Isolation and identification of Isolation and identification of Enteric BacteriaEnteric Bacteria

Experiment five

The family Enterobacteriaceae includes a group oThe family Enterobacteriaceae includes a group o

f bacteria that inhabit the intestinal tracts of humans f bacteria that inhabit the intestinal tracts of humans

and other animals. Some species are normal flora aand other animals. Some species are normal flora a

nd others are pathogens of certain diseases. Commnd others are pathogens of certain diseases. Comm

only used techniques for their isolation and identificonly used techniques for their isolation and identific

ation include biochemical test, antigens present on tation include biochemical test, antigens present on t

heir surfaces and motility. Among the important genheir surfaces and motility. Among the important gen

era included as enterics are Escherichia, Salmonellera included as enterics are Escherichia, Salmonell

a, Shigella, Klebsiella, Proteus and Yersinia. a, Shigella, Klebsiella, Proteus and Yersinia.

Dimidiation of the enterobacteria accordinDimidiation of the enterobacteria according to the fermentation of lactoseg to the fermentation of lactose

• Lactase fermenters:Lactase fermenters: saprophytic and commenssaprophytic and commensalal

Escherichia Klebsiella Citrobater Escherichia Klebsiella Citrobater EnterobacterEnterobacter

• Non lactase fermentersNon lactase fermenters: : pathogens pathogens Salmonella Shigella some Citrobacter Salmonella Shigella some Citrobacter Proteus SerratiaProteus Serratia

Colonial characteristic observation

Specimens isolation Gram Staining

(SS/EMB plate) Serological identification

TSI

Biochemical reaction

Isolation culture and identification procedure

SpecimensSpecimens

• Different specimens should be Different specimens should be taken depending on the kind and taken depending on the kind and the process of the disease.the process of the disease.

blood blood

bone marrowbone marrow

Urine Urine

stoolstool

IsolationIsolation

• Culture medium: S.S agar Culture medium: S.S agar • Method: streak plateMethod: streak plate• Result: Result: Non-pathogenetic colonies: middle sizNon-pathogenetic colonies: middle siz

e, rede, red Suspect colonies: colorless, small, opSuspect colonies: colorless, small, op

aque aque

Biochemical reactions of Salmonella, Biochemical reactions of Salmonella, etcetc

Species bottom slope HSpecies bottom slope H22S motilityS motility E.coli AG AG - +E.coli AG AG - + Salmonella A - +/- + Salmonella A - +/- + Shigella A - +/- -Shigella A - +/- -

bottom: ferment dextrose slope: lactosebottom: ferment dextrose slope: lactose A:acid AG: acid and gasA:acid AG: acid and gas

Items of the experimentItems of the experiment

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

E.coliE.coli: black and typically have : black and typically have a metallic green sheen a metallic green sheen

negative

EMB agar

E.coliE.coli S.typhiS.typhi S.dysenteriaeS.dysenteriae

( ++ )( ++ ) ( - )( - ) ( - )( - )

EMB agar

Items of the experimentItems of the experiment

(2) Salmonella-Shigella Agar(S.S agar )(2) Salmonella-Shigella Agar(S.S agar ) Reagents: Reagents:

– Lactose (Carbohydrate Source)Lactose (Carbohydrate Source)– Neutral red( as an indicator), Neutral red( as an indicator), – Bile salt, Ferric ammonium citrate (Provides for Bile salt, Ferric ammonium citrate (Provides for

inhibition of normal flora coliforms and differeninhibition of normal flora coliforms and differentiationof stool pathogens (e.g. Salmonella anal tiationof stool pathogens (e.g. Salmonella anal Shigella)Shigella)

Interpretation:Interpretation: Lactose non-fermenter: Lactose non-fermenter: colorlesscolorless Lactose fermenter: Lactose fermenter: pink to red coloniespink to red colonies Principle:Principle:

S.S agar

E.coliE.coli S.typhiS.typhi S.dysenteriaeS.dysenteriae

pinkpink colorlesscolorless colorlesscolorless

S.S agar

(3) Indole Broth purpose: Distinguish Enterobacteriaseae based on the abilit

y to produce indole from tryptophan.

principle:

Interpretation

Positive Test - red ring

Negative Test - no color development

tryptophanase indole Kovac’s reagent rosindole tryptophan

(red)

Items of the experimentItems of the experiment

Indole Broth

+ -

E.coliE.coli S.typhiS.typhi S.dysenteriaeS.dysenteriae( + )( + ) ( - )( - ) ( - )( - )

Indole Broth

(4) Double sugar iron (4) Double sugar iron slantslant

main component:main component: Glucose: 0.1%Glucose: 0.1% Lactose: 1%Lactose: 1% Phenol red: as an indicatorPhenol red: as an indicator Ferrous sulfate(FeSOFerrous sulfate(FeSO44) :FeSO) :FeSO44+H+H22S →FeS(black)+HS →FeS(black)+H22SOSO44

purpose:purpose: It provides information about carbohydrateIt provides information about carbohydrate fermentation.fermentation. Principle:Principle:

Items of the experimentItems of the experiment

Only glucose Only glucose fermentingfermenting

Slant: oxidized →neutral →red

Butt: not oxidized →yellow

Lactose Lactose fermentingfermenting

yellow both on the slant

and in the butt

HH22S S productionproduction

FeSO4+H2S

FeS(black)+H2SO4

Gas productionGas production

Double suger iron slantDouble suger iron slant

E.coliE.coli S.typhiS.typhi S.dysenteriaeS.dysenteriaeSlantSlant yellowyellow redred redred

buttbutt yellow yellow yellowyellow yellowyellow

GasGas ( + )( + ) ( - )( - ) ( - )( - )

HH22SS ( - )( - ) ( + )( + ) ( - )( - )

Double suger iron slantDouble suger iron slant