Overview

Anaerobic Columbia Blood Agar — Columbia base supplemented with 5 % defibrinated sheep blood, hemin (5 mg/L) and Vitamin K1 (1 mg/L) — is the universal solid plate medium for primary isolation of obligate anaerobes from clinical specimens. It is the most widely-deployed anaerobic primary-isolation plate in clinical microbiology globally and is the Wadsworth-KTL and ASM Manual of Clinical Microbiology-recommended general-purpose anaerobic plate.

The GMExpression formulation uses the Ellner (1966) Columbia agar base, prepared with Australian DAFF EX188M biosecurity-certified defibrinated sheep blood. The medium permits reading of three independent phenotypes on a single plate: (1) colonial morphology (sub-millimetre Bacteroides & Prevotella through 2–3 mm Clostridium colonies); (2) haemolysis pattern (β-haemolysis of Streptococcus spp., α-haemolysis of viridans streptococci, and the pathognomonic double-zone β-haemolysis of Clostridium perfringens); and (3) pigment production (the bronze-to-black colonies of Prevotella melaninogenica and Porphyromonas gingivalis, the brick-red autofluorescence of Bacteroides ureolyticus group, the chartreuse fluorescence of Veillonella). It differentiates from the existing GMExpression Brucella Agar product line by Columbia base composition (Columbia is richer in beef-heart infusion and peptone), giving more luxuriant colony growth.

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 Anaerobic CBA kit contains:

  • Mixture A — pre-weighed Columbia base + agar (Pantothene-Casein Peptone, Bio-Polytone, beef heart infusion, corn starch, NaCl, agar 13 g/L) for 5 L final volume.
  • Stock H — Hemin stock, 25 mg in 5 mL of 0.05 M NaOH, sealed amber vial.
  • Stock K — Vitamin K1 stock, 50 mg in 5 mL of 95 % ethanol, amber vial, light-protected.
  • Sheep blood pack — 250 mL defibrinated sheep blood (DAFF EX188M certified), shipped at 2–8 °C separately.
  • 50 × Petri plates (90 mm) or 100 × Petri plates (60 mm) if pre-poured option selected.
  • Instruction manual (A5 booklet, v1.0) with phenotype-reading colour atlas annex.

Customisation options on request: selective variants (kanamycin-vancomycin laked blood = KVLB; Bacteroides-Bile-Esculin = BBE — see related product GMNB-BBE01/KVLB01); chocolatised variant (haemolysed at 80 °C for Haemophilus); 5 % horse blood (in place of sheep); supply as pre-poured plates or as bulk powder + blood for in-house pouring.

Composition — per 1 L equivalent unless stated otherwise

Columbia Agar Base (Ellner 1966; BD Difco 211125 reference; per 1 L)

ComponentConcentrationFunction
Pantothene-Casein Peptone (pancreatic digest of casein)10.0 gHigh-quality peptide nitrogen source
Bio-Polytone (peptic digest of animal tissue)5.0 gSupplementary peptide source
Yeast extract5.0 gB-vitamins, growth factors
Beef heart infusion solids3.0 gAmino acids, haematin precursors
Corn starch1.0 gTrace-toxin binding (absorbs autoclave-derived inhibitors)
Sodium chloride (NaCl)5.0 gOsmotic balance
Agar (microbiological grade)13.0 gSolidifying agent (note: Columbia base uses 13 g/L, not the more usual 15 g/L)

Anaerobic supplements (post-autoclave additions)

ComponentFinal concentrationStock and addition
Hemin (CAS 16009-13-5)5.0 mg/L1 mL of 5 mg/mL stock (Stock H) per litre of base
Vitamin K1 (phylloquinone, CAS 84-80-0)1.0 mg/L0.1 mL of 10 mg/mL stock (Stock K) per litre of base
Defibrinated sheep blood (DAFF EX188M)5 % v/v (50 mL/L)Add at 48–52 °C post-autoclave; pour plates immediately

Pre-autoclaving pH: 7.3 ± 0.1 at 25 °C. Adjust with 1 M NaOH or 1 M HCl.

