isolation and identification of enteric bacteria experiment five
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Isolation and identification of Isolation and identification of Enteric BacteriaEnteric Bacteria
Experiment five
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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.
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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
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Colonial characteristic observation
Specimens isolation Gram Staining
(SS/EMB plate) Serological identification
TSI
Biochemical reaction
Isolation culture and identification procedure
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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
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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
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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
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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
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E.coliE.coli: black and typically have : black and typically have a metallic green sheen a metallic green sheen
negative
EMB agar
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E.coliE.coli S.typhiS.typhi S.dysenteriaeS.dysenteriae
( ++ )( ++ ) ( - )( - ) ( - )( - )
EMB agar
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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:
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S.S agar
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E.coliE.coli S.typhiS.typhi S.dysenteriaeS.dysenteriae
pinkpink colorlesscolorless colorlesscolorless
S.S agar
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(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
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Indole Broth
+ -
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E.coliE.coli S.typhiS.typhi S.dysenteriaeS.dysenteriae( + )( + ) ( - )( - ) ( - )( - )
Indole Broth
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(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
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Only glucose Only glucose fermentingfermenting
Slant: oxidized →neutral →red
Butt: not oxidized →yellow
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Lactose Lactose fermentingfermenting
yellow both on the slant
and in the butt
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HH22S S productionproduction
FeSO4+H2S
FeS(black)+H2SO4
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Gas productionGas production
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Double suger iron slantDouble suger iron slant
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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