Overview

Wilkins-Chalgren Anaerobe Medium — broth (WChB) and agar (WChA) — is the CLSI M11 reference medium for the antimicrobial susceptibility testing (AST) of anaerobic bacteria. Originally described by Wilkins & Chalgren in 1976 (Antimicrobial Agents and Chemotherapy 10:926), the medium was specifically designed to deliver reproducible minimum-inhibitory-concentration (MIC) results for clinically important anaerobes — replacing the earlier meat-infusion-based and Brucella-based AST media with a chemically-defined formulation. It remains the global reference broth/agar for clinical and pharma AST workflows on anaerobes.

The GMExpression formulation is supplied with pre-balanced Tryptone, Gelysate Peptone, yeast extract, L-arginine, sodium pyruvate, glucose (1 g/L only, deliberately low to minimise pH drift during AST incubation), NaCl, hemin (5 mg/L), and menadione/Vitamin K₃ (0.5 mg/L). Agar concentration is 10 g/L for broth-grade WChB and 15 g/L for solid WChA. Pre-autoclaving pH is 7.1 ± 0.1 at 25 °C — the tight ± 0.1 specification is one of the reasons WCh delivers more reproducible MICs than BHI-S in inter-laboratory comparisons.

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 Wilkins-Chalgren kit contains:

  • Mixture A — pre-weighed dehydrated WCh base (Tryptone, Gelysate Peptone, yeast extract, L-arginine, sodium pyruvate, NaCl, glucose, hemin, menadione) for 5 L final volume. Reformulated for broth (33 g/L total) or agar (38 g/L total) format.
  • Stock M — Menadione (Vitamin K₃) stock, 1 mg/mL in 95 % ethanol, amber vial, light-protected (for top-up applications).
  • Stock H — Hemin stock, 25 mg in 5 mL of 0.05 M NaOH, sealed amber vial (for top-up applications).
  • 5 × airtight PP storage bags + 5 × heat-resistant rubber bands for PRAS-format dispensing.
  • Instruction manual including a 96-well-plate AST protocol annex (A5 booklet, v1.0).

Customisation options on request: add 5 % laked sheep blood (DAFF EX188M certified) for clindamycin and sulfonamide MIC; add 5 % defibrinated sheep blood (unlaked) for microaerophile AST; configure for 96-well plate pre-dispensing; supply with pre-titrated CLSI antibiotic QC panel.

Composition — per 1 L equivalent unless stated otherwise

WCh Broth (Wilkins & Chalgren 1976; BD Difco 257500 / Oxoid CM0619 reference; per 1 L)

ComponentConcentrationFunction
Tryptone (pancreatic digest of casein)10.0 gAmino-acid / peptide source
Gelysate Peptone (pancreatic digest of gelatin)10.0 gSupplementary peptide source
Yeast extract5.0 gB-vitamins, NAD precursors
D-Glucose1.0 gLimited carbohydrate — minimises pH drift during AST
Sodium chloride (NaCl)5.0 gOsmotic balance
L-Arginine1.0 gDefined nitrogen source; substrate for Bacteroides arginine deiminase
Sodium pyruvate1.0 gDetoxifies H2O2 from oxidative metabolism
Menadione (Vitamin K3, CAS 58-27-5)0.5 mgMenaquinone bypass for haem-requiring anaerobes
Hemin (CAS 16009-13-5)5.0 mgRequired by Bacteroides and other haem-auxotrophs
For broth: agar (semi-solid format)0 g (broth) / 10 g (broth-equivalent)Standard broth has no agar

Pre-autoclaving pH: 7.1 ± 0.1 at 25 °C — adjust with 1 M NaOH or 1 M HCl.

