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Microbiology Testing FAQs — Methods & Media | HiMedia USA
What is Lactose Broth used for?
Lactose Broth is used for presumptive coliform testing in food and water by Multiple Tube (MPN) method. Contains: lactose (fermentable sugar), peptone (nutrients), Durham tube (inverted tube to trap gas). Procedure: (1) Inoculate dilutions into lactose broth tubes, (2) Incubate 24-48 hours at 35°C, (3) Gas production (visible in Durham tube) = presumptive positive for coliforms, (4) Confirm positives with Brilliant Green Bile Broth or EMB/MacConkey. MPN tables used to estimate coliform count from pattern of positive tubes. Classic water testing method, largely replaced by membrane filtration.
What is Brilliant Green Bile Broth used for?
Brilliant Green Bile Broth (BGBB) is selective medium for confirmation of coliforms from presumptive Lactose Broth MPN tests. Contains: (1) Brilliant green and bile salts - inhibit gram-positives and non-coliforms, (2) Lactose - fermented by coliforms, (3) Durham tube - detects gas. Procedure: Transfer gas-positive Lactose Broth tubes to BGBB, incubate 48 hours at 35°C. Gas in Durham tube = confirmed coliforms. Used in water quality testing, food microbiology. Part of standard MPN confirmation process. Also called 2% Brilliant Green Lactose Bile Broth.
What is VRBA used for?
Violet Red Bile Agar (VRBA) is selective medium for enumeration of coliform bacteria in dairy products, food, and water per ISO 4832, APHA methods. Contains: (1) Crystal violet and bile salts - inhibit gram-positives, (2) Lactose - differentiate coliforms, (3) Neutral red - pH indicator. Coliforms produce purple-red colonies with purple halos (acid from lactose fermentation precipitates bile). Used for: (1) Coliform count in milk/dairy, (2) Food quality testing, (3) Process hygiene monitoring. Pour plate or surface spread method. Incubate 24 hours at 35-37°C.
What is CIN Agar used for?
CIN Agar is selective differential medium for isolation of Yersinia enterocolitica from food and clinical specimens. Contains three antibiotics: Cefsulodin - inhibits Pseudomonas, Irgasan (triclosan) - inhibits gram-positives, Novobiocin - inhibits Proteus. Y. enterocolitica produces bull's-eye colonies (dark red center with transparent border) due to mannitol fermentation. Cold enrichment (4°C for 3 weeks) prior to plating enhances recovery from food. Incubate 24-48 hours at 28-30°C (not 35-37°C). Used for outbreak investigations, pork testing. The composition and performance criteria of this medium are as per the specifications laid down in ISO 10273:2017(E)
Why is SS Agar only used for clinical specimens?
SS Agar is highly selective due to high bile salt and brilliant green concentrations. Good for clinical specimens (where Salmonella/Shigella are primary targets and heavy contamination with normal flora). TOO inhibitory for food testing: (1) May suppress stressed or injured Salmonella from food processing, (2) May inhibit Salmonella serovars, (3) Not FDA BAM approved for food testing. For food → use XLD or HE Agar (less inhibitory, better Salmonella recovery). For stool/rectal swabs → SS Agar excellent choice. Choose media selectivity based on sample type.
What is SS Agar used for?
Salmonella-Shigella (SS) Agar is a highly selective differential medium for isolation of Salmonella and Shigella from clinical specimens (stool, rectal swabs). Contains: (1) Bile salts and brilliant green - strongly inhibit gram-positives and most coliforms, (2) Thiosulfate and ferric citrate - detect H2S, (3) Lactose - differentiate fermenters. Salmonella: colorless/transparent colonies with black centers. Shigella: colorless colonies without black centers. Lactose fermenters (coliforms): pink/red colonies but largely inhibited. Too inhibitory for food samples - use XLD or HE instead.
What is SMAC Agar and why is sorbitol used?
Sorbitol MacConkey Agar (SMAC) is used specifically for E. coli O157:H7 detection. Uses sorbitol instead of lactose. E. coli O157:H7 CANNOT ferment sorbitol → produces colorless colonies. Other E. coli CAN ferment sorbitol → produce pink colonies. This differentiates pathogenic O157:H7 from commensal E. coli. Used in: (1) Ground beef testing, (2) Produce testing, (3) Outbreak investigations, (4) Diarrheal stool samples. Colorless colonies are presumptive O157:H7 requiring confirmation with O157 antigen test and molecular methods (Shiga toxin genes stx1/stx2). The composition and performance criteria are in accordance with ISO 16654:2001&/Amd 1:2017.
What is Preston Broth used for?
