Frequently Asked Questions

Both XLD and HE are FDA BAM-approved for Salmonella. XLD: better Salmonella recovery, Salmonella = red with black centers, may have more false positives. HE: More selective, better inhibition of coliforms, Salmonella = blue-green with black centers, may miss some Salmonella. Recommendation: Use BOTH in parallel for maximum Salmonella detection. Many labs plate enrichments on XLD + HE + HiCrome Salmonella for comprehensive detection. Different formulations complement each other's strengths/ weaknesses. In both the media, H2S negative Salmonella may be missed out

Hektoen Enteric (HE) Agar is a selective differential medium for isolation of Salmonella and Shigella species from fecal specimens. More selective than SS Agar, less inhibitory than Bismuth Sulfite. Contains: (1) Bile salts - inhibit gram-positive bacteria, (2) Lactose, sucrose, salicin - differentiate fermenters vs non-fermenters, (3) Ferric ammonium citrate and sodium thiosulfate - detect H2S. Salmonella: blue-green with black centers. Shigella: blue-green without black centers. Coliforms: salmon/orange. FDA BAM approved for Salmonella testing.

XLD colony interpretation: (1) Salmonella: pink/red colonies with black centers (xylose fermentation → acid → decarboxylation → alkali → red; H2S → black), typical size 2-3mm, (2) Shigella: pink/red colonies, no black centers (cannot ferment xylose, no H2S), (3) E. coli and coliforms: yellow colonies (ferment xylose, no lysine decarboxylation), (4) Proteus: black colonies with yellow halos. Medium is originally red. Incubate 18-24 hours at 35-37°C. Do not incubate >48 hours as non-Salmonella may develop similar appearance.

Xylose Lysine Deoxycholate (XLD) Agar is a selective differential medium for isolation of Salmonella and Shigella from clinical and food specimens per FDA BAM, ISO 6579, and AOAC methods. Contains: (1) Deoxycholate - inhibits gram-positive bacteria, (2) Xylose, lactose, sucrose and and lysine - differentiate Salmonella/Shigella from other enterics, (3) Ferric ammonium citrate - detects H2S production. Salmonella: pink/red colonies with black centers (H2S positive). Shigella: pink/red colonies without black centers. Phenol red is the indicator dye Coliforms: yellow colonies.

Listeria Enrichment Broth Base (with selective supplements) is used for enrichment of Listeria species from food and environmental samples per FDA BAM, USDA MLG, and ISO 11290 methods. The base medium is supplemented with: (1) Listeria Selective Supplement (acriflavine, nalidixic acid, cycloheximide) to inhibit competing microflora, (2) Esculin for Listeria detection. Used after primary enrichment in UVM (University of Vermont Medium) or Half-Fraser Broth. Incubate at 30°C or 37°C per method. Black color indicates presumptive Listeria. Fraser Broth Base, Modified (Half Fraser Broth) is recommended as a primary enrichment medium for the isolation and enumeration of Listeria monocytogenes from food and animal feeds. The composition and performance criteria of this media is as per the specification laid down in ISO 11290-1:2017, ISO 11290-2:2017 and 11133:2014 (E) /Amd. :2020. The base medium contains acriflavine and nalidixic acid to inhibit competing microflora. The media after addition of Ammonium Iron citrate aids in detections of Esculin hydrolysis.

Standard Salmonella protocol: (1) Pre-enrichment: 25g sample in 225mL Buffered Peptone Water (BPW), incubate 18-24h at 35°C, (2) Selective enrichment: Transfer 0.1mL from BPW to 10mL RV Broth (1:100 dilution is critical), (3) Incubate RV at 42°C for 18-24 hours, (4) Streak turbid RV broth onto selective agar (XLD, HE, BS, or HiCrome Salmonella), (5) Confirm suspect colonies. RV provides excellent Salmonella recovery while suppressing background microflora. Parallel enrichment in Tetrathionate (TT) broth recommended for maximum sensitivity.

Rappaport Vassiliadis (RV) Broth is a selective enrichment medium for Salmonella from food and environmental samples. Key features: (1) Incubated at 42°C - elevated temperature favors Salmonella, inhibits competitors, (2) Contains malachite green (inhibits gram-positive bacteria and most coliforms), (3) Low pH and high osmolarity create selective pressure, (4) Magnesium chloride increases selectivity. Used as second enrichment step after Buffered Peptone Water in FDA BAM, ISO 6579, and AOAC Salmonella detection methods.

