Gram Negative Bacilli...Classification of Gram Negative Bacilli Enterobacteriace is a family of gram...
Transcript of Gram Negative Bacilli...Classification of Gram Negative Bacilli Enterobacteriace is a family of gram...
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
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.
Prof. Dr. AbD El-GAwAD M. HAsHEM
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.
Prof. Dr. AbD El-GAwAD M. HAsHEM
Salmonella Gram’s Stain
Salmonella: Gram - negative rods of medium length and diameter; single cells are evenly dispersed throughout the field .
Prof. Dr. AbD El-GAwAD M. HAsHEM
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
Prof. Dr. AbD El-GAwAD M. HAsHEM
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
Prof. Dr. AbD El-GAwAD M. HAsHEM
Salmonella on TSI
Salmonella ferment glucose only Alk slant / acid butt.
+Ve H2S production
Prof. Dr. AbD El-GAwAD M. HAsHEM
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
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)
Prof. Dr. AbD El-GAwAD M. HAsHEM
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.
Prof. Dr. AbD El-GAwAD M. HAsHEM
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
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).
Prof. Dr. AbD El-GAwAD M. HAsHEM
Shigella on TSI
Shigella Ferment Glucose only. The picture shows the reaction given by Shigella on TSI.
alk slant/acid butt. - Ve H2S
Prof. Dr. AbD El-GAwAD M. HAsHEM
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
Prof. Dr. AbD El-GAwAD M. HAsHEM
Proteus peritrichous Flagella
Flagella stain showing the peririchous arrangment of flagella in proteus.
Prof. Dr. AbD El-GAwAD M. HAsHEM
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)
Prof. Dr. AbD El-GAwAD M. HAsHEM
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
Prof. Dr. AbD El-GAwAD M. HAsHEM
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.
Prof. Dr. AbD El-GAwAD M. HAsHEM
Pseudomonas aeruginosa on cetrimide agarCetrimide agar
Certimide+ glycerol + MgSo4ConstituentNoIndicator * glycerol + MgSo4 enhance pigment production (pyocyanin) principle
Selective typeisolation of Pseudomonas From clinical specimen Use
Prof. Dr. AbD El-GAwAD M. HAsHEM
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
Prof. Dr. AbD El-GAwAD M. HAsHEM
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
Prof. Dr. AbD El-GAwAD M. HAsHEM
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
Prof. Dr. AbD El-GAwAD M. HAsHEM
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
Prof. Dr. AbD El-GAwAD M. HAsHEM