University of Tabuk

Faculty of Applied Medical Science

Department of Medical Laboratory Technology

Mr.AYMAN.S.YOUSIFM.SC IN Microbiology

&IMMUNOLOGY

Academic Year: (1434-1435-2013-2014)

Bacteria

Gram positive Gram negative

Cocci Bacilli Cocci Rods

Characters of EnterobacteriaceaeAll Enterobacteriaceae.

Gram-negative rods.Ferment glucose with acid production. Reduce nitrates into nitrites.Oxidase negative.

Facultative anaerobic.Motile except shigella and klebsiella . Non-capsulated except Klebsiella.Non-fastidious.Grow on bile containing media

(MacConkey agar).

Enterobacteriaceae

Some Enterobacteriaceae are true pathogensSalmonella spp.Shigella spp.Yersinia spp.Certain strains of E. coli (ETEC, EPEC, EIEC, EHEC)

Most members of the Enterobacteriaceae are opportunistic or cause secondary infections of wounds, the urinary and respiratory tracts, and the circulatory system e.g. E. coli.

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specimens

General Procedure of Bacteriological Diagnosis

Morphologic Identification

Sub culture in the special types of media for confirmation

Biochemical tests ( Identification and Isolation )

Susceptibility Testing ( to select the suitable antibiotics for treatment the pathogenic isolated bacteria from the specimen )

Serological Test

Microscopy & Staining

Cultivation in suitable types of media

Identification of EnterobacteriaceaeGram stain

All Enterobacteriaceae are Gram-negative rodsArranged in single

Classification of Enterobacteriaceae

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 By Growth 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 fermenterscolorless colonies

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Streak-plate techniquefour-area streak plate technique

IV

III 1/5 I1/10

Rotate plate 90

Flame loopRotate 90

Rotate 90III

1/4

Flame loop

Identification of EnterobacteriaceaeDifferentiation between LF & NLF by Growth on MacConkey agar

Method:MacConkey agar is inoculated with tested

organism using streak plate technique.Incubate the plate in incubator at 37 C/24 hrs

Results :LF organism appears as pink colonies (e.g. E.

coli)NLF organism appears as colorless colonies (e.g.

Shigella)

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) agarSS agar is a selective & differential medium used for isolation of Salmonella

and ShigellaThe selective agents are bile salts, and brilliant green dye, which inhibit

gram-positive organismsThe medium contains only lactose as a differential agent and thus

differentiates on the basis of lactose fermentationThe formation of acid on fermentation of lactose causes the neutral red

indicator to make pink colonies.Non lactose fermenting organisms are colorless on the medium.SS agar contains sodium thiosulfate and ferric ammonium citrate allows

the differentiation of organisms that produce H2SLactose fermenters, such as E. coli, have colonies which are pinkShigella appears transparent or amberSalmonella appears transparent or amber with black centers due to H2S

production

LactoseLactose fermenter Acid Neutral red Pink colonies

Ferrous sulfideBlack precipitate

H2S + Ferric ammonium citrate

A. Klebsiella pneumoniaeB. Escherichia coliC: Salmonella sp.D: Proteus mirabilis E: Ps. 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 Enterobacteriaceae on EMB agarConstituents: Nutrients, lactose, neutral red, eosin

and methylene blue.Appearance: A clear red mediumA selective stain for Gram-negative bacteria. It is a

mix of two stains, eosin and methylene blue in the ratio of 6:1.

Acommon application of this stain is in the preparation of EMB agar, a differential microbiological medium, which inhibits the growth of Gram-positive bacteria and provides a colour indicator distinguishing between organisms that ferment lactose (e.g., E. coli) and those that do not (e.g., Salmonella, Shigella).

Eosin Methylene Blue (EMB)Lactose fermentation produces acids,

which lower the pH. This encourages dye absorption by the colonies, which are now coloured purple-black.

Lactose non-fermenters may increase the pH by deamination of proteins. This ensures that the dye is not absorbed.

On EMB if E. coli is grown it will give a distinctive metallic green sheen

Growth of Enterobacteriaceae on EMB agar

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