Isolation and identification of Enteric Bacteria Experiment five Isolation and identification of Enteric Bacteria

The family Enterobacteriaceae includes a group of bacteria that inhabit the intestinal tracts of humans and other animals. Some species are normal flora and others are pathogens of certain diseases. Commonly used techniques for their isolation and identification include biochemical test, antigens present on their surfaces and motility. Among the important genera included as enterics are Escherichia, Salmonella, Shigella, Klebsiella, Proteus and Yersinia.

Dimidiation of the enterobacteria according to the fermentation of lactose Lactase fermenters: saprophytic and commensal Escherichia Klebsiella Citrobater Enterobacter Non lactase fermenters: pathogens Salmonella Shigella some Citrobacter Proteus Serratia

Isolation culture and identification procedure Colonial characteristic observation Specimens isolation Gram Staining (SS/EMB plate) Serological identification TSI Biochemical reaction

Specimens Different specimens should be taken depending on the kind and the process of the disease. blood bone marrow Urine stool

Isolation Culture medium: S.S agar Method: streak plate Result: Non-pathogenetic colonies: middle size, red Suspect colonies: colorless, small, opaque

Biochemical reactions of Salmonella, etc   Species bottom slope H2S motility E.coli AG AG - + Salmonella A - +/- + Shigella A - +/- - bottom: ferment dextrose slope: lactose A:acid AG: acid and gas

Items of the experiment (1) Eosin-Methylene Blue Agar (EMB) Reagents: Eosin and Methylene-blue Carbohydrate Source: Lactose Principle: a precipitate can be formed at acidic pH Purpose: differentiate lactose fermenters from non-lactose fermenters. Interpretation: Lactose fermenter: purple Lactose non-fermenter: colorless

EMB agar E.coli: black and typically have a metallic green sheen negative

EMB agar E.coli S.typhi S.dysenteriae ( ++ ) ( - )

Items of the experiment (2) Salmonella-Shigella Agar(S.S agar ) Reagents: Lactose (Carbohydrate Source) Neutral red( as an indicator), Bile salt, Ferric ammonium citrate (Provides for inhibition of normal flora coliforms and differentiationof stool pathogens (e.g. Salmonella anal Shigella) Interpretation: Lactose non-fermenter: colorless Lactose fermenter: pink to red colonies Principle:

S.S agar

S.S agar E.coli S.typhi S.dysenteriae pink colorless

Items of the experiment (3) Indole Broth purpose: Distinguish Enterobacteriaseae based on the ability to produce indole from tryptophan. principle: Interpretation Positive Test - red ring Negative Test - no color development tryptophanase Kovac’s reagent indole tryptophan rosindole (red)

Indole Broth - +

Indole Broth E.coli S.typhi S.dysenteriae ( + ) ( - )

(4) Double sugar iron slant Items of the experiment (4) Double sugar iron slant main component: Glucose: 0.1% Lactose: 1% Phenol red: as an indicator Ferrous sulfate(FeSO4) :FeSO4+H2S →FeS(black)+H2SO4 purpose: It provides information about carbohydrate fermentation. Principle:

Only glucose fermenting Slant: oxidized →neutral →red Butt: not oxidized →yellow

Lactose fermenting yellow both on the slant and in the butt

H2S production FeSO4+H2S FeS(black)+H2SO4

Gas production

Double suger iron slant

Double suger iron slant E.coli S.typhi S.dysenteriae Slant yellow red butt Gas ( + ) ( - ) H2S