WCh Agar (BD Difco 257600 / Oxoid CM0643 reference; per 1 L)

ComponentConcentrationNotes
WCh broth base (as above)23.0 g (all components above except agar)Identical formulation
Agar (microbiological grade)15.0 gSolid plate format
Laked sheep blood (optional, for clindamycin and sulfonamide MIC)5 % v/vAdd at 50 °C post-autoclave; laking releases thymidine that would otherwise antagonise sulfonamides

→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 Wilkins-Chalgren Anaerobe Medium with the wider YCFA, Chopped Meat Broth, BHI-S, Modified GAM, Anaerobic Columbia Blood Agar, GMM, M2GSC, BBE, KVLB, CCFA, TPY, and Egg Yolk Agar catalogue.

Use and Applications

  • Anaerobic AST — broth microdilution. WCh broth is dispensed in 96-well plates with serial antibiotic dilutions; inoculum standardised to 1 × 106 CFU/mL final concentration; plates incubated in an AAE at 35 ± 2 °C for 42–48 h; MIC read as lowest concentration with no visible turbidity. CLSI M11 reference organisms: B. fragilis ATCC 25285 and B. thetaiotaomicron ATCC 29741.
  • Anaerobic AST — agar dilution. WCh agar with serial antibiotic dilutions; inoculate with a Steers replicator; CLSI M11 standard.
  • E-test® / gradient strip MIC determination on anaerobes — WCh agar is the recommended supporting medium (bioMérieux E-test instructions).
  • Routine anaerobic culture and primary isolation as an alternative to BHI-S or Brucella blood agar; supports virtually all clinically significant anaerobes.
  • Pharma antimicrobial drug-discovery — comparative MIC testing of novel anti-anaerobic candidates; the major non-clinical demand segment.
  • Daily QC plates in clinical microbiology laboratories per CLSI M22 requirements.

Compatible Microorganisms

Reference QC organisms (CLSI M11)

  • Bacteroides fragilis ATCC 25285 — primary AST reference; clindamycin / metronidazole / cefoxitin / carbapenem QC organism
  • Bacteroides thetaiotaomicron ATCC 29741 — secondary reference; β-lactam-resistance QC
  • Eggerthella lenta ATCC 43055 — metronidazole and erythromycin QC
  • Clostridium perfringens ATCC 13124 — penicillin / chloramphenicol QC
  • Clostridioides difficile ATCC 700057 — metronidazole / vancomycin QC
  • Fusobacterium nucleatum ATCC 25586 — β-lactam-susceptible anaerobe AST

General-purpose growth (non-AST)

  • Bacteroides spp., Parabacteroides spp., Prevotella spp., Porphyromonas spp.
  • Clostridium spp., Clostridioides difficile, Peptostreptococcus anaerobius, Finegoldia magna
  • Veillonella spp., Cutibacterium acnes, Actinomyces spp.
  • Fusobacterium spp.

Not optimised for: SCFA-utilising Firmicutes (Faecalibacterium prausnitzii, Roseburia) — these require YCFA Modified Medium for satisfactory recovery.

Preparation

1Weigh. Use the pre-weighed Mixture A: 33.0 g per litre for WCh broth, 38.0 g per litre for WCh agar. Tare a clean autoclavable Schott bottle 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). Heat with frequent agitation to 95–100 °C until completely dissolved (typically 5–8 min). Do not overheat.
3Adjust pH. Cool briefly to ~25 °C and verify pH with a temperature-corrected calibrated meter. Target 7.1 ± 0.1 at 25 °C. Adjust with 1 M NaOH or 1 M HCl as required. The tight ± 0.1 spec is critical for AST reproducibility.
4Bring to final volume. Make up to 1000 mL with distilled water.
5Dispense. Broth tubes: 5–10 mL per Hungate or screw-cap tube. For 96-well AST plates: dispense per-well after autoclaving and cooling — do not autoclave in 96-well plates.
6Autoclave. 121 °C × 15 min (103 kPa). Slow cooling — do not vent rapidly.
7Optional blood supplementation. For laked sheep blood agar: cool to 50 °C, add 50 mL/L of laked sheep blood (prepared by one freeze-thaw cycle of defibrinated blood). Pour plates at 20–25 mL per plate inside a laminar-flow hood.
8QC. Each batch should be QC-tested with B. fragilis ATCC 25285 and B. thetaiotaomicron ATCC 29741 plus a CLSI-recommended antimicrobial QC range per CLSI M11.