Malt Extract Agar is general-purpose medium for cultivation of yeasts and molds. Contains malt extract providing maltose and other nutrients. Acidic pH (5.4-5.6) favors fungi. Used for: (1) General mycology, (2) Fungal culture maintenance, (3) Environmental monitoring for mold, (4) Food mycology, (5) Brewing and fermentation industries. Similar to PDA but uses malt extract instead of potato infusion. Some fungi sporulate better on MEA than PDA. Can add antibiotics (chloramphenicol) for selective isolation. Incubate 25-28°C for 5-7 days.

Membrane filtration procedure: (1) Filter appropriate Water volume through 0.45μm membrane (100mL for drinking Water, diluted samples for contaminated Water), (2) Aseptically transfer membrane to M-Endo Agar pad in petri dish, (3) Incubate 22-24 hours at 35°C, (4) Count colonies with pink-red color and golden metallic sheen, (5) Calculate coliforms per 100mL: (colonies counted × 100) / mL filtered. Colonies without sheen are non-coliforms. Confirm suspect colonies with biochemical tests if required by method.

M-Endo Agar LES (Membrane-Endo Agar Les Endo Substrate) is used for detection and enumeration of coliform bacteria in Water by membrane filtration method per EPA and Standard Methods. After filtering Water through 0.45μm membrane, place filter on M-Endo Agar and incubate 22-24 hours at 35°C. Coliform colonies appear pink/red/dark red with golden metallic sheen (lactose fermentation). This sheen is diagnostic for coliforms. LES formulation has improved selectivity over original Endo formulations. Used for drinking Water, recreational Water, wasteWater testing.

GVPC is antibiotic supplement added to BCYE for selective isolation of Legionella from contaminated environmental samples (Water systems, biofilms). GVPC contains: Glycine - inhibits gram-positive bacteria, Vancomycin - inhibits gram-positive bacteria, Polymyxin B - inhibits gram-negative bacteria except Legionella, Cycloheximide (anisomycin) - inhibits fungi. This combination suppresses background microflora that would overgrow Legionella. Use BCYE with GVPC for environmental samples. Use BCYE without GVPC for clinical specimens (less contamination, antibiotics may inhibit some Legionella strains).

Legionella bacteria are fastidious and will not grow on standard media (TSA, Blood Agar). They have unique requirements: (1) L-cysteine (amino acid) - absolutely essential, cannot synthesize it, (2) Iron (ferric iron) - required for metabolism, (3) Charcoal - absorbs inhibitory compounds produced during growth, (4) Neutral-to-alkaline pH (6.9-7.0) - maintained by ACES buffer. Standard media lack cysteine and adequate buffering. BCYE specifically formulated to meet these requirements. Legionella will not grow without cysteine supplementation.

Buffered Charcoal Yeast Extract (BCYE) Agar is the primary medium for isolation and cultivation of Legionella species from environmental and clinical samples. Legionella pneumophila causes Legionnaires' disease. BCYE contains: (1) L-cysteine and ferric pyrophosphate (required growth factors), (2) Activated charcoal (absorbs toxic metabolites), (3) ACES buffer (maintains pH), (4) Yeast extract (nutrients). Use BCYE with antibiotics (GVPC supplement) for selective isolation from environmental Water samples. Incubate 3-10 days at 35-37°C with 2.5% CO2.

M17 Medium is selective for thermophilic Lactococcus and Streptococcus species used in dairy fermentation. Developed for Streptococcus thermophilus in yogurt. Contains: (1) Disodium glycerophosphate - buffer, (2) Ascorbic acid - reducing agent, (3) Magnesium sulfate - stimulates lactic acid bacteria. Used for: (1) Yogurt starter culture enumeration, (2) Cheese culture testing, (3) S. thermophilus counts in dairy, (4) Probiotic product QC. Incubate anaerobically at 42-43°C for 48 hours (S. thermophilus optimal). Add agar for solid medium. For cultivation of lactic Streptococci and plaque assay of lactic bacteriophages.

Similar to skim milk agar but may contain additional nutrients. Used for dairy microbiology, casein breakdown testing.