Overview

BBE (Bacteroides Bile Esculin Agar, Livingston et al. 1978 JCM 7:448) and KVLB (Kanamycin-Vancomycin Laked Blood Agar, Wadsworth ch. 6) are paired selective and differential plating media for Gram-negative anaerobes. BBE is specifically optimised for the Bacteroides fragilis group; KVLB for Prevotella, Porphyromonas, and the non-fragilis Bacteroides. They are the Wadsworth-tradition primary screening plates for clinical specimens where Gram-negative anaerobes are the suspected pathogens.

BBE uses bile + esculin as its selective and differential principle: 2 % oxgall suppresses bile-intolerant species, esculin hydrolysis (β-glucosidase → aesculetin) chelates iron from ferric ammonium citrate to produce glossy black colonies, and 100 mg/L gentamicin suppresses facultative Gram-negatives. The B. fragilis group are characteristically bile-resistant, esculin-positive, and gentamicin-resistant — black-colony + bile + esculin is the rapid Wadsworth screen.

KVLB uses kanamycin + vancomycin + laked blood: 75 mg/L kanamycin suppresses facultative Gram-negatives; 7.5 mg/L vancomycin suppresses Gram-positives; the laked (freeze-thaw-lysed) sheep blood releases intracellular haematin, which supports haem-auxotrophic Bacteroides / Prevotella / Porphyromonas better than intact erythrocytes. After 5–7 days, pigmented Prevotella / Porphyromonas species produce characteristic black-pigment colonies. Paired use of BBE + KVLB + a non-selective primary plate (anaerobic CBA) is the Wadsworth-recommended primary plating triad for suspected Gram-negative anaerobe infections — intra-abdominal abscess, peritoneal fluid, brain abscess, gynaecological specimens, and dental / head-and-neck infections.

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 BBE + KVLB Selective Plating Set contains:

  • 10 × BBE Agar plates (90 mm) — pre-poured, pre-reduced, individually sealed in oxygen-impermeable foil pouches with oxygen indicator. B. fragilis ATCC 25285 QC release-tested.
  • 10 × KVLB Agar plates (90 mm) — pre-poured, pre-reduced, individually sealed with oxygen indicator. Prevotella melaninogenica ATCC 25845 QC release-tested.
  • Vial G — Gentamicin sulphate stock (100 mg/mL, filter-sterilised), for top-up reinforcement of BBE selectivity (optional; standard plates are already supplemented).
  • Vial KV — Kanamycin (100 mg/mL) + Vancomycin (10 mg/mL) stock, filter-sterilised, for top-up reinforcement of KVLB selectivity (optional).
  • Vial H — Hemin stock (5 mg/mL in 0.05 M NaOH) — for supplementing user-prepared base agar.
  • Vial K — Vitamin K1 stock (10 mg/mL in 95 % ethanol, amber vial).
  • Instruction manual (A5 booklet, v1.0) with full Livingston 1978 BBE protocol, Wadsworth KVLB protocol, QC organism panel, and a Wadsworth plating-triad workflow annex.

Customisation options on request: dehydrated agar base (without supplements, longer shelf life) for in-house preparation; 60 mm contact plates for environmental sampling; bundled triad with anaerobic CBA (A3) for full Wadsworth primary plating; "Non-selective BBE" (esculin-differential only, no gentamicin) for research applications.

Composition — per 1 L equivalent unless stated otherwise

BBE Agar (Livingston et al. 1978 JCM 7:448; BD Difco 297878; per 1 L)

ComponentConcentrationFunction
Tryptic soy agar (TSA) base — tryptone + soytone + NaCl + agar40.0 g (dehydrated TSA powder)Base — tryptone 15 g, soytone 5 g, NaCl 5 g, agar 15 g
Oxgall (dehydrated ox bile)20.0 gSelective agent — 2 % bile concentration
Esculin (6,7-dihydroxycoumarin glucoside)1.0 gDifferential substrate — hydrolysed by β-glucosidase to aesculetin
Ferric ammonium citrate0.5 gDifferential indicator — chelates aesculetin to form black colour
Hemin (post-autoclaving filter-sterilised)5.0 mg/LSupports haem-auxotrophic B. fragilis
Vitamin K11.0 mg/LMenaquinone precursor; fumarate reductase pathway
Gentamicin sulphate (post-autoclaving filter-sterilised)100 mg/LSelective agent — suppresses facultative Gram-negatives

Pre-autoclaving pH: 7.0 ± 0.2 at 25 °C.

