Medical microbiology ii p identification of gram negative bacilli culture characteristics
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University of Tabuk Faculty of Applied Medical Science Department of Medical Laboratory Technology. Medical Microbiology II (p) Identification of Gram Negative Bacilli Culture Characteristics. Mr.AYMAN.S.YOUSIF M.SC IN Microbiology &IMMUNOLOGY. Academic Year: (1434-1435 -2013-2014).

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Medical Microbiology II (p) Identification of Gram Negative Bacilli Culture Characteristics

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Medical microbiology ii p identification of gram negative bacilli culture characteristics

University of Tabuk

Faculty of Applied Medical Science

Department of Medical Laboratory Technology

Medical Microbiology II (p)Identification of Gram Negative Bacilli Culture Characteristics

Mr.AYMAN.S.YOUSIF

M.SC IN Microbiology &IMMUNOLOGY

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


Medical microbiology ii p identification of gram negative bacilli culture characteristics

Classification of Bacteria


Characters of enterobacteriaceae

Characters of Enterobacteriaceae

  • All Enterobacteriaceae.

    • Gram-negative rods.

    • Ferment glucose with acid production.

    • Reduce nitrates into nitrites.

    • Oxidase negative.

  • Facultative anaerobic.

  • Motile exceptshigella and klebsiella .

  • Non-capsulated except Klebsiella.

  • Non-fastidious.

  • Grow on bile containing media (MacConkey agar).


Enterobacteriaceae

Enterobacteriaceae

  • Some Enterobacteriaceae are true pathogens

    • Salmonella 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.


Medical microbiology ii p identification of gram negative bacilli culture characteristics

specimens

Morphologic Identification

Microscopy & Staining

Biochemical tests ( Identification and Isolation )

Sub culture in the special types of media for confirmation

Serological Test

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

General Procedureof Bacteriological Diagnosis

Cultivation in suitable types of media


Identification of enterobacteriaceae

Identification of Enterobacteriaceae

  • Gram stain

    • All Enterobacteriaceae are Gram-negative rods

    • Arranged in single


Classification of enterobacteriaceae

Classification of Enterobacteriaceae

  • There are several selective and differential media used to

    isolate distinguishes between LF & LNF

  • The most important media are:

    • MacConkey agar

    • Eosin Methylene Blue (EMB) agar

    • Salmonella Shigella (SS) agar

    • In addition to Triple Sugar Iron (TSI) agar


D ifferentiation between lf nlf by growth on macconkey agar

Differentiation Between LF & NLF ByGrowth on Macconkey Agar

  • MacConkey agar is selective & differential medium for Enterobacteriaceae

MacConkeyAgar

Contains

Bile salts

Crystal violet

Lactose

Neutral red

pH indicator

Acidic: Pink

Inhibit growth of G+ve bacteria

Cause of differential

Cause of selectivity

Lactose non fermenters

colorless colonies

Lactose feremnters

Pink colonies


Streak plate technique

1/10

II

I

I

1/5

Rotate 90

III

1/4

IV

Streak-plate technique

four-area streak plate technique

Rotate plate 90

Flame loop

Flame loop

Rotate 90


Identification of enterobacteriaceae differentiation between lf nlf by growth on macconkey agar

Identification of EnterobacteriaceaeDifferentiation between LF & NLF byGrowth on MacConkey agar

  • Method:

    • MacConkey agar is inoculated with tested organismusing 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

Growth of Enterobacteriaceae on MacConkey agar

Colorless colonies

Pink colonies

Lactose non feremters

Salmonella, Shigella,

Proteus

Lactose feremters

E. coli, Citrobacter

Klebsiella, Enterobacter

Uninoculated plate


Reaction on salmonella shigella ss agar

Reaction on Salmonella Shigella (SS) agar

  • SS agar is a selective & differential medium used for isolation of Salmonella and Shigella

  • The selective agents are bile salts, and brilliant green dye, which inhibit gram-positive organisms

  • The medium contains only lactose as a differential agent and thus differentiates on the basis of lactose fermentation

  • The 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 H2S

    • Lactose fermenters, such as E. coli, have colonies which are pink

    • Shigella appears transparent or amber

    • Salmonella appears transparent or amber with black centers due to H2S production

Lactose fermenter

Neutral red

Lactose

Acid

Pink colonies

Ferrous sulfide

Black precipitate

H2S + Ferric ammonium citrate


A klebsiella pneumoniae b escherichia coli c salmonella sp d proteus mirabilis e ps aeruginosa

Growth of Enterobacteriaceae on SS agar

A.Klebsiella pneumoniaeB.Escherichia coliC: Salmonella sp.D: Proteus mirabilisE: Ps. aeruginosa.

Bothare lactose fermenters

BothSalmonella sp. & Proteus product H2S

Pseudomonascolonies are nearly colorless


Growth of enterobacteriaceae on emb agar

Growth of Enterobacteriaceae on EMB agar

  • Constituents: Nutrients, lactose, neutral red, eosin and methylene blue.

  • Appearance: A clear red medium

  • A 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

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 agar1

Growth of Enterobacteriaceae on EMB agar

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


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