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Microbiology Testing FAQs — Methods & Media | HiMedia USA
What is TSI Agar used for?
Triple Sugar Iron (TSI) Agar differentiates Enterobacteriaceae based on: (1) Glucose fermentation, (2) Lactose/sucrose fermentation, (3) H2S production, (4) Gas production. Contains glucose (0.1%), lactose (1%), sucrose (1%), ferric ammonium citrate, sodium thiosulfate. Inoculate by stabbing butt and streaking slant. Results interpreted by butt/slant colors and gas/H2S. E. coli: yellow/yellow (ferments all). Salmonella: red/yellow with black (glucose only + H2S). Shigella: red/yellow (glucose only, no H2S). Pseudomonas: red/red (none). Classic identification tool.
How is Ammonia detection done?
Detects ammonia production from peptone, Pale yellow to a dark brown precipitate is indicated by nesslers reagent as a positive reaction. Used in some identification schemes.
Tell us about VP reagents.
Two-part reagent (alpha-naphthol + KOH) for VP test. Add to VP broth after incubation. Cherry /red indicates acetoin production (VP positive).
Explain about MR indicator solution.
Methyl red solution for MR test. Add after 48h incubation in MR-VP broth. Red = MR positive (mixed acid fermentation).
Explain about Indole reagent.
Detects indole from tryptophan breakdown. Add to indole broth after incubation. Red ring = indole positive (E. coli).
What is Oxidase test?
Tests cytochrome c oxidase. Purple color = positive (Pseudomonas, Neisseria). Differentiates oxidase-positive from Enterobacteriaceae (negative).
How is Catalase testing performed?
3% hydrogen peroxide tests catalase enzyme. Bubbles = positive (Staph). No bubbles = negative (Strep). Critical differentiation test.
Tell us about Bile solubility.
For detection of esculin hydrolysis in the presence of bile, for differentiating Group D streptococci from
other Streptococcal groups.
What is Halotolerance testing?
Used for cultivation and differentiation of the enterococcal group D streptococci from nonenterococcal group D streptococci based on salt tolerance.
What is BCP indicator?
Bromocresol purple changes yellow with acid production. Alternative to phenol red. Used in some lactose fermentation tests. Recommended for identification of Escherichia coli and coliform bacteria from water samples.
What is Sucrose fermentation?
Tests sucrose fermentation capability. Yellow color = sucrose fermented. Part of carbohydrate fermentation panels for bacterial ID. Recommended for Sucrose fermentation studies of microorganisms.
Explain Mannitol fermentation test.
Tests mannitol fermentation. Yellow (acid) = positive. Used differentiating Staph aureus (positive) from other Staph in combination with MSA. Recommended for Mannitol fermentation studies of microorganisms.
Explain about the simple glucose fermentation.
Basic glucose fermentation test. Gas in Durham tube + yellow color = glucose fermentation. Baseline test for bacterial identification. Recommended for dextrose fermentation studies of microorganisms.
Explain about Esculin hydrolysis.
Tests esculin breakdown. Black color (esculin + iron) = positive. Esculin Agar is a differential medium for demonstrating esculin hydrolysis by various microorganisms
What is VP test principle?
Tests for acetoin production (2,3-butanediol pathway). Add alpha-naphthol and KOH. Pink/red = positive (Klebsiella, Enterobacter). The 'V' in IMViC.
What is MR test specifics?
Tests for mixed acid fermentation. Add methyl red after incubation. Red = positive (E. coli), Yellow = negative (Enterobacter). The 'M' in IMViC.
What is Malonate test?
Tests ability to use sodium malonate as sole carbon source. Blue color (pH increase) = positive. Differentiates Enterobacter/Klebsiella (positive) from E. coli (negative).
What is tributyrin test?
Tests lipase enzyme (fat breakdown). Tributyrin (glyceryl tributyrate) in agar creates opaque medium. Clear zones around colonies = lipase activity. Used for identifying lipolytic bacteria and fungi.
What is starch hydrolysis test?
Tests amylase enzyme (starch breakdown). After growth, flood with iodine - clear zone around colonies = starch hydrolysis (amylase present). Bacillus species typically positive. For detection of starch hydrolyzing microorganisms.
