TEST REAGENT/MEDIA Catalase test 3% hydrogen peroxide Coagulase test Rabbit’s plasma Mannitol fermentation test 7.5% NaCl HCl precipitation DNase/ Thermonuclease test Dye Method INDICATOR SUBSTRATE Phenol red Pink zone around colonies Clear zone around colonies 0.1% Toluidine Blue Tetramethyl pphenylenediamine dihydrochloride in DMSO dimethylsulfoxide Cefinase Oxidase Test Carbohydrate utilization test Media Bile Esculin Media Tetramethyl pphenylenediamine dihydrochloride Media Cysteine Trypticase Agar Blue color (cell is ALIVE) blue Nitrocefin Leucine-betanaphthylamide Cephalosporinase test Leucine Amino Peptidase (LAP) Cinnamaldehyde test Pyrrolidonyl-alphapnaphthylamide (PYR) test dimethylaminocinnamaldehyde Benzoic acid Ferric chloride Hippurate Hydrolysis test glycine Ninhydrin (rapid test) CAMP (Christie, Atkins, Munch, MediaBAP Peterson) test Known organism S. aureus MediaBAP Reverse CAMP test Known organism S. agalactiae Bile Esculin test NEGATIVE RESULT NO Bubbling/effervescence bubbling/effervescence Clot formation Yellow halo around the Red color colonies 0.1 HCl Methyl Green Modified Oxidase Test POSITIVE result Pink to red color Red No color or slight yellow Red Orange to no color purple Ferric ammonium citrate ENHANCED hemolysis NO enhanced hemolysis ENHANCED hemolysis NO enhanced hemolysis Blackening of media Purple Phenol red Yellow acid pH red Superoxol Test Butyrate Disk Test 30% hydrogen peroxide Bubbling/effervescence Bromo-chloroindolylbutyrate Blue color NO bubbling/effervescence TEST Niacin Test Nitrate Reduction Iron Uptake Test Arylsulfatase Test Detection of rapid growers Pyrazinamidase Test Heat Stable Catalase Test To detect heat labile catalase Mycobacterium like M. tuberculosis BIOCHEMICAL TESTS for Mycobacterium REQUIRED MEDIA REAGENT Strip impregnated with Lowenstein Jensen Media cyanogen bromide n-n dimethyl-l-naphthylamine sulfanilic acid Sodium Nitrate Broth to confirm a (-) result: add zinc dust/zinc powder Lowenstein Ferric Ammonium Citrate Jensen Media Phenolphthalein media 2N Sodium carbonate Pyrazinamidase agar 1% ferrous ammonium sulfate Tween 80 Media Heating of colonies at 68 °C for 20 mins prior to the addition of reagent is required + 30% H2O2 Tween 80 Hydrolysis Test M. kansasii (+) in 3 days ; indiactor neutral red Tellurite Reduction test M. avium Salt Tolerance test 5% NaCl T2H/TCH susceptibility Thiopene-2-carboxylic hydrazide Growth in MacConkey Agar without Crystal Violet For rapid growers and M. triviale Differentiate M. tuberculosis (RESISTANT) and M. bovis (SENSITIVE) Identify M. fortuitum-chelonae complex POSITIVE (+) Result NEGATIVE (-) Result Yellow No color change Red No color change No color change red Rusty brown color No color pink red/pink-colored band Qualitative bubbling No bubbling Quantitative Height of bubbling Height of bubbling > 45 mm < 45 mm Pink No color change Smooth, fine black Gray clumps precipitate (no smoke like action) (smoke like action) Growth No growth Growth No growth Test Gram Staining Catalase Test Coagulase Test CAMP test Positive (+) control S. aureus S. aureus S. aureus S. pyogenes Micrococcus luteus S. agalactiae Bile Esculin Test Enterococcus faecalis Bacitracin Disk test Butyrate Disk test Niacin test Nitrate reduction test Arylsulfatase Test