Brain Heart Infusion

Intended Use

Brain Heart Infusion
Brain Heart Infusion (BHI) is a general-purpose liquid medium used in the cultivation of fastidious and nonfastidious microorganisms, including aerobic and anaerobic bacteria, from a variety of clinical and nonclinical materials. It serves as a base for supplemented media containing 0.1% agar, Fildes enrichment or 6.5% sodium chloride. A supplemented pre-reduced formulation in tubes is especially recommended for the cultivation of anaerobes.
Brain Heart Infusion Agars
Brain Heart Infusion (BHI) Agar is a general-purpose medium suitable for the cultivation of a wide variety of organism types, including bacteria, yeasts and molds. With the addition of 5% or 10% sheep blood, it is used for the isolation and cultivation of a wide variety of fungal species, including systemic fungi,¹ from clinical and nonclinical sources.

Summary and Explanation

Brain Heart Infusion
Rosenow described brain-heart infusion broth prepared by adding pieces of brain tissue to meat infusion or beef extractdextrose broth.¹ A variation of this medium appeared for many years in the National Formulary.² The current formulation is similar to the NF Brain Heart Infusion Broth, but the brain infusion component is composed of solids resulting from the drying of the liquid material and the heart infusion component has been replaced with a peptone of partially digested animal tissue.
BHI broth is used for the cultivation of a wide variety of microorganisms, including bacteria, yeasts and molds. BHI broth, 0.5 mL per tube, is used for the cultivation of bacteria employed in the preparation of inocula for microdilution minimal inhibitory concentration (MIC) and identification (ID) test panels. When a large number of cells are inoculated into the small volume of broth, a bacterial culture rapidly reaches its stationary phase of growth.³ The medium is also used in 5-mL amounts per tube for the preparation of inocula in antimicrobial susceptibility test procedures. This volume and the 8-mL tubes also can be used for general purposes.
Fildes enrichment may be incorporated for the growth of fastidious organisms. With the addition of 0.1% agar, the medium is used for the cultivation of anaerobes. The medium pre-reduced in Hungate tubes is recommended for the cultivation of anaerobic microorganisms, particularly obligate anaerobes. The broth medium that contains 6.5% sodium chloride is used to differentiate the enterococci from nonenterococcal group D streptococci by the 6.5% salt tolerance test.⁴
Brain Heart Infusion without Dextrose is a basal medium used with carbohydrates for fermentation studies.
Brain Heart Infusion, Modified differs from other formulations by the quantities of the ingredients and the substitution of pancreatic digest of casein for pancreatic digest of gelatin.
Brain Heart Infusion Agars
In the early years of bacteriology, meat infusions were utilized as the growth-supporting components in a large number of culture media. Although they were cumbersome to prepare, lacked consistency from batch to batch and were undefined as to their nutritive content, they enabled the cultivation of microorganisms in both solid and liquid media. As the state of the art in enzymology and chemistry advanced, methods were developed for the preparation of peptones that were the result of enzymatic or acid hydrolysis of animal tissues or products and vegetable substances. These peptones currently are the major nutritional additives to culture media formulations, but infusions are still utilized in specific media.
BHI Agar is one formulation in which meat infusion is used, although, unlike in the earlier days, the infusion components are solids resulting from the drying of the liquid infusion material rather than the liquid components themselves. Peptones are also included as sources of nutrients.
Brain Heart Infusion Agar, Modified, the agar form of Brain Heart Infusion, Modified, differs from other formulations by the quantities of the infusion and peptone components utilized. BHI Agar has proven to be effective in the cultivation of a wide variety of microorganisms, including many types of pathogens. BHI Agar can be used as a general medium for aerobic bacteriology and for the primary recovery of fungi from clinical specimens.² Brain Heart Infusion Agar with 10% Sheep Blood can be used to isolate systemic fungi that may grow poorly on the nonenriched medium. Antimicrobial agents, including chloramphenicol, gentamicin, and penicillin in combination with streptomycin, can be incorporated to improve the recovery of pathogenic fungi from specimens heavily contaminated with bacteria (see Selective Brain Heart Infusion Agars).³