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

Use and Applications

  • Primary isolation of obligate anaerobes from clinical specimens — the most-used anaerobic primary plate globally; abscess aspirate, deep-tissue and surgical specimens, blood-culture subculture, intra-abdominal fluid, OB-GYN specimens.
  • Differential plate reading haemolysis and pigment phenotypes alongside colonial morphology — C. perfringens double-zone β-haemolysis is essentially pathognomonic; Prevotella / Porphyromonas bronze-to-black pigmentation enables genus-level presumptive identification at 24–48 h.
  • UV-fluorescence reading under 365 nm Wood's lamp: brick-red fluorescence (Bacteroides ureolyticus group), chartreuse / lime-green (Veillonella), coral-pink (Porphyromonas asaccharolytica).
  • Sub-culture plate for clinical anaerobes after enrichment in BHI-S or Modified Chopped Meat Broth.
  • Base for selective derivatives — KVLB (kanamycin + vancomycin + laked sheep blood, for Gram-negative anaerobes), BBE (with oxgall + esculin, for B. fragilis group), PEA (phenylethyl alcohol, for Gram-positive anaerobes), CCMB (cycloserine-cefoxitin-mannitol, for C. difficile).
  • Compatible with anaerobic gas-jar / AAE / GasPak workflows at 35–37 °C; reading at 24, 48, and 72 h.

Compatible Microorganisms

Obligate anaerobes

  • Bacteroides fragilis ATCC 25285, B. thetaiotaomicron ATCC 29148/29741, B. vulgatus, B. uniformis — grey-white, 2–4 mm, non-haemolytic to weakly α-haemolytic colonies
  • Prevotella melaninogenica (ATCC 25845) — small (1–2 mm) colonies developing bronze-to-black pigment by 72 h
  • Porphyromonas gingivalis (ATCC 33277) — slower-growing (5–7 d), black pigmented; brick-red Wood's-lamp fluorescence
  • Fusobacterium nucleatum (ATCC 25586) — irregular small colonies with breadcrumb morphology
  • Clostridium perfringens (ATCC 13124) — large (3–5 mm) colonies with characteristic double-zone β-haemolysis (inner θ-toxin lytic zone, outer α-toxin partial-lytic zone) — essentially pathognomonic
  • C. sporogenes (ATCC 19404), C. septicum, C. novyi, C. tetani, C. bifermentans
  • Clostridioides difficile (ATCC 9689) — flat irregular ground-glass colonies
  • Peptostreptococcus anaerobius, Finegoldia magna (formerly Peptostreptococcus magnus)
  • Cutibacterium acnes (formerly Propionibacterium acnes; Scholz & Kilian 2016) — small white colonies on extended incubation
  • Veillonella parvula — small grey colonies with chartreuse fluorescence under Wood's lamp
  • Actinomyces israelii, Actinomyces meyeri — molar-tooth or breadcrumb morphology, requires 5–10 d incubation

Facultative anaerobes (read alongside)

  • Staphylococcus aureus, Streptococcus spp., Enterococcus spp., E. coli, Klebsiella spp. — grow well; useful as positive controls.

Preparation

1Weigh. Use the pre-weighed Mixture A: 42.0 g per litre. 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. Heat with frequent agitation to 95–100 °C until completely dissolved (typically 8–12 min — the corn starch component requires extended heating).
3Add pre-autoclave supplements. Pipette 1 mL of Stock H (5 mg/mL hemin) and 0.1 mL of Stock K (10 mg/mL Vitamin K1) directly into the warm solution. Swirl to dissolve.
4Adjust pH. Cool briefly to ~45 °C. Verify pH with a calibrated meter; target 7.3 ± 0.1 at 25 °C. Adjust with 1 M NaOH or 1 M HCl as required.
5Bring to final volume. Make up to 1000 mL with distilled water.
6Autoclave. 121 °C × 15 min (103 kPa). Slow cooling — do not vent rapidly to avoid bumping of the agar-containing base.
7Cool the base. Transfer to a 48–50 °C water bath. This temperature range is critical: too hot (> 55 °C) → blood cells lyse to chocolate appearance and lose differential utility; too cold (< 45 °C) → agar gels before pouring.
8Add sheep blood. Aseptically add 50 mL of pre-warmed (37 °C) defibrinated sheep blood to each 1 L of cooled agar base. Swirl gently — do not agitate vigorously, which causes foaming and chocolatisation.
9Pour plates. Pour 20 mL per 90 mm plate (or 5 mL per 60 mm plate), 25 mL per 100 mm plate. Pour inside a laminar-flow hood. Allow to set on a level surface for 30 min at room temperature.
10Store. Transfer plates to 4 °C in sealed bags. For pre-reduced plates (PRAS), transfer to an AAE while still warm so residual oxygen is scavenged by the hemin/cysteine redox buffer.

