Gram Negative Bacilli...Classification of Gram Enterobacteriace Enterobacteriace is a family of gram...

Gram Negative Bacilli

Enterobacteriace

Prof. Dr. AbD El-GAwAD M. HAsHEM

Classification of Gram EnterobacteriaceEnterobacteriace is a family of gram negative rods inhabiting the intestinal tract of humans and animals.According to their ability to ferment Lactose enterobacteriace are classified into:

Lactose Fermeters: E. coli, Klebsiella, Citrobacter & Enterobacter

Non Lactose: Fermenters: Salmonella, Shigella & ProteusEnterobacteriaceae family members can:

Reduce nitrate to nitrite Ferment glucose Oxidase negative


Biochemical Tests used in Identification of Enterobacteriace

The following test are basic biochemical test used in basic identification of Enterobacteriace.

Sugar Fermentation Test Triple Sugar Iron Test IMViC Test. Urease.


Sugar Fermentation test Enteric bacteria can ferment different types of sugars producing

either acid only or acid and gas. Thus sugar fermentation tests are useful for primary

identification of strains by determining the following. Type of the sugar. End product (acid or acid & gas ) Conditions ( aerobic or anaerobic )

Media used for sugar utilization test usually consist of: Peptone Single fermentable sugar Acid / Base Indicator Durham Tube


Lactose fermentation on MacConkey medium

a) +ve b) -ve

a b

McConkey agarName

Crystal violet + Bile salt + lactoseConstituents

Neutral redIndicator- C.V . + Bile salts inhibit all m.o Except enterobacteriaceae- lactose fermentors (coliform) → produce acid ∴ change color of indicator to pink- non-lactose fermentors (Salmonella, Shigella) → yellow colonies

Principle

Selective, differentialType- isolation of enterobacteriaceae from clinical specimen- differentiate lactose fermentors from non- lactose fermentors

Use


Typical coliform on Eosin Methylene blue showing +ve metallic sheen

Atypical coliform on Eosin Methylene blue showing -ve metallic sheen

Eosin Methylene Blue (EMB)Name

Methylene blue + LactoseConstituentsEosinIndicator- Eosin + M.B. inhibit all m.o Xpt. enterobacteriaceae -typical coliforms produce high conc. of acid enough to

ppt. Eosin ⇒ green metallic sheen- atypical → dark colonies – no sheen

Principle

Selective, differentialType- isolation of enterobacteriaceae- differentiation bet. typical coliforms (as E. coli) & atypical coliforms (as Klebsiella)

Use


Sugar Fermentation test

+ve acid /+ve gas+ve acid : indicated by red color +ve gas: indicated by the gas bubble formed inside durham tube.

+ve acid/-ve gas The organism ferment the sugar and utillized the peptone as carbon source instead.

-ve acid/ -ve gasThe organism couldn't ferment the sugar and utillized the peptone as carbon source instead.Peptone utillization rendered pH alkaline due to its by product

Plain MediaAndrad’s reagentAcid / Base Indic.: ( red in acid, Yellow in Alkaline pHInverted Durham Tube to indicate produced gas

When phenol red is used as indicator


IMViC TestIMViC test is a group of four biochemical test collectively used for primary identification of enteric bacteria.

Indole Methyl Red. Vogeus proskuer. Citrate Utillization.


IMViCCitrateVogus

proskauerMethyl redIndole

Agar+ citrate + NaCl Simmon’s medium

Glucose po4 medium

Glucose po4 medium

Tryptone H2O Constituent

Bromothymol blue

Barrit’s Alc. α-naphthal +KOH

MR Kovac’s+ HCl+amylalcohol

Reagents or indicator

Citrate pyruvate + Nacl + Co2 Na2Co3

Ghucose a acetion change color of indicator pH>4.4

Glucose → acid change color of indicator

pH<4

Tryptophan →indole +reageat red color in organic layer

Principle

Test ability of m.o. to utilize citrate as the sole carbon source

Orfurther utilize acid →acetion

Test ability of m.o. to utilize glucose →acid

Test ability of m.o. to convert trp. → Indole

Use

Prof. Dr. Abd El-Gawad M. Hashem

IMViC: + + - -

a) +ve b) +ve c) - ve d) –ve

Eschrechia coli

IMViC: - - + +

a) -ve b) -ve c) + ve d) +ve

Klebsiella pneumonia


Indole Test Indole Test: This reaction test the abillity of

microorganism to break down Tryptophan to indole.