KVLB Agar (Wadsworth ch. 6; BD BBL™ 297877; per 1 L)

ComponentConcentrationFunction
Brucella agar base — tryptone, peptamin, yeast extract, NaCl, dextrose, agar, sodium bisulfite43.0 g (or 42.5 g Columbia agar base alternative)Rich peptone base supporting fastidious anaerobes
defibrinated sheep blood50 mL (5 % v/v)Critical — freeze-thaw-lysed; releases haematin (better than intact RBC)
Hemin (pre-autoclaving stock)5.0 mg/LSupports haem-auxotrophic Bacteroides / Prevotella
Vitamin K1 (pre-autoclaving ethanolic stock)1.0 mg/LMenaquinone precursor
Kanamycin sulphate (post-autoclaving)75 mg/LSelective agent — suppresses facultative Gram-negatives
Vancomycin hydrochloride (post-autoclaving)7.5 mg/LSelective agent — suppresses Gram-positives

Pre-autoclaving pH: 7.3 ± 0.2 at 25 °C (matches Brucella base).

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

Use and Applications

  • Clinical primary plating for intra-abdominal abscess fluid, peritoneal fluid, surgical wound exudate, brain abscess, post-surgical abdominal infection, and gynaecological specimens. Paired use of BBE + KVLB + anaerobic CBA is the Wadsworth-recommended primary plating triad.
  • Rapid identification of Bacteroides fragilis group — BBE black-colony + bile-resistant + esculin-positive is the Wadsworth rapid screen.
  • Identification of pigmented anaerobes — KVLB black-pigment colonies after 5–7 days identify Prevotella melaninogenica, P. intermedia, P. nigrescens, P. corporis, Porphyromonas gingivalis, P. asaccharolytica, P. endodontalis.
  • Gut-microbiome culturomics — BBE is the standard selective plate for Bacteroides isolation from faecal samples; KVLB enables enrichment of Prevotella-dominant communities (the "Prevotella enterotype" research; plant-fibre-diet microbiome work).
  • Oral / periodontal microbiology — KVLB is the reference medium for recovery of black-pigmented oral anaerobes (periodontitis pathogens P. gingivalis, P. intermedia).
  • Veterinary anaerobe identification — BBE has been validated for canine, equine, bovine, porcine intestinal Bacteroides identification (selective profile is largely host-species-independent).
  • Bacteraemia and CSF anaerobe workup — secondary plating from positive anaerobic blood-culture bottles to identify Bacteroides bacteraemia and Prevotella CSF infections.

Compatible Microorganisms

BBE — designed targets (positive growth, black colonies)

  • Bacteroides fragilis (ATCC 25285) — large, raised, glossy black colonies after 24–48 h. Reference QC organism.
  • Bacteroides thetaiotaomicron (ATCC 29148) — black colonies; bile-stimulated growth.
  • B. ovatus, B. uniformis, B. vulgatus, B. caccae — all B. fragilis group; positive on BBE.
  • Parabacteroides distasonis (formerly B. distasonis; ATCC 8503) — black colonies, slightly weaker esculin reaction.
  • Bilophila wadsworthia — bile-loving sulphur-utiliser; characteristic black colonies (H2S + iron, not esculin).

BBE — non-target / suppressed

  • Fusobacterium mortiferum — bile-tolerant; grows but esculin-negative (non-pigmented).
  • Enterococcus faecalis — bile-tolerant but gentamicin-sensitive; suppressed. QC organism for the gentamicin selectivity check.
  • E. coli, Klebsiella, Pseudomonas — bile-tolerant facultatives; suppressed by gentamicin.

KVLB — designed targets (positive growth, pigment after 5–7 days)

  • Prevotella melaninogenica (ATCC 25845) — black-pigmented colonies after 5–7 days. Reference QC organism.
  • Prevotella intermedia (ATCC 25611), P. nigrescens, P. denticola, P. corporis, P. tannerae — black-pigmented.
  • Porphyromonas gingivalis (ATCC 33277), P. asaccharolytica (ATCC 25260), P. endodontalis — slower pigment formation (7–14 days).
  • Fusobacterium nucleatum (ATCC 25586) — non-pigmented; characteristic lobed small colonies.
  • Bacteroides spp. — recoverable but not differentiated from Prevotella; BBE is preferred for B. fragilis group.

