Mannitol Salt Agar (MSA)

Purpose: Selective isolation of staphylococcus (and some salt-tolerant strains of micrococcus and enterococcus)Component: Beef extract, D-mannitol, and salt concentration NaCl of 7.5% (inhibits most bacteria)Indicator: phenol red indicator (red- pH 8.4, yellow-pH 6.8)Result:Yellow colonies grow and ferment mannitol (S.aureus)Small red colonies - grow but do not ferment mannitol (most coagulase-negative strains of Staphylococcus)

Original color: RED

Eosin Methylene Blue (EMB)

Purpose: Selective isolation of gram negative bacteria and differentiation of lactose-fermenting and nonlactose fermenting enteric bacilliComponent: Peptone base with lactose and sucroseIndicator: Eosin and methylene blue (inhibits Gram+)Result:Purple lactose is fermented, precipitated eosin & methylene are absorbedWith green metallic sheen Escherichia coliWith fish eye appearance Enterobacter aerogenes (rapid lactose fermenters)Colorless- non-lactose fermenters

Original color: RED

MacConkey agar (MAC)

Purpose: Selective isolation of gram negative bacteria and differentiation of lactose-fermenting and nonlactose fermenting enteric bacilliComponent: Peptone base with lactose, crystal violet and bile salt (inhibits Gram+)Indicator: Neutral red (together with crystal violet further inhibits Gram+); brown in pH 6.8-8.0 and red-pink at pH less than 6.8Result:Pink-Red lactose are fermented, bile salts are precipitated. The precipitated dye is absorbedBrown lactose not fermented

Original color: RED

Hektoen Enteric Agar (HEA) Purpose: Selective and differential for Salmonella and Shigella from other Gram (-) enteric bacilliComponent: Lactose, sucrose and salicin as C source. Bile salt (inhibits Gram+)Indicator: Bromthymol blue (Blue at pH greater than 7.6, Green at pH 6.0-7.6, and Yellow at pH less than 6.0)Result:Orange- 1,2 or 3 carbohydrates are fermentedGreen- non fermentersBlack centered colonies presence of sodium thiosulfate (H2S + ferric ammonium citrate = ferric sulfide[FeS])

Original color: BLUEXylose-Lysine Deoxycholate (XLD)

Purpose: Selective and differential for Salmonella and Shigella from other Gram (-) enteric bacilliComponent: Yeast extract agar with lysine, xylose, lactose, sucrose and ferric ammonium citrate,Sodium deoxycholate (inhibits Gram+ and the swarming of proteus, partially inhibits E.coli )Indicator: Phenol redResult:Yellow fermentation of xylose (Enterobacteriaciaceae except Shigella)Red colonies cannot ferment xylose (Shigella cant ferment lactose too)*Lysine positive first produce yellow as xylose is fermented then lysine decarboxylation (yellow)Black centers reaction of H2S with ferric ammonium citrate

Salmonella-Shigella Agar

Purpose: Selective for Shigella and SalmonellaComponents: Peptone base with lactose, ferric citrate, and sodium citrate. Brilliant green (inhibits negative coliform) and bile salt (inihibits gram+)Indicator: Neutral RedResult: Pink-red lactose fermenters (normal coliforms)Colorless- non-lactose fermenters (Shigella)Black centered H2S production (Salmonella clear colonies)

Original color: BROWN/ORANGEBismuth Sulfite Agar (BSA)

Purpose: Selective for SalmonellaComponents: bismuth sulfite and brilliant green (inhibits gram+, most lactose fermenters and Shigellae). Ferrous sulfite (reactive with hydrogen sulfide to produce ferric sulfide, deposited as black insoluble precipitate)Indicator: Brilliant greenResult:Black with metallic sheen Salmonella typhiLight green S. gallinarum, S. choleraesuis, and S. paratyphi

Thiosulfate Citrate Bile Salt Sucrose (TCBS)

Purpose: Selective and differential for vibriosComponents: Sodium citrate, sodium thiosulfate, and oxygall (inhibits Gram+ cocci and Gram- rods) and sucrose. High pH enables growth of vibrio*Aeromonas or Pseudomonas can growIndicator: Bromthymol blue Result:Yellow colonies: sucrose fermenters (V.cholerae and V. alginolyticus)Blue-green colonies: sucrose non-fermenters (V. parahaemolyticus and V. vulnificus) Black center produce hydrogen sulfide

Original: Green

Triple Sugar Iron (TSI) agar

Purpose: screening medium for identification of enterobacteriaceaeComponent: Glucose 1%, Lactose 10%, Sucrose 10%Indicator: Phenol RedH2S indicator: ferrous sulfate

Slant portion- exposed to oxygen Alkaline (due to oxidative decarboxylation of peptides and AA); REDDeep portion- anaerobic Acidic (minimal degradation of AA and small quantity of acid produced because few amines are being formed from AA)

Alkaline Slant/Acid butt (K/A)- ferment glucose but not lactose or sucroseProduce only small quantities of acid and cant counteract degradation of AA in slant which results in alkaline pH due to oxidative decarboxylation.

