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

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Gram Negative Bacilli Enterobacteriace

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

Gram Negative Bacilli

Enterobacteriace

Classification of Gram Negative Bacilli

Enterobacteriace 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

Prof. Dr. AbD El-GAwAD M. HAsHEM

Enterobacteriaceae

II- Non-Lactose FermentersA- Salmonella

I- Basic Characters

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

Biochemical Reactions Lactose & sucrose non-fermenters Ferment Glucose, Maltose, Mannose & mannite

Salmonella typhi production of acid only. Salmonella paratyphi produce acid & gas

Reaction on TSI: Alkaline slant, acid Buttom with H2S production.

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Basic Characters cont. Culture Characteristics Nutrient Media

N. Agar: Circular, convex and small smooth colonies. Selective Diffrential Media

MacConkeys medium: Pale colonies indicating non-lactose fremintation

Eosin Methylene blue (E.M.B): colorless colonies Bismuth sulphite agar (Wilson andBlair medium): Salmonella

gives black colonies with metallic sheen due to H2S Deoxycholate Citrate Agar (Leifson’s): Simillar to Macconkey’s

with black center Taylor’s Xylose Lysine Deoxycholate: Red colonies with black

center. Enrichment Media:

Tetrathionate, Selenite F Broth.

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II- Microscopic examination

Salmonella Gram’s Stain

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

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III- Culture characteristics.

Salmonella typhi colonies on MacConkey’s medium

The Salmonella typhi colonies are Rough and flat, and have an

irregular edge, which in places is effuse and spreading.

Note the pink lactose fermenters (coliforms)

(24 hours at 370C.)

Salmonella

Coliform

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Bismuth sulphite

BiSo3 + Brilliant green Constituents

FeSo4Indicator

Bi, brilliant green inhibit all m.o. xpt Salmonella whichchanges So3 → H2S Fe+2 + H2S → FeS↓ (Black ppt. On colonies)

Pr inciple

Selective , differentialType

- isolation of Salmonelladifferentiation bet. Salmonllatyphi & other sp.)

Use

a- negative b- positive

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IV- Biochemical Reaction

Salmonella on TSI

Salmonella ferment glucose only Alk slant / acid butt.

+Ve H2S production

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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 + gas2* Lactose → acid + gas3* Oxidative decarboxylation of peptone → alkaline roducts4* H2S+Fe →FeS (black ppt.)

principle

Differentiate bet. Enterobacteriaceae type

E. Coli : acid slope , acid butt , + ve gas Salmonella: alk slope , acid butt , +ve H2S , + ve gas Shigella: alk slope, acid butt , - Ve H2S

Use

Prof. Dr. AbD El-GAwAD M. HAsHEM

IV-Serological Identification

Identification of a salmonella by slide agglutination

(Widal) This test can be used in identification and differentiation of different

Salmonella species using Specific H- and O- antisera.

Widal test

Direct Cell agglutinationType of reaction

O somatic Ag of S. typiH Flagellar Ag of S. typiAH Flagellar Ag of paratyphi ABH flagellar Ag of paratyphi B

Reagent

Cellular Ags of SalmonellaFrom attenuated or killed bacteria

Clinical Significance

Detection of serum Ab against S. typhi or paratyphiCause of enteric fever -present from 2nd week

(Use)

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Enterobacteriaceae

II- Non-Lactose FermentersB- Shigella

I- Basic Characters Morphology

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

Biochemical Reactions All strains are Lactose & sucrose non-fermenters Non- Mannitol Fermenters:

Shigella Dysenteriae which ferment glucose only. Mannitol Fermentors:

Sh. Flexneri, Sh. Boydii, Sh. Sonii ferment glucose and mannitol Growth on TSI: Acid buttom /alkaline slant without H2S

production.

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Basic Characters cont.

Culture Characteristics Nutrient Media

N.Agar: smooth greyish or colorless translucent colonies. Enrichment Media:

Selenite F Broth. Selective Diffrential Media

MacConkeys medium: Pale yellowish indicating non-lactose frementing colonies

Bismuth sulphite agar (Wilson and Blair medium): Shigella dosent grow on this media.

Deoxycholate Agar (Leifson’s): Translucent colonies without black center differ from those of salmonella.

Prof. Dr. AbD El-GAwAD M. HAsHEM

Shigella Gram’s Stain

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

Prof. Dr. AbD El-GAwAD M. HAsHEM

II- Culture characteristics.

Shigella sonnei colonies on Macconkesy’s medium

The S. sonnei; colonies are fairly smooth and conical (‘coolie hat’ shaped), and entire edges.

