Overview

Brain Heart Infusion (Supplemented) — BHI-S — is the universal, general-purpose enrichment broth for fastidious aerobic, facultatively anaerobic, and obligately anaerobic bacteria. It is the most-cited general bacterial-growth medium in clinical microbiology globally (≥ 30 000 PubMed-indexed citations of "Brain Heart Infusion"). The GMExpression formulation provides pre-weighed BHI base supplemented with hemin (5 mg/L), Vitamin K1 (1 mg/L), and L-cysteine·HCl (0.5 g/L) for direct rehydration to a CLSI M22 / Wadsworth-aligned anaerobic enrichment broth.

BHI-S serves as the primary enrichment broth for fastidious anaerobes recovered from clinical specimens (blood, abscess aspirate, intra-abdominal fluid, deep wound, gynaecological / OB-GYN surgical specimens), as the inoculum broth for anaerobic antimicrobial susceptibility testing (AST) per CLSI M11 where Wilkins-Chalgren is unavailable, and as the cell-bank holding medium for Clostridium, Bacteroides, Bifidobacterium, and Faecalibacterium in pharma / FMT / live biotherapeutic product manufacturing workflows. It is also the base for BHI-blood agar and BHI-chocolate agar when supplemented with agar and 5 % defibrinated sheep blood. Australian DAFF EX188M biosecurity-certified bovine source; customs-cleared into 20+ jurisdictions.

We also have

YCFA Modified Medium · YCFA Full Recipe · YCFA Base · Modified Chopped Meat Broth (ATCC 1490) · Chopped Meat with Carbohydrates · Beef Granules

Package Contents

Each GMExpression BHI-S kit contains:

  • Mixture Base — pre-weighed BHI base (calf-brain + beef-heart infusion solids, proteose peptone, dextrose, NaCl, Na2HPO4) for 5 L final volume.
  • [Optional] Stock H — Hemin stock, 25 mg in 5 mL of 0.05 M NaOH, sealed amber vial, ready-to-add.
  • [Optional] Stock K — Vitamin K1 stock, 50 mg in 5 mL of 95 % ethanol, amber vial, light-protected.
  • [Optional] Mixture L — L-cysteine·HCl·H2O 2.5 g (= 0.5 g/L final), N2-flushed PP bag.
  • [Along with Stocks] 5 × airtight PP storage bags + 5 × heat-resistant rubber bands for PRAS-format dispensing.
  • Instruction manual (A5 booklet, v1.0).

Customisation options on request: add 5 % defibrinated sheep blood (DAFF EX188M certified), add NaHCO3 2 g/L for AAE-CO2 buffering, omit Vitamin K1 for Clostridium-only workflows, add 15 g/L agar for solid-phase BHI-S agar.

Composition — per 1 L equivalent unless stated otherwise

Mixture A — Base BHI broth (per 1 L; ATCC Medium 44 / BD Difco 237500 reference)

ComponentConcentrationFunction
Calf-brain infusion (solids from 200 g brain)7.7 gComplex peptides, vitamins, growth factors
Beef-heart infusion (solids from 250 g heart)9.8 gHaematin precursors, amino acids
Proteose Peptone (pancreatic digest)10.0 gPeptide nitrogen source
Dextrose (D-glucose)2.0 gCarbohydrate energy source
Sodium chloride (NaCl)5.0 gOsmotic balance
Disodium phosphate (Na2HPO4)2.5 gPhosphate buffer

Pre-autoclaving pH: 7.4 ± 0.2 at 25 °C (adjust with 1 M NaOH or 1 M HCl as required).