Critical control points

  • Blood-addition temperature. The agar base must be at 48–52 °C when blood is added. Outside this range either chocolatisation (> 55 °C) or premature gelling (< 45 °C) destroys the plate's utility.
  • Sheep blood lot QC. Each new blood lot should be tested with C. perfringens ATCC 13124 and S. aureus ATCC 25923 to verify the double-zone β-haemolysis and β-haemolysis patterns respectively before clinical use.
  • Wood's-lamp pigment reading. Read pigment fluorescence at 24, 48, and 72 h — Prevotella bronze pigment can take 5–7 d to develop fully; do not read pigment under fluorescent room-light alone, use a 365 nm UV (Wood's) lamp in a dark room.

Cautions

Bovine biosecurity. Defibrinated sheep blood is a bovine-derived biological product. Customs in EU, Japan, and Canada may apply TSE/BSE checks. GMExpression's Australian DAFF EX188M zoosanitary documentation pipeline addresses this.
Chocolatisation of the medium. If the agar base is too hot at blood addition (> 55 °C) the red cells lyse and the plate becomes chocolate (brown) rather than red. Chocolate plates support fastidious organisms (Haemophilus, Neisseria) but lose haemolysis differential utility for anaerobes.
Hemin precipitation. Free hemin precipitates in neutral water. Use the alkaline-NaOH stock (Stock H) and add to warm medium (≥ 45 °C). Black flecks in the prepared plate indicate hemin precipitation.
Plate drying. Anaerobic plates lose differential utility (haemolysis becomes hard to read) when the agar surface dries. Store plates in sealed bags at 4 °C; bring to room temperature before inoculation.
Sheep blood expiry. Defibrinated sheep blood has a maximum 28-day shelf life at 4 °C. Plates poured with end-of-life blood may show suboptimal haemolysis patterns.

Storage and Expiry · Safety

  • Dehydrated powder (Mixture A): 15–30 °C in original packaging. Shelf life 36 months.
  • Stock H (hemin): 4 °C, protected from light. Stable 8 weeks.
  • Stock K (Vitamin K1): 4 °C, protected from light. Stable 6 months.
  • Defibrinated sheep blood: 2–8 °C. Shelf life 28 days from collection.
  • Prepared plates, sealed in bags at 4 °C: 4 weeks routine; 6 weeks if double-bagged.
  • PRAS plates, vacuum-sealed in AAE + oxygen absorber: 3 months for primary-isolation use.

Safety notes. Defibrinated sheep blood is a BSL-2 biological. Handle in a Class II biosafety cabinet, particularly when preparing plates for culturing BSL-3 pathogens. Hemin in alkaline NaOH is corrosive; pipette with care. Vitamin K1 ethanolic stock is flammable. SDS available on request.

References

  1. Ellner PD, Stoessel CJ, Drakeford E, Vasi F. (1966). A new culture medium for medical bacteriology. American Journal of Clinical Pathology 45(4): 502–504.
  2. Jousimies-Somer HR et al. (2002). Wadsworth-KTL Anaerobic Bacteriology Manual, 6th ed. Star Publishing — chapter 6.
  3. Carroll KC, Pfaller MA, Landry ML, McAdam AJ, Patel R, Richter SS, Warnock DW (eds). (2019). Manual of Clinical Microbiology, 12th ed. ASM Press — anaerobe bacteriology chapters.
  4. Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH. (eds). (current ed.) Manual of Clinical Microbiology, anaerobe bacteriology chapter.
  5. Scholz CFP, Kilian M. (2016). The natural history of cutaneous propionibacteria: Propionibacterium acnes reclassified as Cutibacterium acnes. IJSEM 66: 4422–4432.
  6. Lawson PA et al. (2016). Reclassification of Clostridium difficile as Clostridioides difficile. Anaerobe 40: 95–99.
  7. BD Difco™ Columbia Agar Base product information sheet 211125; BBL™ Columbia II Agar 297715.
  8. Oxoid Manual 9th ed., CM0331 Columbia Agar Base.

