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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(
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)
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
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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 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