Preston Campylobacter Enrichment Broth is selective enrichment for Campylobacter from food (especially poultry) per ISO 10272. Contains antibiotics (polymyxin, rifampicin, trimethoprim, cycloheximide) inhibiting competing microflora. Procedure: Add 25g sample to Preston broth, incubate microaerobically at 41.5°C for 24-48h, plate onto Campylobacter-selective agar (CCDA). Microaerobic atmosphere critical (5% O2, 10% CO2, 85% N2). For selective enrichment and cultivation of Campylobacter species from clinical and nonclinical specimens.
What supplement does Baird Parker Agar need?
Baird Parker Agar Base requires Egg Yolk Tellurite Emulsion (added after autoclaving at 45-50°C). The emulsion provides: (1) Egg yolk - lecithin for lecithinase test, (2) Potassium tellurite - selective agent producing black colonies. Add 50mL supplement per liter of base. Without supplement, medium is not selective for S. aureus. Many labs prefer pre-supplemented RTU plates for consistency and safety (raw eggs). For isolation and enumeration of coagulase positive Staphylococci from food and clinical samples.
What is Alkaline Peptone Water used for?
Alkaline Peptone Water is enrichment for Vibrio species (V. cholerae, V. parahaemolyticus) from food and clinical specimens. High pH (8.5-9.0) favors Vibrio while inhibiting competitors. Used as primary enrichment: inoculate sample, incubate 6-8 hours at 35-37°C, plate onto TCBS. Essential for cholera surveillance, seafood testing. The alkaline pH is critical - Vibrio tolerates it while most bacteria don't.
What is UVM Broth?
UVM (University of Vermont Medium) is primary selective enrichment for Listeria from food per USDA MLG and ISO 11290. Contains nalidixic acid (inhibits most gram-negatives) and acriflavine (inhibits gram-positives except Listeria). Less selective than Fraser (allows initial Listeria recovery from stressed cells). Two-stage enrichment: (1) Primary: UVM at 30°C for 24h, (2) Secondary: Fraser Broth at 35-37°C for 24-48h. UVM's gentler selectivity aids recovery of injured Listeria from frozen foods, heat-treated foods. Used in combination with Fraser for maximum sensitivity. For selective isolation and cultivation of Listeria monocytogenes.
What is Oxford Listeria Agar?
Oxford Listeria Agar is selective differential medium for Listeria isolation from food samples. Contains: (1) Lithium chloride, acriflavine, colistin, cefotetan, cycloheximide - selective agents (Oxford supplement), (2) Esculin and ferric ammonium citrate - Listeria hydrolyzes esculin producing black colonies with black halos. Used after Fraser enrichment per FDA BAM. L. monocytogenes and L. ivanovii appear black. Less selective than PALCAM (more competitors grow) but still effective. Incubate 24-48 hours at 30-35°C. Confirm suspect colonies with biochemical tests and serology.
What is Tetrathionate Broth used for?
Tetrathionate Broth is selective enrichment for Salmonella. Contains: (1) Tetrathionate - selective agent (Salmonella can reduce it, others inhibited), (2) Brilliant green and bile salts - additional selectivity, (3) Iodine solution added before use. Used in parallel with RV broth for comprehensive Salmonella detection per FDA BAM. Tetrathionate works differently than RV - complementary mechanisms. Procedure: Add iodine solution to prepared base, inoculate from pre-enrichment, incubate 24h at 35-43°C, plate onto selective agar. Some Salmonella serovars recover better in TT than RV and vice versa.
What is Selenite Broth used for?
Selenite Broth (Selenite F) is selective enrichment medium for Salmonella from fecal and food specimens. Contains sodium selenite (toxic to most bacteria except Salmonella/Shigella). Used after pre-enrichment in BPW. Procedure: Transfer 0.1-1.0mL from BPW to 10mL Selenite, incubate 18-24 hours at 35-37°C (some protocols 43°C), plate onto selective agar. Less selective than Rappaport-Vassiliadis but useful for foods where RV may be too harsh. Selenite is toxic - handle carefully, autoclave before disposal. Many labs use RV (better selectivity) or parallel RV + Selenite for maximum recovery.
What is Buffered Peptone Water used for?
Buffered Peptone Water (BPW) is non-selective pre-enrichment medium for resuscitation of stressed or injured bacteria from food samples. Used as first step before selective enrichment in Salmonella, Listeria, Campylobacter testing per FDA BAM, USDA, ISO methods. Procedure: (1) Add 25g food sample to 225mL BPW (1:10 dilution), (2) Homogenize/stomaching, (3) Incubate 18-24 hours at 35-37°C, (4) Transfer to selective enrichment (RV, Fraser, etc.). Phosphate buffer maintains pH during bacterial growth. Gentle resuscitation allows detection of low levels or stressed pathogens.
What is TCBS Agar used for?
