophilic or salt-tolerant microorganisms.
• Disodium phosphate: This is a salt that acts as a buffer to maintain the pH of the medium at around 7.4. Disodium phosphate also provides some phosphorus for the microorganisms.
• Agar: This is a polysaccharide extracted from seaweed that serves as a solidifying agent for the medium. Agar provides a firm surface for the microorganisms to grow and form colonies.

The final concentration of each ingredient in BHI Agar per liter of distilled water is as follows:

BHI Agar can be further supplemented with blood or antibiotics to enhance its performance for specific purposes. For example, adding sheep blood can provide hemin and other growth factors for fastidious organisms, while adding chloramphenicol or streptomycin can inhibit bacterial growth and select for fungi.

BHI Agar has a final pH of 7.4 ± 0.2 at 25°C after sterilization by autoclaving. The pH can be adjusted by adding small amounts of sodium hydroxide or hydrochloric acid before autoclaving if needed.

BHI Agar is available commercially in dehydrated form or in ready-to-use plates or tubes. It should be stored in a cool and dry place away from direct sunlight and moisture. It should also be checked for signs of deterioration such as discoloration, contamination or loss of gel strength before use.

Brain Heart Infusion (BHI) Agar is a highly nutritious medium that supports the growth 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.

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.

BHI Agar can be further enriched or rendered selective by adding different antibiotics or other supplements. For example, chloramphenicol, cycloheximide, penicillin and streptomycin can be used to inhibit a range of gram-positive and gram-negative bacteria and saprophytic fungi, while allowing the recovery of pathogenic fungi and Actinomycetales. Yeast extract can also be added to serve as a growth stimulant.

BHI Agar is thus a versatile medium that can be modified according to the specific needs of the microorganisms to be isolated or cultivated.

To prepare BHI agar, you will need the following ingredients per liter of medium:

• Brain heart infusion solids: 6.0 g
• Casein peptone: 14.5 g
• Meat peptone: 7.0 g
• Sodium chloride: 5.0 g
• Disodium phosphate: 2.5 g
• Dextrose: 2.0 g
• Agar: 15.0 g
• Demineralized water: 1000 ml

The steps for preparing BHI agar are as follows:

1. Suspend the ingredients in demineralized water and heat with frequent agitation to dissolve them completely.
2. Sterilize the medium by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
3. Cool the medium to 45-50°C and mix well.
4. Pour the medium into sterile Petri plates and let it solidify.

If you want to enrich the medium with blood or make it selective with antibiotics, you can add them after autoclaving and before pouring. For example, you can add:

• 5-10% sheep blood for isolation of dimorphic fungi and Actinomyces.
• 50 mg/l chloramphenicol or 40 mg/l streptomycin or a mixture of 50 mg/l gentamicin and 50 mg/l chloramphenicol for inhibition of bacteria and isolation of pathogenic systemic fungi.
• 20 units of penicillin and 40 µg streptomycin per ml of the medium for selective isolation of fungi.
• 2-3% sodium chloride for cultivation of halophilic Vibrio spp.

To use BHI agar, you need to streak the specimen onto the surface of the medium using a sterile inoculating loop. Depending on the type of microorganisms you want to isolate, you may need to use different incubation conditions and additional media. For example:

• For aerobic bacteria, incubate the plates at 35-37°C for 18-24 hours in an inverted position.
• For fungi, incubate the plates at 25-30°C in an inverted position with increased humidity for up to several weeks. You may also need to use a duplicate set of plates incubated at 35-37°C for isolation of fungi causing systemic mycoses.
• For Actinomyces, incubate the plates anaerobically at 35-37°C for up to two weeks.

After sufficient incubation, examine the plates for fungal and/or bacterial colonies exhibiting typical color and morphology. You may need to perform further biochemical tests and/or microscopic or serological procedures to confirm your findings.

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.

Some examples of common microorganisms that can grow on BHI Agar are:

• Streptococcus pneumoniae: small, alpha-hemolytic, greenish colonies
• Trichophyton mentagrophytes: white to cream-colored, cottony colonies with a red pigment on the reverse
• Candida albicans: white to cream-colored, smooth, yeast-like colonies
• Listeria monocytogenes: small, grayish-white, non-hemolytic colonies
• Shigella flexneri: colorless, translucent colonies

Some examples of biochemical tests that can be used to differentiate microorganisms on BHI Agar are:

• Triple sugar iron agar (TSI): a differential medium that detects glucose fermentation, lactose fermentation, sucrose fermentation, and hydrogen sulfide production. Salmonella spp. produce acid from glucose only and hydrogen sulfide, resulting in a yellow butt and a black precipitate. Shigella spp. produce acid from glucose only but not hydrogen sulfide, resulting in a yellow butt and no black precipitate.
• Lysine iron agar (LIA): a differential medium that detects lysine decarboxylation, lysine deamination, and hydrogen sulfide production. Salmonella spp. produce lysine decarboxylase and hydrogen sulfide, resulting in a purple slant and a purple butt with a black precipitate. Shigella spp. do not produce lysine decarboxylase or hydrogen sulfide, resulting in a red slant and a yellow butt.
• Catalase test: a simple test that detects the presence of catalase enzyme, which breaks down hydrogen peroxide into water and oxygen. Staphylococcus spp. produce catalase and bubble when exposed to hydrogen peroxide. Streptococcus spp. do not produce catalase and do not bubble when exposed to hydrogen peroxide.
• Coagulase test: a test that detects the presence of coagulase enzyme, which causes plasma to clot. Staphylococcus aureus produces coagulase and forms clots in plasma. Other Staphylococcus spp. do not produce coagulase and do not form clots in plasma.

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.

It is also used for the cultivation of fastidious pathogenic bacteria, such as streptococci, meningococci and pneumococci, as well as yeasts and molds from clinical and non-clinical samples.

BHI Agar is a highly nutritious and non-selective medium that can support the growth of a wide range of microorganisms. However, this also means that it can be easily overgrown by normal flora or contaminants from the specimens, making it difficult to isolate the pathogens of interest. Therefore, it is advisable to use BHI Agar in conjunction with other selective or differential media to enhance the isolation and identification of the target organisms.

BHI Agar may not be suitable for some fastidious organisms that require specific growth factors or conditions that are not provided by the medium. For example, some anaerobic bacteria may not grow well on BHI Agar due to the presence of oxygen or the lack of reducing agents. Some obligate intracellular parasites or viruses may not grow at all on BHI Agar as they need living host cells to replicate. Some bacteria may produce pigments or hemolysis that are not visible on BHI Agar due to its dark color. Therefore, it is important to know the nutritional and environmental requirements of the organisms being cultured and to use appropriate media accordingly.

BHI Agar is a general purpose medium that does not provide any diagnostic information about the organisms growing on it. It does not differentiate between gram-positive and gram-negative bacteria, nor does it indicate their metabolic or biochemical characteristics. Therefore, further tests and procedures are necessary to confirm the identity and characteristics of the isolates. These may include gram staining, catalase test, oxidase test, coagulase test, indole test, sugar fermentation test, antibiotic susceptibility test, serological test, molecular test, etc. Depending on the type and complexity of the tests, they may require additional time, equipment, reagents and expertise to perform and interpret.

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