Gram-negative rodsGram-negative rods

EnterobacteriaceaeEnterobacteriaceae

Bacteria

Gram positive Gram negative

Cocci Bacilli Cocci Rods

Characters of Characters of EnterobacteriaceaeEnterobacteriaceaeAll All EnterobacteriaciaeEnterobacteriaciae– Gram-negative rodsGram-negative rods– Ferment glucose with acid production Ferment glucose with acid production – Reduce nitrates into nitritesReduce nitrates into nitrites– Oxidase negativeOxidase negative

Facultative anaerobicFacultative anaerobic

Motile Motile exceptexcept ShigellaShigella and and KlebsiellaKlebsiella

Non-capsulated except Non-capsulated except KlebsiellaKlebsiella

Non-fastidiousNon-fastidious

Grow on bile containing media (MacConkey agar) Grow on bile containing media (MacConkey agar)

EnterobacteriaceaeEnterobacteriaceaeSome Some Enterobacteriaceae Enterobacteriaceae are true pathogensare true pathogens– SalmonellaSalmonella spp. spp. – ShigellaShigella spp. spp. – Yersinia Yersinia spp. spp. – Certain strains of Certain strains of E. coli E. coli (ETEC, EPEC, EIEC, EHEC) (ETEC, EPEC, EIEC, EHEC)

Most members of the Most members of the Enterobacteriaceae Enterobacteriaceae are are opportunistic or cause secondary infections of opportunistic or cause secondary infections of wounds, the urinary and respiratory tracts, and wounds, the urinary and respiratory tracts, and the circulatory system e.g. the circulatory system e.g. E. coliE. coli..Enterobacteriaceae divided into TWO main Enterobacteriaceae divided into TWO main groups according to action on LACTOSEgroups according to action on LACTOSE– Lactose Fermenters (LF)Lactose Fermenters (LF)

E. coli, Citrobacter, Klbesiella, EnterobacterE. coli, Citrobacter, Klbesiella, Enterobacter– Lactose Non-Fermenters (LNF)Lactose Non-Fermenters (LNF)

Salmonella, Shigella, Proteus, YersiniaSalmonella, Shigella, Proteus, Yersinia

Identification of Identification of EnterobacteriaceaeEnterobacteriaceae

Gram stainGram stain– All All EnterobacteriaceaeEnterobacteriaceae are Gram-negative rods are Gram-negative rods– Arranged in singleArranged in single

Identification of Identification of EnterobacteriaceaeEnterobacteriaceaeBiochemical reactionsBiochemical reactions

Oxidase testOxidase test

– All members of All members of EnterobacteriaceaeEnterobacteriaceae are oxidase negative are oxidase negative

– PseudomonasPseudomonas is oxidase positive is oxidase positive

O/F testO/F test

– All members of All members of EnterobacteriaceaeEnterobacteriaceae are O+/F+ are O+/F+

– PseudomonasPseudomonas is O+/F- is O+/F-

Nitrate reductaseNitrate reductase

– All members of Enterobacteriaceae are nitrate reductase positiveAll members of Enterobacteriaceae are nitrate reductase positive

– PseudomonasPseudomonas is nitrate reductase negative is nitrate reductase negative

See & compare these tests under See & compare these tests under Pseudomonas LabPseudomonas Lab

Classification of Classification of EnterobacteriaceaeEnterobacteriaceae

Enterobacteriaceae

Lactose fermentersE. coli, Citrobacter,

Klebsiella, Enterobacter

Non-lactose fermenterSalmonell, ShigellaProteus, Yersinia

There are several selective and differential media used to isolate distinguishes between LF & LNFThe most important media are:

MacConkey agarEosin Methylene Blue (EMB) agarSalmonella Shigella (SS) agarIn addition to Triple Sugar Iron (TSI) agar

Differentiation between LF & NLF byDifferentiation between LF & NLF by Growth on MacConkey agarGrowth on MacConkey agar

MacConkey Agar

Contains

Bile salts Crystal violet Lactose Neutral red

MacConkey agar is selective & differential medium for MacConkey agar is selective & differential medium for EnterobacteriaceaeEnterobacteriaceae

Inhibit growth of G+ve bacteria

Cause of selectivity

Cause of differentialpH indicatorAcidic: Pink

Lactose feremntersPink colonies

Lactose non feremnterscolorless colonies

Classification of Classification of EnterobacteriaceaeEnterobacteriaceae according to according to lactose fermentation (growth on MacConkey Agar)lactose fermentation (growth on MacConkey Agar)

Enterobacteriaceae

Lactose Fermenters Lactose Non-Fermenters

Escherichia coliKlebsiella sppEnterobacter sppCitrobacter spp

Salmonella sppSchigella sppProteus sppYersinina spp

Pink coloniesColorless colonies

AcidNeutral red

No acid

Identification of Identification of EnterobacteriaceaeEnterobacteriaceaeDifferentiation between LF & NLF byDifferentiation between LF & NLF by Growth on MacConkey agarGrowth on MacConkey agar

Method:Method:– MacConkey agar is inoculated with tested organismMacConkey agar is inoculated with tested organism

using streak plate techniqueusing streak plate technique– Incubate the plate in incubator at 37 C/24 hrsIncubate the plate in incubator at 37 C/24 hrs

Results:Results:– LF organism appears as pink colonies (e.g. LF organism appears as pink colonies (e.g. E. coliE. coli))– NLF organism appears as colorless colonies (e.g. NLF organism appears as colorless colonies (e.g.