Tween 80 Hydrolysis Test Tellurite Reduction Test Salt Tolerance Test 5% NaCl T2H/TCH susceptibility Moraxella catarrhalis Biochemical Tests for Mycobacterium M. tuberculosis M. tuberculosis M. fortuitum M. gordonae M. kansasii M. avium M. fortuitum M. bovis Negative (-) control E. coli S. pyogenes S. epidermidis S. agalactiae S. aureus S. pyogenes E. coli S. pyogenes N. gonorrhoea M. intracellulare M. intracellulare M. tuberculosis M. intracellulare M. tuberculosis M. gordonae M. tuberculosis Photochromogens M. kansasii M. marinum M. asiaticum M. simae M. intermedium M. novocastrense ++-E. coli Y. enterocolitica P. vulgaris Edwardsiella tarda Morganella morganii Scotochromogen M. szulgai M. scrofulaceum M. gordonae/M. aquae M. xenopi M. flavescens M. interjectum M. heckeshornense M. europeaeum M. lentiflavum M. mantenii M. palustre M. parmense M. tusciae M. kubicae M. cookie M. hiberniae --++ Klebsiella pneumoniae Klebsiella ozaenae Enterobacter spp. Non-Photochromogens M. avium complex M. celatum M. ulcerans M. lacus M. malmoense M. genovense M. haemophilum M. heidelbergense M. shimoidei M. simiae M. terrae complex IMViC Result -+--+-+ Shigella dysenteriae Citrobacter Shigella sonnei freundii Salmonella typhi P. mirabilis Y. pestis Rapid Growers M. abscessus subp. Abscessus M. fortuitum M. chelonei M. phlei M. smegmatis ++-+ Providencia rettgeri Providencia stuarti + - ++ Klebsiella oxytoca -V++ Serratia marcescens Hafnia alvei BIOCHEMICAL TESTS for Enterobacterales REQUIRED MEDIA INDICATOR TEST pH phenol red Carbohydrate Fermentation test Triple Sugar Iron H2S sodium thiosulfate Ferrous sulfate POSITIVE (+) Result Acid Yellow Alkaline Red NEGATIVE (-) Result Blackening Splitting of media, cracks on media, pulling away of media ONPG O-nitrophenyl-beta-dgalactopyranoside Gelatin Hydrolysis Test Urease Test Deaminase Test Indole IMViC Indole-Methyl RedVogues ProskauerCitrate Methyl Red VP Citrate Detect LLF Differentiate Citrobacter (+) from Salmonella (-) Yellow color (presence of β-galactosidase) Colorless (absence of enzyme) Nutrient Gelatin media Christensen Urea Phenol red Agar/Urea Broth Color developer 10% ferric chloride Uses lysine, tryptophan, phenylalanine SIM Media Reagent: Tryptophan broth KOVAC’s or Erlich MRVP Broth Methyl red Clark Lubbs Broth MRVP Broth Alpha Naphthol and KOH Clark Lubbs Broth Liquefaction of gelatin Pink; red After 4 hours orange Phenylalanine agar green Tryptophan agar brown Solidification at 4 °C Simmon Citrate Agar Bromthymol Bblue Red ring Distinct red color; pH less than 4.5 Pink to red color Blue yellow No color development No color change/ Yellow No color change/ Yellow Green *Yellow prolonged incubation TEST REQUIRED MEDIA Motility Test SIM Media Malonate Utilization Test H2S production Bromthymol blue TSI, LIA, SIM, HEA; SIM most sensitive Moeller’s Broth Decarboxylase test TEST that will separate SALMONELLA & SHIGELLA NEGATIVE (-) INDICATOR POSITIVE (+) Result Result Growth OUTSIDE Growth is at the the line of streak line of streak Lysine Iron Agar HEA, SSA, LIA ferric ammonium citrate Bromcresol purple Cresol red Ferric ammonium citrate Blue green Blackening Purple yellow Deaminase red/burgundy Deaminase purple Decarboxylase Purple Decarboxylase yellow SALMONELLA SHIGELLA + - + - + - K/K K/A