Critical control points

  • pH measurement temperature. WCh's tighter pH specification (± 0.1) requires the pH meter to be temperature-corrected to 25 °C. Measuring at autoclave-exit temperature (~80 °C) will give a falsely low reading by 0.2–0.3 pH units due to the temperature coefficient of the phosphate / amine buffer system.
  • Menadione light-sensitivity. Vitamin K3 photoisomerises to its trans-form (inactive) under fluorescent or sunlight exposure. Protect both the stock and the prepared plates from extended light exposure; wrap WCh agar plates in aluminium foil for long-term storage.
  • AST inoculum standardisation. Per CLSI M11, the inoculum is prepared by suspending colonies in WCh broth (or saline), adjusting to 0.5 McFarland (1.5 × 108 CFU/mL), and diluting 1:10 in WCh broth to give the 1.5 × 107 CFU/mL AST inoculum. The final in-well density is 1 × 106 CFU/mL after 1:15 dilution. Inoculum-density deviations are the single most common source of unreproducible MICs.

Cautions

Maintain CLSI-mandated pH and inoculum density. WCh's reproducibility advantage hinges on pH 7.1 ± 0.1 and inoculum standardisation. Do not substitute for BHI-S or Brucella unless the substitution is documented in the QA file.
Glucose content is deliberately low. WCh has only 1 g/L glucose (vs 2 g/L in BHI). Adding extra glucose will lower pH during AST incubation and invalidate MIC results. Do not supplement.
Light protection. Menadione is light-sensitive. Store powders and prepared plates in light-protected packaging; for prolonged storage, wrap plates in aluminium foil.
Blood-product safety. Laked sheep blood is a biological hazard. Handle in a Class II biosafety cabinet. DAFF EX188M-certified zoosanitary documentation available.
Lot-to-lot QC. Each batch of prepared medium should be tested against the CLSI M11 reference organisms and antibiotic QC ranges before being released for clinical AST use.

Storage and Expiry · Safety

  • Dehydrated powder (Mixture A): store sealed at 15–30 °C away from direct sunlight. Shelf life 36 months from manufacture.
  • Stock M (menadione in ethanol): store at 4 °C protected from light. Stable 6 months.
  • Stock H (hemin in NaOH): store at 4 °C protected from light. Stable 8 weeks.
  • Prepared broth, aerobic 4 °C: 4 weeks routine; 6 weeks if not subject to repeated opening.
  • Prepared agar plates, aerobic 4 °C, foil-wrapped: 4 weeks; blood-supplemented agar: 2 weeks.
  • Prepared broth, anaerobic (vacuum-sealed + oxygen absorber): 6 months.

Safety notes. WCh is largely defined; the principal biosafety concerns are (i) the BSL-2 organisms being tested (handle in Class II BSC) and (ii) the menadione ethanol stock (flammable). Laked sheep blood is bovine-derived (DAFF EX188M certified). SDS available on request.

References

  1. Wilkins TD, Chalgren S. (1976). Medium for use in antibiotic susceptibility testing of anaerobic bacteria. Antimicrobial Agents and Chemotherapy 10(6): 926–928. doi: 10.1128/AAC.10.6.926.
  2. CLSI M11, current edition. Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved Standard. Wayne, PA: Clinical and Laboratory Standards Institute.
  3. CLSI M100, current edition. Performance Standards for Antimicrobial Susceptibility Testing.
  4. Jousimies-Somer HR et al. (2002). Wadsworth-KTL Anaerobic Bacteriology Manual, 6th ed., chapter 7.
  5. BD Difco™ Wilkins Chalgren Anaerobe Broth (257500) and Agar (257600) product information sheets.
  6. Oxoid Manual 9th ed., CM0619 (broth) and CM0643 (agar).
  7. Carroll KC et al. (eds). (2019). Manual of Clinical Microbiology, 12th ed. ASM Press.