Anaerobic supplements (added per the supplied stock vials)

ComponentFinal concentrationStock and gradeNotes
Hemin (porphyrin-Fe complex; CAS 16009-13-5)5.0 mg/L5 mg/mL in 0.05 M NaOH (Stock H)Required by Bacteroides (haem-auxotroph)
Vitamin K1 (phylloquinone; CAS 84-80-0)1.0 mg/L10 mg/mL in 95 % ethanol (Stock K); protect from lightMenaquinone precursor; required by Bacteroides fragilis fumarate reductase
L-Cysteine·HCl·H2O (CAS 7048-04-6)500 mg/L (0.05 % w/v)Solid (Mixture C); add directly OR pre-dissolve as 5 % stock in oxygen-free waterReductant; final Eh < −150 mV
NaHCO3 (optional, for AAE-CO2)1–4 g/LFilter-sterilised 100 mg/mL post-autoclavepH stabilisation in CO2-equilibrated AAE
Defibrinated sheep blood (optional)5 % v/vSterile commercial; add at 50 °C post-autoclaveProvides X (hemin) and V (NAD) factors

For BHI-S agar: add 15 g/L bacteriological-grade agar before autoclaving.
For BHI-blood agar: add 50 mL/L sterile defibrinated sheep or horse blood at 48–50 °C post-autoclave and pour plates immediately.

→Culturomics and Media Navigator

Need help matching this medium to your microorganism — or starting from a target organism and finding the medium that fits? Use the GMExpression Culturomics and Media Navigator: a curated, cross-referenced tool that lets you (i) start from a medium and view the bacterial taxa it is recommended for, or (ii) start from a target organism (genus, species, or DSMZ/ATCC strain ID) and view the panel of GMExpression media that will support it. The Navigator integrates this product with the wider YCFA, Chopped Meat Broth, Wilkins-Chalgren, BHI-S, GAM, GMM, M2GSC, BBE, KVLB, CCFA, TPY, and Egg Yolk Agar catalogue.

Use and Applications

  • Primary isolation & enrichment of fastidious anaerobes from clinical specimens (blood, abscess aspirate, intra-abdominal fluid, deep wound, OB-GYN surgical specimens) per CLSI M22 and Wadsworth Manual ch. 6.
  • Cell-bank holding and working-cell-bank propagation for Clostridium, Bacteroides, Bifidobacterium, and Faecalibacterium in pharma / FMT / live biotherapeutic product manufacturing.
  • Antimicrobial susceptibility testing inoculum broth — CLSI M11-acceptable alternative when Wilkins-Chalgren is unavailable; supplementation with laked sheep blood produces "BHI-blood broth" used for Streptococcus pneumoniae and other capsular pathogens.
  • Maintenance medium for Streptococcus, Enterococcus, Staphylococcus, Listeria, Neisseria, Haemophilus (with supplementation), and other fastidious facultative anaerobes.
  • Blood-culture subculture broth for follow-up identification of positive blood-culture bottles.
  • Base medium for chocolate agar, BHI-blood agar, and BHI-chocolate agar when supplemented with 5 % defibrinated sheep blood (haemolysed for chocolatising) at 50 °C post-autoclave.
  • Microaerophile culture — Campylobacter, Helicobacter pylori (with selective antibiotic supplements) under 5–10 % CO2 + 5–10 % O2 atmosphere.

Compatible Microorganisms

Obligate anaerobes

  • Bacteroides fragilis (ATCC 25285), B. thetaiotaomicron (ATCC 29148), B. vulgatus (ATCC 8482), B. uniformis (ATCC 8492); whole B. fragilis group
  • Parabacteroides distasonis (ATCC 8503), P. merdae (ATCC 43184)
  • Prevotella melaninogenica (ATCC 25845), P. intermedia (ATCC 25611), P. bivia, P. disiens
  • Porphyromonas gingivalis (ATCC 33277), P. asaccharolytica (ATCC 25260)
  • Fusobacterium nucleatum (ATCC 25586), F. necrophorum (ATCC 25286), F. varium
  • Clostridium perfringens (ATCC 13124), C. sporogenes (ATCC 19404), C. tetani, C. novyi, C. septicum, C. bifermentans
  • Clostridioides difficile (ATCC 9689; reclassified Lawson 2016 Anaerobe 40:95)
  • Peptostreptococcus anaerobius (ATCC 27337), Peptoniphilus spp., Anaerococcus spp., Finegoldia magna (ATCC 29328; formerly Peptostreptococcus magnus)
  • Veillonella parvula (ATCC 10790), V. atypica
  • Eubacterium limosum (ATCC 8486); E. lentum reclassified as Eggerthella lenta (ATCC 25559)
  • Cutibacterium acnes (ATCC 6919; formerly Propionibacterium acnes, Scholz & Kilian 2016 IJSEM 66:4422)
  • Lactobacillus spp., Bifidobacterium spp. (TPY medium preferred for Bifidobacterium)