Frequently Asked Questions

Q1. How does this differ from the existing GMExpression Brucella Agar product?
The base composition differs. Brucella agar uses Brucella base (Tryptone, NaCl, dextrose, yeast extract, NaHSO3); Columbia uses a richer peptone-and-heart-infusion base (Pantothene-Casein Peptone, Bio-Polytone, beef heart infusion, corn starch). In side-by-side comparisons, Columbia gives larger, more luxuriant colonies — preferred for colonial-morphology-based presumptive identification. Brucella is preferred where the bisulfite component is wanted for additional reduction. Both are CLSI/Wadsworth-acceptable; the choice is laboratory preference.
Q2. Why is the agar concentration 13 g/L instead of the more standard 15 g/L?
This is Ellner's (1966) original specification. The slightly softer agar gives better recovery of small fastidious anaerobic colonies (which can be partially submerged into a softer agar) and improves haemolysis-zone visibility through the thinner gel. Standard 15 g/L agar can be used as an alternative if a firmer gel is preferred, but the formulation reverts to a more generic 'blood agar' rather than the Ellner Columbia formulation.
Q3. Why are some Prevotella colonies not showing pigment by 48 h?
Prevotella melaninogenica pigment (a protohaem-derived bronze-to-black polymer) requires (a) full haem availability from the blood, (b) extended anaerobic incubation (often 5–7 d for full pigment), and (c) absence of glucose excess (high glucose suppresses pigmentation via catabolite repression of the heme-utilisation pathway). Standard Columbia base is glucose-poor, so pigmentation should develop by 72 h. If not, verify the anaerobic atmosphere (resazurin tab inside the jar/AAE) and extend incubation to 7 d. Some Prevotella species are genuinely non-pigmenting (P. bivia, P. disiens).
Q4. Can I use this plate for Helicobacter or Campylobacter?
Not without modification. Helicobacter pylori requires microaerophilic atmosphere (5–10 % O2) plus a specific selective supplement (Skirrow or Dent supplement: vancomycin + cefsulodin + trimethoprim + amphotericin B). Campylobacter requires the Skirrow / Karmali / Preston selective supplements. The unmodified Columbia blood plate will support Campylobacter growth under microaerophilic atmosphere but is not selective; selective variants are available as custom orders.
Q5. What does Clostridium perfringens double-zone β-haemolysis look like?
Two concentric clearing zones around each colony. The inner narrow zone is complete β-haemolysis caused by θ-toxin (perfringolysin O, a cholesterol-dependent cytolysin). The outer wider zone is partial / incomplete haemolysis caused by α-toxin (phospholipase C, sphingomyelinase activity). Side-by-side, the pattern looks like a colony surrounded by a dark inner ring and a paler outer ring. This pattern is essentially pathognomonic for C. perfringens within the Clostridium genus, and combined with Gram-positive box-car bacilli morphology, lecithinase-positive Egg Yolk Agar reading, and 'reverse-CAMP' test, gives a definitive presumptive identification.
Q6. Why use 5 % sheep blood rather than horse blood?
Sheep blood is the CLSI / Wadsworth standard. It contains no thymidine (which would interfere with sulfonamide AST), gives clear β-haemolysis with most pathogens, and is widely available DAFF-certified in Australia. Horse blood is an acceptable substitute but contains some thymidine and gives slightly altered haemolysis patterns for some Streptococcus species. Use sheep blood unless a specific protocol calls for horse.
Q7. How are the plates packaged for shipment?
Pre-poured plates: 50 × 90 mm plates per sealed bag with oxygen absorber, double-bagged, shipped in insulated container at 2–8 °C with temperature monitoring. Bulk powder + blood configuration: powder at room temperature; blood pack at 2–8 °C separately. Plate shelf life is verified by GMExpression release-testing using C. perfringens double-zone haemolysis and Bacteroides fragilis growth.
Q8. Are the plates pre-reduced?
Standard configuration: not pre-reduced (saves cost; user reduces on receipt by transfer to AAE 24 h before inoculation). PRAS option: pre-reduced (vacuum-sealed in AAE with O2 absorber); ready for direct inoculation but adds AUD ~50/kit cost. For routine clinical-microbiology workflows the non-PRAS option is generally adequate. For research-grade pure-culture studies of strict anaerobes, choose the PRAS option.