Frequently Asked Questions

Q1. Why is the pH specification ± 0.1 instead of the more usual ± 0.2?
WCh was specifically engineered for reproducible inter-laboratory MIC determination. AST results — particularly for ionisable antibiotics such as β-lactams, tetracyclines, and macrolides — depend on pH because the protonation state of the drug determines its membrane permeability. A pH variation of 0.2 units can shift the MIC of an ionisable drug by one doubling dilution, the smallest unit detectable in serial-dilution AST. The ± 0.1 spec is therefore not a quality marker — it is a methodological requirement of CLSI M11.
Q2. Can I use WCh broth in place of BHI-S for routine anaerobic enrichment?
Yes. WCh broth supports the same broad range of anaerobes as BHI-S and is occasionally preferred where the lower glucose content (1 g/L vs 2 g/L) reduces pH drift in long enrichment incubations. The reverse substitution — using BHI-S in place of WCh for AST — is permitted by CLSI M11 only as an alternative when WCh is unavailable, and AST reports should explicitly state which medium was used.
Q3. Why does the agar contain menadione but not Vitamin K1?
Wilkins and Chalgren (1976) showed that menadione (VK3) is cheaper, more chemically stable, and equivalently effective at supporting Bacteroides menaquinone biosynthesis as Vitamin K1 (phylloquinone). Bacteroides spp. salvage menadione via a single methylation step into its menaquinone pool. Vitamin K1 use is therefore a BHI-S convention but not the CLSI M11 reference.
Q4. How do I add the laked sheep blood for clindamycin MIC?
Laking is one freeze-thaw cycle of defibrinated sheep blood: aliquot defibrinated blood into sterile bottles, freeze at −20 °C for ≥ 4 h, thaw at 37 °C, and add 50 mL per 1 L of WCh agar that has cooled to 50 °C. Laking releases intracellular thymidine, which would otherwise antagonise sulfonamides and the diaminopyrimidines (trimethoprim). Without laking, the blood retains intact erythrocytes and is unsuitable for MIC determinations involving these drug classes.
Q5. Is WCh compatible with the GMExpression Anaerobic Preparation Kit?
Yes. The APK workflow (vacuum deoxygenation, N2 + oxygen-absorber packing) applies identically to WCh broth and to WCh agar plates. For AST use, however, the 96-well plates are typically prepared fresh and used within 24 h, so APK packaging is most relevant for the bulk broth supply and for stored agar plate stock.
Q6. Can I include resazurin in WCh broth as an anaerobic indicator?
Standard CLSI M11 WCh broth does not contain resazurin (unlike YCFA). Adding 1 mg/L resazurin would convert the broth into a coloured medium that complicates visual MIC reading (the 0.5 McFarland turbidity threshold is read against a clear-medium background). Resazurin is therefore not recommended for AST-grade WCh broth. For bulk anaerobic-enrichment use the resazurin-supplemented variant can be supplied on request as a custom order.
Q7. What is the expected colour of correctly prepared WCh agar?
Plain WCh agar: pale amber, transparent. WCh agar with laked sheep blood: dark red-brown to mahogany (the laked blood is fully haemolysed, so the agar is uniformly coloured rather than showing intact red cells). If the laked-blood agar shows a granular or speckled appearance, the laking cycle was incomplete — repeat the freeze-thaw before incorporation.
Q8. Is the ATCC 29741 reference strain the same as ATCC 29148?
No. B. thetaiotaomicron ATCC 29741 is the type strain and the CLSI M11 reference organism for AST. ATCC 29148 is a different B. thetaiotaomicron isolate (VPI-5482, sequenced reference genome by Xu et al. 2003 Science 299:2074). Use ATCC 29741 for AST QC; ATCC 29148 is the genomics-research reference strain.