What is gelatin liquefaction test?
Tests gelatinase enzyme production. Organism liquefies gelatin. Used for identifying Serratia, Pseudomonas, Bacillus (positive) from non-gelatinase producers. For cultivation and identification of Vibrio species.
What is decarboxylase testing?
Tests ability to decarboxylate amino acids (lysine, ornithine, arginine). Purple color = positive (amine production raises pH). Used for Enterobacteriaceae differentiation. With addition of appropriate L-amino acid, it is used to differentiate bacteria on the basis of their ability to decarboxylate the amino acids.
What is SIM Medium?
Combination medium testing: Sulfide production (S), Indole production (I), Motility (M). Single stab inoculation tests 3 characteristics. Black = H2S, Red ring with Kovac's = indole, Diffuse growth = motility. For determination of hydrogen sulphide production, indole formation and motility of enteric bacilli.
What is ONPG test?
ONPG (o-nitrophenyl-β-D-galactopyranoside) test detects beta-galactosidase enzyme. Organisms with beta-galactosidase cleave ONPG producing yellow o-nitrophenol. Used to differentiate late lactose fermenters from non-fermenters. Example: Citrobacter and some Salmonella possess beta-galactosidase but ferment lactose slowly on MacConkey. ONPG positive within hours. Shigella lacks enzyme (negative). Procedure: Inoculate heavy suspension in ONPG broth, incubate 35°C up to 24h. Yellow = positive. Used in enteric bacteria identification, helps differentiate Salmonella (usually negative) from Citrobacter (positive).
What is Phenylalanine Agar used for?
Phenylalanine Agar tests phenylalanine deaminase enzyme production. Organism deaminates phenylalanine to phenylpyruvic acid. After incubation on slant, add 10% ferric chloride - green color indicates phenylpyruvic acid (positive). Used for identifying: Proteus, Morganella, Providencia (all positive). Differentiates these from other Enterobacteriaceae (negative). Particularly useful for identifying Proteus species - positive reaction within 1-5 minutes of adding ferric chloride. Part of enteric bacteria identification schemes. Inoculate slant heavily, incubate 18-24h at 35°C.
What is DNase Test Agar used for?
DNase Test Agar tests production of deoxyribonuclease enzyme that degrades DNA. Contains DNA in agar. After incubation, flood plate with 1N HCl - unhydrolyzed DNA precipitates (cloudy/opaque). Clear zone around colonies = DNase positive. Used for: (1) S. aureus identification (positive), differentiates from coagulase-negative Staph (negative), (2) Serratia marcescens (positive), (3) Enterococcus faecalis testing. Clinical labs use for presumptive S. aureus - faster than coagulase test. Can also use with toluidine blue (no acid needed).
What is Xylose Agar?
Xylose Agar tests xylose fermentation - useful for differentiating Enterobacteriaceae. Contains xylose and phenol red indicator. Yellow = xylose fermentation (E. coli positive within 4h, Shigella negative or slow). Used for rapid E. coli presumptive identification from stool cultures. Most E. coli ferment xylose rapidly while Shigella does not or very slowly (24-48h). Can help differentiate these pathogens before full biochemical testing. Part of some clinical lab rapid ID protocols. For selective isolation and enumeration of Salmonella typhi and other Salmonella species from clinical and non-clinical samples.
What is Nitrate Broth used for?
Nitrate Broth tests ability to reduce nitrate (NO3) to nitrite (NO2) or nitrogen gas (N2). Contains: peptone, beef extract, potassium nitrate. After 24-48h incubation: Add sulfanilic acid + alpha-naphthylamine (Griess reagent). Red color = nitrite present (nitrate reduced). No color = either (a) no reduction, or (b) complete reduction to N2. Add zinc dust: Red color develops = no nitrate reduction (zinc reduces nitrate). No color = complete reduction to N2. Important for: Enterobacteriaceae ID, Mycobacterium ID, Pseudomonas testing.
What is Indole Nitrite Medium used for?
