Upload
lekuta-kimojino
View
184
Download
0
Tags:
Embed Size (px)
Citation preview
Gram-negative rodsGram-negative rodsEnterobacteriaceaEnterobacteriacea
Clinical MicrobiologyClinical Microbiology
MESHACK JUMAMESHACK JUMA
MEDICAL MICROBIOLOGY DEPT (UONMEDICAL MICROBIOLOGY DEPT (UON((
Msc. Med Micro (UON), PGD-RM(UONMsc. Med Micro (UON), PGD-RM(UON(, (,
Bsc. Med Micro(JKUAT), Dip MLS, HND Dx MicroBsc. Med Micro(JKUAT), Dip MLS, HND Dx Micro..
Family Family EnterobacteriaceaeEnterobacteriaceae often referred to as often referred to as
“enterics“enterics””..
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– Catalase positiveCatalase positive
Facultative anaerobicFacultative anaerobic
All motile All motile exceptexcept Shigella, Klebsiel Shigella, Klebsiel and and YersiniaYersinia
Non-capsulated except Non-capsulated except KlebsiellaKlebsiella
Non-fastidiousNon-fastidious
Grow on bile containing media (MacConkey agar( Grow on bile containing media (MacConkey agar(
Microscopic and Colony Microscopic and Colony MorphologyMorphology
Gram negative bacilli or coccobacilliGram negative bacilli or coccobacilliNon-spore formingNon-spore formingColony morphology on BA or MH of little Colony morphology on BA or MH of little value, as they look the same, except for value, as they look the same, except for KlebsiellaKlebsiellaSelective and differential media are used Selective and differential media are used for initial colony evaluation (ex. for initial colony evaluation (ex. MacConkey, Hektoen Enteric (HE), MacConkey, Hektoen Enteric (HE), Xylose lysine deoxycholate (Xylose lysine deoxycholate (XLDXLD(( agars agars((
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 EnterobacteriaceaeEnterobacteriaceaeBased on Biochemical reactionsBased on Biochemical reactions
Can be grouped into threeCan be grouped into three:-:-
A) Ability to ferment carbohydrates to acid or A) Ability to ferment carbohydrates to acid or Acid and gasAcid and gas..
B) Ability to utilize a substrate as the sole B) Ability to utilize a substrate as the sole source of carbonsource of carbon
C) Ability to enzymaticaly degrade a C) Ability to enzymaticaly degrade a compoundcompound
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
Oxidative/Fermentation testOxidative/Fermentation test
– All members of All members of EnterobacteriaceaeEnterobacteriaceae are O+/F+ are O+/F+
– PseudomonasPseudomonas is Oxidative+ / Fermentation - is Oxidative+ / Fermentation -
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 for See & compare these tests for Pseudomonas spp Pseudomonas spp in the Labin the 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. ShigellaShigella))
Flame & Cool
Flame & Cool
Flame & Cool
1 23
45
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 The formation of acid on fermentation of lactose causes the neutral red indicator to make pink coloniesred indicator 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 ferric ammonium citratecitrate allows the differentiation of organisms that produce H2S allows the differentiation of organisms that produce H2S– Lactose fermenters, such as Lactose fermenters, such as E. coliE. coli, have colonies which are , have colonies which are
pinkpink– ShigellaShigella appears transparent or amber appears transparent or amber– SalmonellaSalmonella appears transparent or amber with black centers appears transparent or amber with black centers
due to H2S productiondue to H2S production
Lactose Lactose fermenter Acid Neutral red Pink coloniesFerrous 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
Flame & Cool
Flame & Cool
Flame & Cool
1 2
3
4
5
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 sp. & Proteus Salmonella sp. & 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 (0.1%)Glucose (0.1%)
Lactose (1%)Lactose (1%)
Sucrose (1%)Sucrose (1%)
– 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 color Slant color H2S
Yellow RedNegative A/Alk/-
(Glucose fermented)LNF
e.g. Shigella
Yellow RedPositive
black in buttA/Alk/+
(Glucose fermented with H2S)LNF e.g. Salmonella &
Proteus
Yellow Yellow NegativeA/A/-
(three sugars are fermented)LF e.g. E. coli,
Klebsiella,Enterobacter
Red Red NegativeAlk/Alk/-
(No action on sugars)Non fermenter e.g.
Pseudomonas
Results
Practicle WorkPracticle Work
Gram stainGram stain
Oxidase testOxidase test
Indole testIndole test
O/F test *O/F test *
Nitrate reductase test*Nitrate reductase test*
Growth on MacConkey’s agarGrowth on MacConkey’s agar
Growth on EMB agar*Growth on EMB agar*
Growth on SS agarGrowth on SS agar
Reaction on TSIReaction on TSI