The three pink colonies are lactose fermenters (coliforms).

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III- Biochemical Reactions.

Shigella on TSI

Shigella Ferment Glucose only. The picture shows the reaction given by Shigella on TSI.

alk slant/acid butt. - Ve H2S

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Enterobacteriaceae

II- Non-Lactose FermentersC- Proteus

I- Basic Characters Morphology

Shape: gram Negative Rods Arrangement: Single Non spore forming, non Capsulated Motile with peritrichous flagella.

Biochemical Reactions Lactose & sucrose non-fermenters Pvulgar is, P. mirabilis +ve H2S Urease Positive

Culture Media: Grow on ordinary media producing concentric growth zone ( swarming) On XLD show black colonies due to H2S production

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II- Microscopic examination

Proteus peritrichous Flagella

Flagella stain showing the peririchous arrangment of flagella in proteus.

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III- Culture characteristics.

Swarming of Proteus on N. Agar P. mirabilis and P. vulgar is both

swarm over the surface of the common laboratory medium.

Most characteristically, this produces a series of contour lines around a point of inocula-tion, which results from alternate periods of growth and of swarming, as shown here.

These zones are less obvious on moist media, over which the organisms swarm without stopping.

(Digest agar B, 18 hours at 370C)

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IV- Biochemical Reaction

Urease reactions

a- positive b- negative

Urease testUrea + Peptone+glucose + Agar (pH 6.8)

Constituent

Phenol redIndicator - urea urease NH3 + Co2NH3→Indicator pink

principle

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

Use

a b

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Gram Negative Bacilli

II-Pseudomonas

I- Basic Characters Morphology

Shape: gram Negative Rods Arrangement: Single Non spore forming, non Capsulated & motile Produce different exopigments with different colors

Biochemical Reactions Cant ferment sugar but oxidize glucose to form acids Oxidase + Ve Citrate + Ve

Culture Characteristics Bluish green coloration on nutrient agar as result of pigment

production. Heamolysies on blood agar.

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II- Microscopic examination

Gram stain Pseudomonas

Gram negative rods

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III- Culture characteristics.

Pseudomonas aeruginosa on cetrimide agarCetrimide agar

Certimide+ glycerol + MgSo4ConstituentNoIndicator * glycerol + MgSo4 enhance pigment production (pyocyanin) principle

Selective typeisolation of Pseudomonas From clinical specimen Use

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III- Biochemical Reactions.

Oxidase testOxidaseIf oxidase enz. Is present it changes color of redox. indicator [tetra methyl –p-phenylene diamine di HCl]

principle

Test for cytochrome oxidase enz. eg. PseudomonasPrecaution ! Use glass rod or Platinum loop To avoid false +ve

Use

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Oxidation and fermentation of glucose (O/F)

Oxidation & fermentation of glucose

a) +ve oxid. & -ve ferm. b) +ve ferm. & -ve oxid.

) + id & + f

Oxidation & fermentation of glucoseName1% Glucose + 0.5% agar (semisolid ?) + tryptone(stab inoculation)

Constituents

Bromocresol purpleIndicatorPseudomonas is an obligate Aerobe(aerobic tube turns yellow)

Principle

Test for metabolic activity of m.o.according to O2 requirement

Use

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Gelatinase activity of Pseudomonas

1- positive

2- negative

Gelatinase test

N. Broth + gelatin (12-15%)Constituent

NoIndicator Gelatin is liquified by m.o. producing gelatinaseprinciple

Normaltype

Test for gelatinase production by m.o Examples: Bacillus, Pseudomonas, Staph. aureus

Use

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Gram Negative Bacilli

III- Vibrio cholerae

Gram staining of Vibrio cholerae

Gram negative curved rods, motile with single polar flagella

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Culture of Vibr io cholerae on thiosulphate citrate- bile salt sucrose

TCBS agar

Na Thiosulphate – Na Citrate Bile salt – Sucrose

Constituents

Thymol blue & Bromothymol blue Indicator

* Bile salt inhibit all m.o xpt Vibrio *pH is 8.6 – sucrose fermented by Vibrio cholera → acid cause ind. →yellow around colonies

Principle

Selective , differentialType

isolation of Vibrios & differentiation between Vibr io cholera & other species

Use

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Cholera red testCholera red testName

Alkaline peptone + nitrateConstituents

Vibrio colera produce indole from tryptopophan and reduce nitrate to nitrite, additino of sulphuric acid leeds to formation of red nitrosoindole

Principle

Identification of Vibrio choleraUse

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