Microaerophiles (with appropriate atmosphere)

  • Streptococcus pyogenes (ATCC 19615), S. agalactiae, S. pneumoniae, viridans group streptococci
  • Campylobacter jejuni, C. coli (with selective supplements)
  • Helicobacter pylori (with selective supplements; specialised Helicobacter media preferred)

Facultative anaerobes (aerobic incubation)

  • Staphylococcus aureus, S. epidermidis, coagulase-negative staphylococci
  • Enterococcus faecalis, E. faecium
  • Escherichia coli, Klebsiella, Enterobacter (no selective agents present)
  • Neisseria meningitidis, N. gonorrhoeae (chocolatised + CO2)
  • Haemophilus influenzae (chocolatised)
  • Listeria monocytogenes

Preparation

1Weigh. Use the pre-weighed Mixture A (37.0 g for a 1 L batch). Tare a clean autoclavable Schott bottle or Erlenmeyer flask of at least 1.5× final volume.
2Suspend & dissolve. Add Mixture A to 950 mL of distilled or deionised water (Type II reagent water, > 1 MΩ·cm). Swirl, then heat in a 95–100 °C water bath or to gentle boil on a stirrer-hot-plate until completely dissolved (5–10 min).
3Add anaerobic supplements pre-autoclaving. Pipette 1 mL of Stock H (= 5 mg/L hemin), 0.1 mL of Stock K (= 1 mg/L Vitamin K1), and 0.5 g of L-cysteine·HCl from Mixture C directly to the warm solution. Swirl to dissolve.
4Adjust pH. Cool briefly to ~45 °C, check pH with a calibrated meter; adjust to 7.4 ± 0.2 at 25 °C with 1 M NaOH or 1 M HCl. Solution should be straw-yellow to light amber.
5Bring to final volume. Make up to 1000 mL with distilled water.
6Dispense. Broth tubes (Hungate or screw-cap): 5–10 mL per tube. Bulk: leave in autoclavable bottle, cap one-quarter turn loose for pressure equilibration.
7Autoclave. 121 °C × 15 min (103 kPa). Slow cooling — do not vent rapidly (loss of dissolved CO2, bumping/foaming).
8Cool & reduce. Transfer to 50 °C water bath for plate pouring, or to 4 °C for storage. For PRAS tubes, move to AAE while still warm so residual oxygen is scavenged by the cysteine/hemin redox buffer.
9Post-autoclave sterile additions. If using NaHCO3 (filter-sterilised 100 mg/mL) or defibrinated blood, add after the medium reaches 50 °C; swirl gently and dispense or pour immediately.

Critical control points

  • L-cysteine added pre-autoclaving partially oxidises to cystine, lowering reductant capacity. For maximum reducing power, prepare a 5 % (w/v) stock in oxygen-free water (boiled 10 min and cooled under N2), filter-sterilise (0.22 µm), and add 10 mL/L post-autoclave inside an AAE.
  • Hemin must be fully in solution before autoclaving; undissolved hemin will not redissolve in the cooled medium and will appear as black flecks. Use the alkaline-stock approach (5 mg/mL in 0.05 M NaOH); pre-warm the medium to ≥ 45 °C before adding.
  • Vitamin K1 in ethanol stock can crash out of solution if added to cold medium; add to still-warm medium (≥ 45 °C) with rapid mixing.