KVLB — suppressed (selective check)

  • Facultative Gram-negatives (kanamycin-sensitive): E. coli, Klebsiella, Enterobacter, Pseudomonas.
  • Gram-positives (vancomycin-sensitive): Streptococcus, Staphylococcus, Enterococcus (most), Lactobacillus (most), Clostridium (most). N.B.: a small fraction of Lactobacillus and Pediococcus spp. are intrinsically vancomycin-resistant and may grow.

Preparation

1Use pre-poured plates (recommended). Pre-poured pre-reduced GMExpression BBE and KVLB plates are released from QC ready to inoculate. Remove from foil pouch inside the AAE or just before inoculation; equilibrate at room temperature for 10 min. Verify the oxygen indicator inside the pouch shows reduced state (colourless / blue → red transition indicates O2 exposure).
2If preparing from dehydrated base (BBE). Weigh 40 g dehydrated TSA base + 20 g oxgall + 1 g esculin + 0.5 g ferric ammonium citrate. Combine in 1000 mL distilled water.
3BBE: heat-dissolve. 95–100 °C with stirring until clear (8–10 min; oxgall is the slow-dissolving component). Adjust pH to 7.0 ± 0.2 at 25 °C.
4BBE: autoclave and cool. 121 °C × 15 min. Cool in a water bath to 50 °C.
5BBE: add post-autoclave supplements. 1 mL of Vial H (5 mg/mL hemin) = 5 mg/L final; 0.1 mL of Vial K (10 mg/mL VK1) = 1 mg/L final; 1 mL of Vial G (100 mg/mL gentamicin sulphate, filter-sterilised) = 100 mg/L final. Critical: gentamicin is the selective agent — do NOT autoclave.
6BBE: pour plates. 20–25 mL per 90 mm plate. Allow to set 30 min at room temperature. Bag and store at 4 °C.
7If preparing from dehydrated base (KVLB) — prepare laked blood in advance. Freeze 100 mL of defibrinated sheep blood at −20 °C overnight; thaw at 37 °C; repeat one freeze-thaw cycle. Result: dark-red suspension with no intact erythrocytes (microscopic check: no cell membranes visible).
8KVLB: dissolve base + hemin. Weigh 43 g Brucella agar base (or 42.5 g Columbia agar base). Combine with 1000 mL distilled water. Add 1 mL of 5 mg/mL hemin stock pre-autoclaving. Heat-dissolve at 95–100 °C until clear. Adjust pH to 7.3 ± 0.2.
9KVLB: autoclave and cool. 121 °C × 15 min. Cool to 50 °C in a water bath.
10KVLB: add VK1, laked blood, and antibiotics. Add 0.1 mL of Vial K (10 mg/mL VK1); 50 mL of pre-warmed laked sheep blood (to room temperature, not cold); 0.75 mL of 100 mg/mL kanamycin stock from Vial KV (= 75 mg/L); 0.75 mL of 10 mg/mL vancomycin stock from Vial KV (= 7.5 mg/L).
11KVLB: mix and pour. Mix gently by swirling (avoid foaming) and pour 20–25 mL per 90 mm plate immediately. Bag and store at 4 °C protected from light.

Critical control points

  • Gentamicin / kanamycin / vancomycin solubility and stability. All three antibiotics are water-soluble at the required stock concentrations. Filter-sterilise (0.22 µm). Store at 4 °C for up to 1 month (kanamycin, gentamicin) or 2 weeks (vancomycin); −20 °C for up to 6 months. Reconstituted stocks must be re-titre-tested if used after 2 weeks.
  • Antibiotic temperature sensitivity. Vancomycin and gentamicin tolerate brief exposure to 50 °C but prolonged (> 30 min) causes activity loss. Add antibiotics last, mix, and pour plates within 15 min.
  • Laked-blood preparation completeness. Freeze-thaw lysis must be complete — incompletely-lysed blood gives granular appearance and uneven Prevotella pigment. Two cycles typically sufficient; verify microscopically.
  • Plate uniformity and pH. Pour at a level surface; ferric ammonium citrate precipitates at low pH producing visible specks. If BBE pH drops below 6.8, re-dissolve and adjust.