Principles of the Procedure

Brain Heart Infusion
BHI Broth is a nutritious, buffered culture medium that contains infusions of brain and heart tissue and peptones to supply protein and other nutrients necessary to support the growth of fastidious and nonfastidious microorganisms. In the formulation containing 6.5% sodium chloride, the salt acts as a differential and/or selective agent by interfering with membrane permeability and osmotic and electrokinetic equilibria in salt-intolerant organisms. Fildes enrichment (peptic digest of sheep blood) is incorporated into one tubed formulation for the cultivation of fastidious microorganisms, such as Haemophilus influenzae.^5,6 The addition of 0.1% agar aids in the cultivation of anaerobic microorganisms because its consistency yields conditions of reduced oxygen tension. The pre-reduced medium in Hungate tubes is based on Hungate methods of culturing anaerobic microorganisms outside of an anaerobic chamber.⁷ The tubes provide a reduced medium in a self-contained, anaerobic tube sealed using a Hungate screw cap. The cap contains a butyl rubber septum stopper that permits inoculation and incubation without exposing the medium to air.
Brain Heart Infusion Agars
BHI Agar derives its nutrients from the brain heart infusion, peptone and dextrose components. The peptones and infusion are sources of organic nitrogen, carbon, sulfur, vitamins and trace substances. Dextrose is a carbohydrate source that microorganisms utilize by fermentative action. The medium is buffered through the use of disodium phosphate.
When defibrinated sheep blood is added to the basal medium, it provides essential growth factors for the more fastidious fungal organisms.

User Quality Control

NOTE: Differences in the Identity Specifications and Cultural Response testing for media offered as both Difco™ and BBL™ brands may reflect differences in the development and testing of media for industrial and clinical applications, per the referenced publications.
Identity Specifications
Bacto™ Brain Heart Infusion
Dehydrated Appearance: Light tan, free-flowing, homogeneous.
Solution:                        3.7% solution, soluble in purified water upon
                                     boiling. Solution is light to medium amber, clear.
Prepared Appearance:     Light to medium amber, clear.
Reaction of 3.7%
Solution at 25°C:            pH 7.4 ± 0.2
BBL™ Brain Heart Infusion
Dehydrated Appearance: Fine, homogeneous, free of extraneous material.
Solution:                        3.7% solution, soluble in purified water upon
                                     boiling. Solution is light to medium, yellow
                                     to tan, clear to slightly hazy.
Prepared Appearance:    Light to medium, yellow to tan, clear to
slightly hazy.
Reaction of 3.7%
Solution at 25°C:            pH 7.4 ± 0.2
Difco™ Brain Heart Infusion Agar
Dehydrated Appearance: Beige, free-flowing, homogeneous.
Solution:                        5.2% solution, soluble in purified water upon boiling.
                                     Solution is light to medium amber, slightly
                                     opalescent to opalescent with a flocculent precipitate.
Prepared Appearance:     Light to medium amber, slightly opalescent to
                                     opalescent with a flocculent precipitate.
Reaction of 5.2%
Solution at 25°C:            pH 7.4 ± 0.2
BBL™ Brain Heart Infusion Agar
Dehydrated Appearance: Fine, homogeneous, free of extraneous material.
Solution:                        5.2% solution, soluble in purified water upon
                                     boiling. Solution is medium to dark, yellow
                                     to tan, trace to moderately hazy.
Prepared Appearance:    Medium to dark, yellow to tan, trace to moderately hazy.
Reaction of 5.2%
Solution at 25°C:            pH 7.4 ± 0.2
Cultural Response
Bacto™ Brain Heart Infusion 
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 18-48 hours.

BBL™ Brain Heart Infusion 
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C under appropriate atmospheric conditions for 7 days (incubate C. albicans at 20-27°C).

Difco™ Brain Heart Infusion Agar
Prepare the medium per label directions without (plain) and with 5% defibrinated sheep blood (SB). Inoculate and incubate at 35 ± 2°C with 5-10% CO2 for 18-48 hours (incubate A. brasiliensis aerobically at 30 ± 2°C for 18-72 hours).

BBL™ Brain Heart Infusion Agar
Prepare the medium per label directions without (plain) and with 5% defibrinated sheep blood (SB). Inoculate and incubate at 35 ± 2°C under appropriate atmospheric conditions for 48 hours (incubate S. rimosus at 23-27°C for up to 7 days if necessary).