Cautions

Hemin solubility. Free hemin (Fe3+-protoporphyrin IX chloride) is essentially insoluble in neutral aqueous solution because the propionate side chains exist as the carboxylic-acid form and the iron-porphyrin core stacks via π–π interactions. Dissolution requires alkaline pH (0.05 M NaOH), triethanolamine co-solvent, or an Fe3+-stabilising ligand. In YCFA Mixture B this has been documented as the "black-fleck precipitate" failure mode; the same chemistry applies to BHI-S. Use Stock H directly and store at 4 °C protected from light; remake every two weeks.
L-cysteine oxidation during autoclaving. L-cysteine·HCl autoxidises to cystine under autoclaving conditions, particularly in the presence of trace iron (which it has, via the hemin). Loss of free thiol = loss of reducing power = higher Eh = poorer growth of strict anaerobes. Post-autoclave addition of filter-sterilised cysteine restores reducing capacity if maximum performance is required.
Maillard / browning reactions. The combination of high-protein peptone and reducing sugars (dextrose) in BHI undergoes mild Maillard browning at 121 °C, producing a slight darkening. This is normal. Excessive browning (dark amber → brown) indicates either overlong autoclaving (> 20 min) or elevated dextrose. Limit autoclave time to 15 min.
pH drift on storage. Prepared BHI-S broth stored aerobically at 4 °C will gradually lose dissolved CO2 and rise in pH by 0.1–0.2 units over 4 weeks. Store under N2 + oxygen-absorber in vacuum-sealed bags (GMExpression Anaerobic Preparation Kit technique); use within 3 months when sealed.
Bovine-derived components and biosecurity. BHI contains calf-brain and beef-heart infusion solids. Although fully denatured during processing, EU, Japan, and Canada (CFIA) customs may apply TSE/BSE prion-risk checks. GMExpression uses the established Australian DAFF EX188M zoosanitary certificate pipeline; documentation packs available on request via support@gmexpression.com.
Excessive foaming. BHI is rich in surface-active peptides and foams vigorously on autoclaving. Do not fill containers more than half full; leave caps one-quarter turn loose; consider antifoam B (silicone-based, food-grade) at 0.05 mL/L for fermenter-scale batches.

Storage and Expiry · Safety

  • Dehydrated powder (Mixture A, C): store sealed at 15–30 °C in original packaging away from direct sunlight. Shelf life 36 months from manufacture.
  • Stock H (hemin in NaOH): store at 4 °C protected from light. Stable 8 weeks at supplied concentration; prepare fresh 5 mg/mL working stock every 2 weeks for maximum performance.
  • Stock K (Vitamin K1 in ethanol): store at 4 °C protected from light. Stable 6 months.
  • Prepared broth, aerobic 4 °C: 4 weeks routine; 8 weeks if not subject to repeated opening.
  • Prepared broth, anaerobic (vacuum-sealed + oxygen absorber): 6 months for strict-anaerobe use; up to 12 months for facultative-anaerobe use after cysteine depletion.

Safety notes. BHI is a bovine-derived medium. Handle in a Biosafety Class II cabinet when culturing BSL-2 pathogens. L-cysteine in solid form is a mild eye irritant; wear nitrile gloves and safety glasses during weighing. Vitamin K1 ethanolic stock is flammable; keep away from open flame. Hemin in alkaline NaOH is corrosive; pipette with care. SDS available on request.

References

  1. Rosenow EC. (1919). Studies on elective localization. Journal of Dental Research 1: 205–267. [Original BHI formulation]
  2. CLSI M22 (current ed.). Quality Assurance for Commercially Prepared Microbiological Culture Media; Approved Standard. Wayne, PA: CLSI.
  3. CLSI M11-A8 / A9 / current ed. Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria. Wayne, PA: CLSI.
  4. Jousimies-Somer HR, Summanen P, Citron DM, Baron EJ, Wexler HM, Finegold SM. (2002). Wadsworth-KTL Anaerobic Bacteriology Manual, 6th ed. Belmont, CA: Star Publishing — Chapter 6.
  5. Carroll KC, Pfaller MA, Landry ML, McAdam AJ, Patel R, Richter SS, Warnock DW (eds). (2019). Manual of Clinical Microbiology, 12th ed. Washington, DC: ASM Press — chapter 60.
  6. BD Difco™ Brain Heart Infusion product information sheet 237500 / 237200; BBL™ BHI sheet 211059.
  7. Oxoid Manual 9th ed., CM1135 Brain Heart Infusion Broth.
  8. ATCC Microbiology Catalogue: ATCC Medium 44 Brain Heart Infusion Agar/Broth.