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Growth of Enterobacteriaceae on MacConkey agar

Uninoculated plate Lactose non feremtersSalmonella, Shigella,

Proteus

Lactose feremtersE. coli, Citrobacter

Klebsiella, Enterobacter

Colorless colonies Pink colonies

Reaction on Salmonella Shigella (SS) agarReaction on Salmonella Shigella (SS) agarSS agar is a selective & differential medium used for isolation of SS agar is a selective & differential medium used for isolation of SalmonellaSalmonella and and ShigellaShigellaThe selective agents are The selective agents are bile saltsbile salts, and , and brilliant green dyebrilliant green dye, which , which inhibit gram-positive organismsinhibit gram-positive organismsThe medium contains only The medium contains only lactoselactose as a differential agent and thus as a differential agent and thus differentiates on the basis of lactose fermentationdifferentiates on the basis of lactose fermentationThe formation of acid on fermentation of lactose causes the neutral red The formation of acid on fermentation of lactose causes the neutral red indicator to make pink coloniesindicator to make pink coloniesNon lactose fermenting organisms are colorless on the mediumNon lactose fermenting organisms are colorless on the mediumSS agar contains SS agar contains sodium thiosulfatesodium thiosulfate and and ferric ammonium citrateferric ammonium citrate allows the differentiation of organisms that produce H2Sallows the differentiation of organisms that produce H2S– Lactose fermenters, such as Lactose fermenters, such as E. coliE. coli, have colonies which are pink, have colonies which are pink– ShigellaShigella appears transparent or amber appears transparent or amber– SalmonellaSalmonella appears transparent or amber with black centers due to appears transparent or amber with black centers due to

H2S productionH2S production

LactoseLactose fermenter

AcidNeutral red

Pink colonies

Ferrous sulfideBlack precipitate

H2S + Ferric ammonium citrate

Identification of Identification of EnterobacteriaceaeEnterobacteriaceaeDifferentiation between LF & NLF byDifferentiation between LF & NLF by Growth on SS agarGrowth on SS agar

Method:Method:

– SS agar is inoculated with tested organismSS agar is inoculated with tested organism using using

streak plate techniquestreak plate technique

– Incubate the plate in incubator at 37 C/24 hrsIncubate the plate in incubator at 37 C/24 hrs

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AA.. Klebsiella pneumoniaeKlebsiella pneumoniaeBB.. Escherichia coliEscherichia coliCC: : Salmonella spSalmonella sp..DD: : Proteus mirabilisProteus mirabilis EE: : Ps. aeruginosaPs. aeruginosa . .

BothBoth are lactose fermentersare lactose fermenters

BothBoth Salmonella spSalmonella sp. . & Proteus & Proteus product H2Sproduct H2S

PseudomonasPseudomonas colonies are nearly colorlesscolonies are nearly colorless

Growth of Enterobacteriaceae on SS agar

Growth of Growth of EnterobacteriaceaeEnterobacteriaceae on on EMB agarEMB agar

Coli-type colonies are very dark, almost black e.g. E. coli

Reaction on Triple Sugar Iron (TSI) AgarReaction on Triple Sugar Iron (TSI) Agar

TSI containsTSI contains– Three different types of sugarsThree different types of sugars

Glucose (1 part)Glucose (1 part)

Lactose (10 part)Lactose (10 part)

Sucrose (10 part)Sucrose (10 part)

– Phenol red (acidic: Yellow)Phenol red (acidic: Yellow)

TSI dispensed in tubes with equal butt & slantTSI dispensed in tubes with equal butt & slant

PrinciplePrinciple – To determine the ability of an organism to attack a specific To determine the ability of an organism to attack a specific

carbohydrate incorporated into a basal growth medium, with or carbohydrate incorporated into a basal growth medium, with or

without the production of gas, along with the determination of without the production of gas, along with the determination of

possible hydrogen sulphide production. possible hydrogen sulphide production.

Reaction on TSIReaction on TSI

Method:Method:– Inoculate TSI medium with an organism by Inoculate TSI medium with an organism by

inoculating needle by stabbing the butt and inoculating needle by stabbing the butt and streaking the slantstreaking the slant

– Incubate at 37°C for 24 hoursIncubate at 37°C for 24 hours

Reaction on TSIResult

Example

Butt colorSlant colorH2S

Yellow RedNegativeA/Alk/- (Glucose fermented)

LNF e.g. Shigella

YellowRedPositive

black in buttA/Alk/+

(Glucose fermented with H2S)

LNF e.g. Salmonella &

Proteus

YellowYellowNegativeA/A/- (three sugars are fermented)

LF e.g. E. coli, Klebsiella,

Enterobacter

RedRedNegativeAlk/Alk/-

(No action on sugars)Non fermenter e.g.

Pseudomonas

Results

Practicle WorkPracticle Work

Gram stainGram stain

Oxidase testOxidase test

O/F testO/F test

Nitrate reductase testNitrate reductase test

Growth on MacConkey’s agarGrowth on MacConkey’s agar

Growth on EMB agarGrowth on EMB agar

Growth on SS agarGrowth on SS agar

Reaction on TSIReaction on TSI