Media: Tryptophane rich peptone water.

Reagent: Kovac’s reagent which give red color in organic layer indicating indole production.

From left to right: Control medium; Escherichia coli culture,

positive reaction ; Enterobacter cloacae culture,

negative reaction. Positive Negative

Negative Positive


Methyl Red

Methyl Red: This is a qualitative test of the acidity produced by the growth of a bacterium in phosphate-buffered glucose peptone water

Escher ichia coli produces a pH of about 5, and hence has a red colour after addition of methyl red.

K.pneumonia the pH never drops so low; it appears yellow after addition of methyl red Escherichia coli culture

(positive reaction) Klebsiella pneumonia culture

(negative reaction).

This is a qualitative test of the acidity produced by the growth of a bacterium in phosphate-buffered glucose peptone water

Glucose phosphate peptone water, 24 hours at 37ºCPositive Negative

Negative Positive


Voges-Proskauer.

Media: Glucose phosphate peptone water

Reagent: alpha naphthol and KOH added and shaken for one minute, then allowed to stand for five minutes.

Klebsiella culture (positive reaction)

Escherichia coli culture (negative ).

From carbohydrate some bacteria produce acetylmethylcarbinol, which in the presence of KOH and air is oxidised to diacetyl. This reacts with alpha- naphthol and a breakdown product of the arginine in the peptone to produce a red colour.

Positive Negative

Negative Positive


Citrate Utillization.

Growth on the slant of Simmon’s citrate agar and conversion of the bromthy-mol blue indicator to an alkaline blue color indicates that the organism can utilize sodium citrate as the sole source of carbon

The development of turbidity of the medium is sufficient evidence of growth. It is essential to use a small inoculum, which will not itself render the medium turbid Koser’s Medium

A test for the ability of an organism to grow in a medium in which citrate is the sole source of carbon.

Positive Negative

Negative Positive


Urease TestUrease psitive bacteria hydrolyse urea to ammonia ; this turns the Phenol Phethaline indicator a characteristic red-violet colour.

a) +ve b) -ve

a b Urease Test

Urea + Peptone + glucose + Agar (pH 6.8)

Constituent

Phenol redIndicator

urea urease NH3 + CO2NH3 Indicator pink

principle

Test the abihity of m.o to produce urease enzyme- rapid urease results within 4-6 hours(differentiate Proteus spp. from other enterobacteriaceae)

Use


Nitrate reduction: a) +ve b) -ve

Nitrate reduction

a b

Nitrate reduction

N. Broth + KnO3 ConstituentSulphanilic acid +α naphthyl amine

Indicator Or reagent

Nitrate reductase NO3- → NO2-Form azodye with indicator

Principle

Test for the presence of Nitrate reductase enzyme in Enterobacteriace

Use


Reactions in triple sugar iron agar

This medium, and others employing similar principles, are extensively used for the preliminary identification of gram- negative enteric pathogens.

TSI : a) acid slant & acid buttb) alkaline slant & acid buttc) alkaline slant + H2S

a b c

TSI agar(Triple sugar Iron agar)

0.1% glucole-1% sucrose 1% lactose - Fe (NH4) SO4 - Na thiosulphate-peptone (pH 7.4)

Constituent

Phenol red Indicator

1* Glucose fermentation → acid + gas

2* Lactose → acid + gas3* Oxidative decarboxylation of peptone → alkaline roducts4* H2S+Fe →FeS (black ppt

principle

Differentiate bet. Enterobacteriaceae Usea) E. coli: acid slope, acid butt, + ve gas

c) Salmonella: Alkaline slope, acid butt , +ve H2S, + ve gas b) Shigella: alkaline slope, acid butt


API

Several biochemical reactions carried in microtubules (cupules)


Enterobacteriaceae

I- Lactose FermentersA- E. coli


I- Basic Characters

Morphology Shape: gram Negative Rods Arrangement: Single Non spore forming, non Capsulated & motile

Culture Characteristics N.Agar: Circular, convex and small smooth colonies. MacConkeys medium: Rose pink colonies indicating

lactose fremintation Eosin Methylene blue (E.M.B): Metallic Sheen colonies


II- Microscopic examination


E. coli Gram’s Stain

E. coli Gram - negative rods of medium length and diameter; single cells are evenly dispersed throughout the field .

Mixture consists of the gram-positive Staphylococcus albus and the gram negative Escherichia coil


III- Culture characteristics.