Cautions

Gentamicin lot variability (BBE). Gentamicin sulphate is a complex mixture (gentamicin C1, C1a, C2, C2a); active titre varies by manufacturer. Use a single supplier (Sigma, GIBCO, Roche, Merck) and verify titre by Enterococcus faecalis ATCC 29212 zone of inhibition.
Oxgall (bile) batch variability (BBE). Dehydrated ox bile varies in bile-acid composition (taurocholate, glycocholate, deoxycholate ratios) between commercial suppliers; this affects selective stringency. The Livingston 1978 reference used Difco "Oxgall" specifically; alternatives should be QC'd against B. fragilis (positive growth) and E. coli (suppression on supplemented BBE — gentamicin is the operative agent, since E. coli is bile-resistant).
Esculin photoinstability (BBE). Esculin slowly photoreduces under fluorescent light; protect prepared plates from light during storage. The aesculetin product is also light-sensitive; the iron-aesculetin black colour is stable.
Black-colony false positives (BBE). Some Bilophila wadsworthia and rare Fusobacterium strains produce H2S that reacts with ferric ammonium citrate to give a black appearance without esculin hydrolysis. Mitigation: sub-culture suspicious colonies to non-selective anaerobic CBA + esculin disc test; the true B. fragilis black colour is glossy (aesculetin-iron complex), while H2S black is matt black-grey (ferric sulfide).
Pigment failure with fresh blood (KVLB). Too-fresh blood (< 24 h old, still containing functional erythrocytes) gives less haem than aged laked blood. Use blood that has been laked for 12–48 h before incorporation. For ambiguous pigment-recognition, supplement the agar with 1 g/L haemoglobin solution.
Shelf life. BBE: 4 weeks at 4 °C (gentamicin loses ~10 % activity per month at 4 °C). KVLB: 4 weeks at 4 °C (vancomycin loses ~15 % activity per month at 4 °C). Use within these windows for selective-grade work; for primary-isolation work without strict selective demands, plates remain usable up to 8 weeks.

Storage and Expiry · Safety

  • Pre-poured plates (sealed in foil pouches with O2 indicator): 4 °C, light-protected. Shelf life 8 weeks for selective-grade work (BBE: gentamicin-limited; KVLB: vancomycin-limited).
  • Dehydrated agar base (BBE / KVLB powder): 15–30 °C, sealed in original packaging. Shelf life 30 months.
  • Vial G (Gentamicin) / Vial KV (Kanamycin + Vancomycin): 4 °C for 1 month; −20 °C for 6 months. Aliquot to avoid freeze-thaw cycling.
  • Vial H (Hemin) / Vial K (Vitamin K1): 4 °C, light-protected. Hemin: 8 weeks. VK1: 6 months.
  • Prepared plates, opened pouch: use within 24 h once oxygen-exposed.

Safety notes. Sheep blood is a Class 2 biological material — handle inside BSC; assume universal precautions. Gentamicin, kanamycin, vancomycin are antibiotics — handle as Category 2 chemicals; avoid dust formation; use face mask when weighing. SDS available on request.

References

  1. Livingston SJ, Kominos SD, Yee RB. (1978). New medium for selection and presumptive identification of the Bacteroides fragilis group. Journal of Clinical Microbiology 7(5): 448–453. [BBE primary reference]
  2. Finegold SM, Sutter VL, Mathisen GE. (1983). Normal indigenous intestinal flora. In: Human Intestinal Microflora in Health and Disease. Academic Press, pp. 3–31. (KVLB usage in gut-microbiome work.)
  3. Jousimies-Somer HR et al. (2002). Wadsworth-KTL Anaerobic Bacteriology Manual, 6th ed., chapter 9 (selective media for anaerobes). [Wadsworth KVLB protocol reference]
  4. CLSI M22, current edition. (QC for prepared media.)
  5. Carroll KC et al. (eds). (2019). Manual of Clinical Microbiology, 12th ed., chapter 60. ASM Press.
  6. BD Difco™ / BBL™ product information sheets 297878 (BBE Agar) and 297877 (KVLB Agar).
  7. Oxoid Manual 9th ed., CM1080 (BBE Agar) and CM0653 + SR0108 (KVLB).
  8. ATCC Microbiology Catalogue: Medium 2127 (BBE).