Frequently Asked Questions

Q1. Can I use the YCFA Anaerobic Preparation Kit (APK-Lite / APK-Plus) for BHI-S preparation?
Yes. The APK vacuum-deoxygenation workflow is medium-agnostic. Steps 8–11 of the YCFA Modified Medium Instructions (Hungate-tube vacuuming, oxygen-absorber storage) apply directly to BHI-S in the same Hungate-tube format. The 30–60 min rest period to verify the resazurin colour change is the same.
Q2. Why is my BHI-S broth more yellow than the YCFA broth?
BHI base does not contain resazurin (the redox indicator that makes YCFA pink/red when oxidised and colourless/pale yellow when reduced). The natural colour of BHI broth is straw-yellow to amber. If you wish to use resazurin as a visual oxygen indicator, add 1 mg/L resazurin to the formulation (this matches the YCFA Mixture A spec). The standard CLSI / Wadsworth BHI-S does not include resazurin.
Q3. Can I use BHI-S as a replacement for YCFA when culturing gut microbiota?
Only partially. BHI-S supports the majority of gut anaerobes, but it lacks the short-chain fatty acid (SCFA) supplementation that makes YCFA the gold standard for butyrate-producing Firmicutes (Faecalibacterium prausnitzii, Roseburia intestinalis, Eubacterium rectale). For broad-spectrum gut-microbiome culturomics where SCFA-utilising Firmicutes are part of the target, YCFA remains preferred. BHI-S is appropriate when isolating clinical anaerobes from non-gut sources (wound, abscess, blood culture) or when the butyrate-producer enrichment is not specifically required.
Q4. How long can I store autoclaved BHI-S broth?
In aerobic storage at 4 °C with the screw-cap fully tightened: 4 weeks for routine use, 8 weeks if not subject to repeated opening. In anaerobic storage (vacuum-sealed with an oxygen absorber as per the YCFA Anaerobic Preparation Kit workflow): 6 months. After this point the L-cysteine reductant is largely consumed and the medium will not adequately support strict anaerobes; the medium remains suitable for facultative-anaerobe use for up to 12 months.
Q5. Do I need to add Vitamin K1 if I am only culturing Clostridium species?
Clostridium spp. do not require Vitamin K1 for growth (they generate their own menaquinones via a different biosynthetic pathway), but the supplement does not inhibit them and the standardised BHI-S formulation includes it. Omitting Vitamin K1 is acceptable for Clostridium-only workflows but introduces a deviation from the CLSI / Wadsworth reference formulation, which may complicate later regulatory submissions or method-comparison studies. The recommendation is to retain Vitamin K1 as standard.
Q6. Can I autoclave the post-autoclave cysteine stock separately and store it for later use?
L-cysteine·HCl in solution is autoxidised to cystine and an array of disulfide / sulfonic species over a period of days at room temperature in the presence of dissolved oxygen. The recommended practice is to prepare the 5 % stock fresh in oxygen-free water (boiled 10 min and cooled under N2 flush, or prepared inside an AAE), filter-sterilise (0.22 µm), and use within 14 days when stored at 4 °C. For repeated use, prepare in 5 mL aliquots and freeze at −20 °C; thaw individual aliquots as required. Avoid freeze-thaw cycling.
Q7. Why does my prepared BHI-S agar show a dark brown discoloration after autoclaving?
Two possible causes: (a) Maillard browning from autoclaving > 15 min at 121 °C; (b) hemin precipitation / aggregation (visible as fine black or dark-brown flecks). Confirm by visual inspection — diffuse uniform brown is Maillard (no functional impact); localised dark flecks are hemin (will not affect growth but may concern users). If hemin-flecking is observed, the root cause is incomplete hemin dissolution at the stock-preparation step; remake the hemin stock in 0.05 M NaOH and use within 2 weeks.
Q8. Is BHI-S an acceptable substitute for Wilkins-Chalgren broth in anaerobic AST?
BHI is listed as an alternative CLSI M11 AST medium for anaerobes when the Wilkins-Chalgren reference broth is unavailable, but it is not the reference medium. For published, peer-reviewed AST data BHI-S is acceptable; for clinical reporting that follows CLSI M11, Wilkins-Chalgren broth and agar are the preferred reference media and BHI-S is a backup.