Formula

Bacto™ Brain Heart Infusion
Approximate Formula* Per Liter
Calf Brains, Infusion from 200 g.................................... 7.7 g
Beef Heart, Infusion from 250 g.................................... 9.8 g
Proteose Peptone....................................................... 10.0 g
Dextrose..................................................................... 2.0 g
Sodium Chloride.......................................................... 5.0 g
Disodium Phosphate..................................................... 2.5 g
BBL™ Brain Heart Infusion
Approximate Formula* Per Liter
Brain Heart, Infusion from (solids)................................. 6.0 g
Peptic Digest of Animal Tissue....................................... 6.0 g
Pancreatic Digest of Gelatin......................................... 14.5 g
Dextrose..................................................................... 3.0 g
Sodium Chloride.......................................................... 5.0 g
Disodium Phosphate..................................................... 2.5 g
Difco™ Brain Heart Infusion Agar
Approximate Formula* Per Liter
Calf Brains, Infusion from 200 g.................................... 7.7 g
Beef Heart, Infusion from 250 g.................................... 9.8 g
Proteose Peptone....................................................... 10.0 g
Dextrose..................................................................... 2.0 g
Sodium Chloride.......................................................... 5.0 g
Disodium Phosphate..................................................... 2.5 g
Agar.......................................................................... 15.0 g
BBL™ Brain Heart Infusion Agar
Approximate Formula* Per Liter
Brain Heart, Infusion from (solids)................................. 8.0 g
Peptic Digest of Animal Tissue....................................... 5.0 g
Pancreatic Digest of Casein......................................... 16.0 g
Dextrose..................................................................... 2.0 g
Sodium Chloride.......................................................... 5.0 g
Disodium Phosphate..................................................... 2.5 g
Agar.......................................................................... 13.5 g
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

Bacto™ Brain Heart Infusion
1. Suspend the powder in 1 L of purified water:
Bacto™ Brain Heart Infusion – 37 g;
BBL™ Brain Heart Infusion – 37 g;
Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
3. Autoclave at 121°C for 15 minutes.
4. Test samples of the finished product for performance using stable, typical control cultures.
Bacto™ Brain Heart Infusion Agar
1. Suspend the powder in 1 L of purified water: Difco™ Brain Heart Infusion Agar – 52 g;
BBL™ Brain Heart Infusion Agar – 52 g;
Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
3. Autoclave at 121°C for 15 minutes.
4. Before use, agitate gently to distribute the precipitate uniformly throughout the medium.
5. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

Bacto™ Brain Heart Infusion
With liquid specimens, tubed media should be inoculated with 1-2 drops of the specimen using a sterile pipette. Swab specimens may be inserted into broth after inoculation of plated media.
Liquid media for anaerobic incubation should be reduced prior to inoculation by placing the tubes, with caps loosened, under anaerobic conditions for 18-24 hours prior to use. An efficient and easy way to obtain suitable anaerobic conditions is through the use of BD GasPak™ EZ anaerobic systems or an alternative anaerobic system. Alternatively, liquid media may be reduced immediately prior to use by boiling with caps loosened and cooling with tightened caps to room temperature before inoculation.
Before inoculating Hungate tubes, disinfect the septum of the cap. To inoculate, insert needle of syringe containing specimen through the septum and inject the specimen into the medium. Withdraw the needle slowly to avoid introducing air into the tube. For use in antimicrobial susceptibility testing, refer to appropriate references.^8-10
Bacto™ Brain Heart Infusion Agar
Prepare plated medium from tubed agar deeps by liquefying the medium in boiling water, cooling to 45-50°C and pouring into sterile Petri dishes. Additives (e.g., blood) can be used as desired.
Use standard procedures to obtain isolated colonies from specimens. Since many pathogens require carbon dioxide on primary isolation, plates of plain BHI may be incubated in an atmosphere containing approximately 5-10% CO2. Incubate plates at 35 ± 2°C for 24-48 hours.
For isolation of fungi from potentially contaminated specimens, a selective medium should be inoculated along with the nonselective medium. Incubate the plates at 25-30°C in an inverted position (agar side up) with increased humidity. For isolation of fungi causing systemic mycoses, two sets of media should be inoculated, with one set incubated at 25-30°C and a duplicate set at 35 ± 2°C. All cultures should be examined at least weekly for fungal growth and should be held for 4-6 weeks before being reported as negative.
BHI Agar slants primarily are used for the cultivation and maintenance of pure cultures of microorganisms.

Expected Results

Bacto™ Brain Heart Infusion
Growth in the tubes is indicated by the presence of turbidity compared to an uninoculated control. If growth appears, cultures should be examined by Gram stain and subcultured onto appropriate media; e.g., a Trypticase™ Soy Agar with 5% Sheep Blood and/or Chocolate II Agar plate, EMB Agar or MacConkey II Agar plate. If anaerobes are suspected, subcultures should be incubated anaerobically, as in a GasPak EZ anaerobic system.
Enterococci will grow in the 6.5% NaCl broth within 24-48 hours. Nonenterococcal group D streptococci fail to grow in the medium after 48 hours of incubation.³
Bacto™ Brain Heart Infusion Agar
After sufficient incubation, the plates should show isolated colonies in streaked areas and confluent growth in areas of heavy inoculation. When culturing for fungi, examine plates for fungal colonies exhibiting typical color and morphology. Biochemical tests and serological procedures should be performed to confirm findings.
Slant cultures may be used as sources of inocula for additional studies or for organism maintenance purposes.

*Store at 2-8° C.

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