Colonies of Escher ichia colion MacConkey medium

MacConkey’s media media is a selective differential media Bile Salt Render media

selective to enteric bacteria On MacConkey’s media

E.coli give rose pink colonies indicating lactose positive Fermintation


Biochemical Reactions


Sugar Fermentation Test E. coli ferment glucose, Lactose

maltose & sucrose to produce acid & gas

Media: Sugar fermentation media Result

Red colour + ve acid production Gas bubble in durham tube indicate

gas production.


IMViC Test For E. Ecoli

Four tubes demonstrating the indole (I), methyl red (MR), Voges-Proskauer (VP), and citrate (C) tests. These four tests when performed together constitute the classic IMViC reactions.

The formation of indole from tryptophan is indicated by a red color upon the addition of Kovac’s reagent (far left tube ).

The development of a red color in the MR tube indicates a drop in pH to 4.4 or below, (second tube from left,).

No red color in the VP tube indicates the absence of acetoin (acetylmethylcarbinol) formed from pyruvate in the butylene glycol metabolic pathway (third tube from left ).

Absence Growth on the slant of Simmon’s citrate agar indicates that the organism can’t utilize sodium citrate as the sole source of carbon (last tube on right,) shows the reactions for E. coli

(+ + - - ).


E. Coli on triple sugar iron Media

E. coli can ferment Glucose, Lactose and sucrose so on growth on TSI it give the shown reaction Acid ( yellow) : slant Acid ( yellow ) : buttom Abundant gas production

indicated by the crack formation.


Gram Negative Bacilli

II- Lactose FermentersB- Klebsiallat


I- Basic Characters Morphology

Shape: gram Negative Rods Arrangement: Single Capsulated, Non spore forming & non-motile

Culture Characteristics N. Agar: Mucoid, convex and smooth colonies. MacConkeys medium: Rose pink colonies indicating

lactose fermentation Biochemical Reaction:

IMViC : --++ TSI: Acid but / Acid Slant with gas production


II- Microscopic examination


Klebsiella gram stain

The slide Cleary show the capsule surrounding the klebsiella.

Capsule appear as clear zone surrounding the cells.

Gram - negative rods of medium length and diameter; single cells are evenly dispersed throughout the field .

Klebsiella Capsule Stain


III- Culture characteristics.


Colonies of Klebsiella aerogenes on MacConkey agar

This species ferments lactose but does not greatly reduce the pH on MacConkey agar.

Moreover, a reversion of pH quickly occurs in the area of confluent growth. Isolated colonies are large, domed, smooth and viscid.

Compare the pale pink of these isolated colonies with the deep red of Escherichia coli colonies on the same medium.

(MacConkey agar B, 18 hours at 370C; reflected light, X 1/2.)


IV-Biochemical Reactions


IMViC Test For Klebsiella

Tubes From Left To right: Indole: -ve formation of indole from

tryptophan is indicated by a yellow color upon the addition of Kovac’s reagent (far left tube ).

Methyl Red: absence of a red color in the MR tube indicates a negative test

VP: Red color in the VP tube indicates the presence of acetoin (acetylmethylcarbinol) formed from pyruvate in the butylene glycol metabolic pathway (third tube from left ).

Citrate : Blue color & Growth on the slant of Simmon’s citrate agar indicates that the organism can utilize sodium citrate as the sole source of carbon (last tube on right,).

Four tubes demonstrating the indole (I), methyl red (MR), Voges-Proskauer (VP), and citrate (C) tests. These four tests when performed together constitute the classic IMViC reactions.

The reactions for Klebsiella (- - + +)


Gram Negative Bacilli...Classification of Gram Enterobacteriace Enterobacteriace is a family of gram...

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