Frequently Asked Questions

Q1. Why are BBE and KVLB sold together as a paired set?
Because they are used together in the Wadsworth-tradition clinical workflow. BBE targets the Bacteroides fragilis group (bile-resistant, esculin-positive); KVLB targets the pigmented anaerobes Prevotella / Porphyromonas and the haem-auxotrophic Gram-negatives. Neither plate alone covers the full Gram-negative anaerobe spectrum; the bundled set simplifies ordering for hospital and research laboratories that follow the Wadsworth Manual workflow.
Q2. Why are my Bacteroides colonies on BBE not turning black?
The black colour requires the esculin → aesculetin → iron-aesculetin complex to form. Loss of any link breaks the colour. Common causes: (a) the iron in ferric ammonium citrate has precipitated (visible as brown sediment in the plate base) — check pH and re-prepare with adjustment; (b) the isolate is genuinely esculin-negative (rare in B. fragilis group but possible — Parabacteroides distasonis is often weak); (c) the incubation is too short (require 48 h, not 24 h). Confirmatory: spot a 30 µL drop of 50 % ferric ammonium citrate solution adjacent to the colony — if the esculin in the agar has hydrolysed, the spot will turn black.
Q3. Can I use BBE for Bacteroides fragilis species identification alone?
BBE black-colony + bile-resistant + gentamicin-resistant is presumptive for B. fragilis group; species-level identification requires further testing (16S rRNA gene sequencing, MALDI-TOF, or a B. fragilis-specific PCR). The Wadsworth tradition treats BBE positive + correct morphology + correct Gram stain as sufficient for hospital reporting at the fragilis-group level; species-level identification is reserved for atypical or treatment-failing cases.
Q4. Why does KVLB not give pigment for some of my Prevotella isolates?
Black-pigment formation requires both genetic capacity (the hmuR or related haem-uptake system, which most pigmented Prevotella / Porphyromonas have) and adequate haem in the medium. Cofactors that block pigment: (a) sub-optimal anaerobiosis (Eh > +50 mV) suppresses pigment expression; (b) too-fresh blood (< 24 h, still containing functional erythrocytes) gives less haem than aged laked blood — use blood laked 12–48 h before incorporation; (c) some Prevotella species (e.g., P. catoniae, P. oralis) are simply non-pigmented by genetic determinism. For ambiguous pigment-recognition, supplement the agar with 1 g/L haemoglobin solution.
Q5. What incubation time and atmosphere are required?
Both BBE and KVLB are incubated at 35–37 °C in anaerobic atmosphere (< 0.5 % O2; typically 80 % N2 + 10 % H2 + 10 % CO2 or 90 % N2 + 10 % H2). BBE: 48–72 h for B. fragilis group identification; KVLB: 5–7 days for Prevotella pigment formation, up to 14 days for Porphyromonas pigment.
Q6. Are BBE and KVLB CLSI-recommended media?
CLSI M22 (Quality Assurance for Commercially Prepared Microbiological Culture Media) recognises both BBE and KVLB as commercially-prepared selective media subject to CLSI QC protocols. CLSI M11 (anaerobe AST) and CLSI M100 do not specify a primary-isolation medium, deferring to the Wadsworth Manual and ASM-MCM for plating choices. The Wadsworth Manual (the de facto US clinical standard for anaerobic bacteriology) explicitly recommends both BBE and KVLB.
Q7. Can I prepare BBE or KVLB without the antibiotic supplement?
BBE without gentamicin: the bile + esculin selectivity is retained but the medium loses its facultative-Gram-negative suppression; E. coli and Klebsiella may overgrow. The "non-antibiotic" variant is sometimes used for non-clinical research where E. coli contamination is not a concern. KVLB without kanamycin + vancomycin: loses essentially all selectivity and becomes simply "laked-blood anaerobic CBA"; not generally useful.
Q8. Can BBE be used for veterinary samples?
Yes. BBE has been validated for canine, equine, bovine, and porcine intestinal Bacteroides group identification. The selective profile (bile, esculin, gentamicin) is largely independent of host species. Note that veterinary specimens are more likely to contain bile-resistant Enterococcus and Pseudomonas spp., which may overgrow if the gentamicin concentration is reduced from the standard 100 mg/L. Maintain full gentamicin selectivity for veterinary work.