Acid Slant/Acid butt (A/A)- ferment glucose and sucrose and/or lactoseLarge quantities of acid produced overcoming the alkaline reaction of the slant

H2S (+)- production of H2SSodium thiosulfate (sulfur source) provides sulfur atoms to detect productuin of H2SReacts with iron salts (ferrous sulfate or ferric ammonium citrate)

Gas (encircled A)- crack or pulling away in the medium

A/AGPantoea agglomeransEscherichia coliEnterobacter aerogenesKlebsiella pneumonia

A/AG or K/AGSerratia marcescensCitrobacter koseriHafnia alvei

A/AG+ or K/AG+Citrobacter freundiiSalmonella

K/AG+Proteus vulgarisProteus mirabilisEdwardsiella tardaSalmonella paratyphiSalmonella choleraesuis

A/A or K/AYersinia enterocoliticaShigella sonnei

K/AShigella dysenteriaeShigella flexneriShigella boydiiLysine Iron Agar (LIA)

Purpose: Identification of Salmonella, Proteus, Providencia, and MorganellaAbility of organism to deaminate lysine, decarboxylase lysine and produe H2s*Deaminase positive Proteus, Providencia, and MorganellaComponents: lysine, peptones, a small amount of glucose, ferric ammonium citrate, and sodium thiosulfate.Indicator: bromcresol purpleH2s indicator: ferric ammonium citrate

1st Glucose fermentation yellow butt2nd Lysine decarboxylation produces alkaline cadaverine purple butt3rd Lysine deamination in the presence of oxygen in slant red slantH2S production black ppt in the butt when H2S reacts with ferric ammonium citrate

Lysine decarboxylase positive:Purple/Purple (K/K)Lysine decarboxylase negative:Puple/Yellow (K/A)Deaminase positive:Red/Yellow (R/A)H2S positive:Blackening

K/KKlebsiella pneumoniaEscherichia coliEnterobacter aerogenesSerratia marcescensHafnia alvei

K/ACitrobacter koseriPantoea agglomeransShigella dysenteriaeShigella flexneriShigella boydiiShigella sonnei

R/AProteusProvidencia Morganella

K/K+Salmonella typhimuriumSalmonella arizonaeSalmonella typhiSalmonella paratyphiSalmonella choleraesuis

IMViC Test

Indol Production Test

Principle: The organism oxidized tryptophan to indol by the enzyme tryptophanaseMedium: Tryptone brothReagent: Ehrlichs or Kovacs (paraaminobenzaldehyde)Result: (+) Red ring(-) yellow/brown ring

Methyl Red Test

Principle: The organism ferments glucose to pyruvic acid resulting in mixed acid products leading to a decreased pH (4.5-5.5) Medium: Methyl red voges proskauer medium (MRVP)Reagent: Methyl red indicatorResult:(+)red solution(-) yellow solution

Voges Proskauer Test

Principle: The organism ferments glucose producing acetylmethylcarbinol(acetoin) and converted to diacetyl in the presence of oxygen and KOHMedium: MRVPReagent: VP reagent A (Alpha naphthol in absolute methyl alcohol)VP reagent B (KOH with creatine)Result:(+) Pink to red color(-) yellow color/ copper-like

Citrate Utilization Test

Principle: the organism utilized the citrate producing ammonia and converted to ammonium hydroxide. This alkaline compound raises the pH of the medium and takes the blue colorMedium: Simmon Citrate Agar (SCA)Indicator: Bromthymol blueResult:(+) Prussian blue(-) Green

Urease Test

Principle: Urease splits the urea molecule into ammonia, carbon dioxide and water. Ammonia reacts in solution to form an alkaline compound, ammonium carbonate, which results in an increased pH of the medium and a color change to pink-redMedium: Urea brothIndicator: Phenol redResult:(+) pink-red(-) no color change*Rapid Urease Production (2-4 hrs) Proteus, Providencia, Morganella*LUP (24 hrs) Citrobacter, Klebsiella, Enterobacter, Yersinia, Serratia

Sulfide Indol Motility (SIM) Medium

Medium: SIMReagent: Ehlirchs or Kovacs

Motile: growth pattern is away from the inoculation line (test tube brush appearance)Non-motile: growth pattern is confined on the inoculation lineH2S production: blackening of buttIndol production (red ring)

*Non-motile- Klebsiella and Shigella

Blood Agar (BA)

Purpose: detection of hemolytic patternComponents: Human blood or 5% defibrinated sheep blood added to a base either trypticase soy agar or nutrient agar.*Blood provides serum ingredients as well as erythrocyteResult:Alpha hemolysis incomplete hemolysis (green)Beta hemolysis complete hemolysis (colorless) due to diffusion of hemoglobin into the surrounding mediumGamma hemolysis non-hemolytic

Chocolate Agar (CA)

Purpose: Utilization of fastidious bacteriaComponent: Lysed RBC added to molten agar base (nutrient agar or trypticase soy agar)*Lysis releases the intracellular nutrients such as hemoglobin, hemin (X factor) and coenzyme NAD (V factor)

Phenylethyl alcohol agar (PEA) inhibits Gram (-) bacteria