TCBS Agar is highly selective medium for isolation of Vibrio species, particularly V. cholerae and V. parahaemolyticus from seafood and clinical specimens. Contains: (1) Bile salts and high pH (8.6) - inhibit most bacteria, (2) Thiosulfate and citrate - selective agents, (3) Bromothymol blue and thymol blue - pH indicators. V. cholerae: yellow colonies (sucrose fermentation). V. parahaemolyticus: blue-green colonies (no sucrose fermentation). Used for seafood testing, cholera surveillance. Do not autoclave - boil to dissolve. Incubate 18-24 hours at 35-37°C.
What is CCDA used for?
Campylobacter Charcoal Differential Agar (CCDA) is selective medium for isolation of Campylobacter jejuni and C. coli from food and clinical specimens. Contains: (1) Charcoal - absorbs toxic oxygen metabolites, (2) Cefoperazone and amphotericin - selective agents, (3) Blood-free formulation. Campylobacter appears as gray, flat, moist colonies with metallic sheen. Requires microaerophilic atmosphere (5% O2, 10% CO2, 85% N2) and 42°C incubation for 48 hours. Used for: poultry testing, diarrheal stool samples. Campylobacter is leading cause of bacterial gastroenteritis worldwide. The composition and performance criteria of this medium are as per the specifications laid down in ISO 10272-1&2:2017.
What is PALCAM Agar used for?
PALCAM (Polymyxin Acriflavine LiCl Ceftazidime Esculin Mannitol) Agar is selective differential medium for isolation of Listeria monocytogenes and Listeria species from food samples. Contains multiple selective agents inhibiting competing microflora. Listeria species hydrolyze esculin producing black colonies with black halos on gray-green background. L. monocytogenes ferments mannitol. Used after enrichment in Fraser or UVM broth. Part of FDA BAM and ISO 11290 methods. More selective than Oxford Agar. Incubate 48 hours at 35-37°C.
What is Fraser Broth used for?
Fraser Broth is selective enrichment medium for Listeria species from food per FDA BAM, USDA, ISO 11290. Two-step enrichment: (1) Half-Fraser Broth (half-strength selective supplement) - primary enrichment 24h at 30°C, (2) Fraser Broth (full-strength) - secondary enrichment 24h at 35-37°C. Contains esculin - Listeria hydrolyzes to esculetin causing black color. Acriflavine and nalidixic acid inhibit competing bacteria. Black color = presumptive Listeria. Plate onto PALCAM, Oxford, or HiCrome Listeria Agar for isolation. Essential for detecting low levels of Listeria in RTE foods.
How is Bile Esculin used for Water quality testing?
In Water quality testing, Bile Esculin Azide Agar is used for confirmation of fecal streptococci (enterococci) from membrane filtration. Procedure: (1) Filter Water sample through 0.45μm membrane, (2) Place membrane on Bile Esculin Azide Agar, (3) Incubate 48 hours at 35°C, (4) Count colonies with black halos or blackening of medium. These are presumptive enterococci. Fecal streptococci/enterococci are indicators of fecal contamination in recreational Water, drinking Water. EPA Method 1600 uses similar approach. Presence indicates possible fecal pollution.
What is Bile Esculin Azide Agar used for?
Bile Esculin Azide Agar is a selective differential medium for isolation and presumptive identification of Enterococcus species and Group D Streptococcus. Also used for Listeria species. Contains: (1) Sodium azide - inhibits gram-negative bacteria, (2) Bile - inhibits most gram-positives except enterococci and Listeria, (3) Esculin and ferric citrate - detect esculin hydrolysis. Positive organisms (Enterococcus, Listeria, Group D Strep): black colonies or colonies with black halos. Medium turns brown/ black. Used in Water quality testing, clinical microbiology, food testing.
What is Bismuth Sulfite Agar used for?
Bismuth Sulfite (BS) Agar is a highly selective medium specifically for Salmonella typhi and other Salmonella species. Most selective Salmonella medium, but also most inhibitory. Contains bismuth sulfite producing black colonies from H2S. S. typhi produces characteristic black colonies with metallic sheen and black surrounding agar (diagnostic). Other Salmonella: brown/gray/black colonies. Most other organisms inhibited. Use BS when: (1) Suspect typhoid fever, (2) Need maximum selectivity (heavily contaminated samples), (3) Confirmation of Salmonella from enrichment. Do not overheat during preparation.
What is the difference between XLD and HE Agar for Salmonella?
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
What is Hektoen Enteric Agar used for?
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.
How do I interpret colony colors on XLD Agar?
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.
What is XLD Agar used for?
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.
What is Listeria Enrichment Broth used for? What is Fraser Broth Base, Modified (Half Fraser Broth) used for?
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.
How do I use RV Broth in Salmonella detection protocol?
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.
What is Rappaport Vassiliadis (RV) Broth used for?
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.