Indole Nitrite Medium tests two biochemical reactions: (1) Indole production from tryptophan, (2) Nitrate reduction to nitrite. Part of bacterial identification. After incubation: For indole - add Kovac's reagent, red ring = positive (E. coli positive, Klebsiella negative). For nitrite - add sulfanilic acid and alpha-naphthylamine, red color = nitrate reduced to nitrite. Combined test saves tubes and time. Used for: Enterobacteriaceae identification, differentiating E. coli from Enterobacter. The 'I' in IMViC tests.
What is MR-VP Broth used for?
MR-VP Broth tests for two reactions: (1) Methyl Red (MR) test - detects mixed acid fermentation, (2) Voges-Proskauer (VP) test - detects acetoin production. Part of IMViC tests for Enterobacteriaceae. After incubation at 35°C for 48h: MR test - add methyl red indicator, red = positive (E. coli). VP test - add alpha-naphthol and KOH, pink/red = positive (Enterobacter, Klebsiella). E. coli: MR+ VP-. Enterobacter: MR- VP+. Critical for differentiating coliforms. The MR and VP in IMViC (Indole, Methyl Red, Voges-Proskauer, Citrate).
What is Lysine Iron Agar used for?
Lysine Iron Agar (LIA) tests for: (1) Lysine decarboxylase, (2) Lysine deaminase, (3) H2S production. Used with TSI for Enterobacteriaceae identification. Inoculate by stabbing butt and streaking slant. Results: Purple butt = lysine decarboxylase (Salmonella, most E. coli). Red slant = lysine deaminase (Proteus, Providencia). Black = H2S production (Salmonella, Citrobacter). Yellow = glucose fermentation only (Klebsiella). Critical for differentiating Salmonella (purple/purple or purple/red with black) from other enterics. Used in conjunction with TSI in identification schemes.
What is Urea Agar used for?
Urea Agar (Christensen) tests urease enzyme production. Urease hydrolyzes urea to ammonia, raising pH and turning phenol red indicator pink/magenta. Used for: (1) Identifying Proteus species (rapid urease, pink in 4-6 hours), (2) Differentiating Klebsiella (positive) from E. coli (negative), (3) Identifying Cryptococcus (yeast), (4) H. pylori identification (rapid urease test in GI diagnosis). Inoculate slant surface heavily. Proteus: pink in hours. Klebsiella: 1-2 days. E. coli/Shigella: remain yellow/tan (negative). Critical for enteric bacteria differentiation.
What is Simmons Citrate used for?
Simmons Citrate Agar tests ability to utilize citrate as sole carbon source. Part of IMViC tests for Enterobacteriaceae identification. Contains: (1) Citrate (sole carbon source), (2) Bromothymol blue (pH indicator), (3) Minimal nutrients. Inoculate slant surface only (light inoculum), incubate 24-48 hours. Citrate-positive: growth + blue color (pH increase from ammonia). Citrate-negative: no growth, medium remains green. E. coli: negative, Enterobacter/Klebsiella: positive, Salmonella: most positive. The 'C' in IMViC tests (Indole, Methyl Red, Voges-Proskauer, Citrate).
How is motility testing performed?
Motility Test Medium (semi-solid agar 0.4%) detects bacterial motility. Procedure: (1) Inoculate by single stab to medium center, (2) Incubate 24-48 hours at optimal temperature, (3) Observe growth pattern. Motile organisms: diffuse, turbid growth spreading from stab line. Non-motile organisms: growth only along stab line. Can add TTC (triphenyltetrazolium chloride) for better visualization - motile organisms produce red diffuse growth. Used for differentiating: E. coli (motile) from Shigella (non-motile), Salmonella (motile) from Klebsiella (non-motile). Part of IMViC tests.
What is OF Medium used for?
Oxidation-Fermentation (OF) Medium determines how organisms metabolize glucose: oxidatively (with O2) or fermentatively (without O2). Procedure: inoculate duplicate tubes by stabbing, overlay one with mineral oil (creates anaerobic), incubate both. Results: Fermentation - both tubes yellow (acid), Oxidation - open tube yellow, sealed tube green/blue (no growth), Oxidation only - open tube yellow, sealed tube no change. Non-saccharolytic - both tubes no change. Used for identifying Pseudomonas (oxidizer), Enterobacteriaceae (fermenter), Acinetobacter. Hugh-Leifson's OF test.