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Experiment five
Isolation and identification of
Enteric Bacteria
The family Enterobacteriaceae includes a group
of bacteria that inhabit the intestinal tracts of
humans and other animals. Some species are
normal flora and others are pathogens of certain
diseases. Commonly used techniques for their
isolation and identification include biochemical test,
antigens present on their surfaces and motility.
Among the important genera included as enterics
are Escherichia, Salmonella, Shigella, Klebsiella,
Proteus and Yersinia.
Dimidiation of the enterobacteria
according to the fermentation of lactose
• Lactase
fermenters:
commensal
Escherichia
Enterobacter
saprophytic
Klebsiella
• Non lactase fermenters:
Salmonella
Proteus
Shigella
Serratia
Citrobater
pathogens
some Citrobacter
and
Isolation culture and
identification procedure
Colonial characteristic observation
Specimens
isolation
Gram Staining
(SS/EMB plate)
Serological identification
TSI
Biochemical reaction
Specimens
• Different specimens should be taken
depending on the kind and the process
of the disease.
blood
bone marrow
Urine
stool
Isolation
• Culture medium: S.S agar
• Method: streak plate
• Result:
Non-pathogenetic colonies: middle size,
red
Suspect colonies: colorless, small,
opaque
Biochemical reactions of Salmonella,
etc
Species
motility
E.coli
Salmonella
Shigella
bottom
AG
A
A
bottom: ferment dextrose
A:acid
AG: acid and gas
AG
-
slope
+/+/-
H2S
+
slope: lactose
+
-
Items of the experiment
(1)
Eosin-Methylene Blue Agar (EMB)
Reagents: Eosin and Methylene-blue
Carbohydrate Source: Lactose
Principle: a precipitate can be formed at acidic pH
Purpose: differentiate lactose fermenters from
non-lactose fermenters.
Interpretation:
Lactose fermenter: purple
Lactose non-fermenter: colorless
EMB agar
E.coli: black and typically have a
metallic green sheen
negative
EMB agar
E.coli
S.typhi
S.dysenteriae
( ++ )
(-)
(-)
Items of the experiment
(2) Salmonella-Shigella Agar(S.S agar )
Reagents:
– Lactose (Carbohydrate Source)
– Neutral red( as an indicator),
– Bile salt, Ferric ammonium citrate (Provides for
inhibition of normal flora coliforms and
differentiationof stool pathogens (e.g. Salmonella
anal Shigella)
Interpretation:
Lactose non-fermenter: colorless
Lactose fermenter: pink to red colonies
Principle:
S.S agar
S.S agar
E.coli
S.typhi
S.dysenteriae
pink
colorless
colorless
Items of the experiment
(3) Indole Broth
purpose: Distinguish Enterobacteriaseae based on the
ability to produce indole from tryptophan.
principle:
tryptophan
tryptophanase
indole
Kovac’s reagent
rosindole
(red)
Interpretation
Positive Test - red ring
Negative Test - no color development
Indole Broth
+ -
Indole Broth
E.coli
S.typhi
S.dysenteriae
(+)
(-)
(-)
Items of the experiment
(4) Double sugar iron slant
main component:
Glucose: 0.1%
Lactose: 1%
Phenol red: as an indicator
Ferrous sulfate(FeSO4) :FeSO4+H2S →FeS(black)+H2SO4
purpose: It provides information about carbohydrate
fermentation.
Principle:
Only glucose fermenting
Slant: oxidized →neutral →red
Butt: not oxidized →yellow
Lactose fermenting
yellow both on the slant
and in the butt
H2S production
FeSO4+H2S
FeS(black)+H2SO4
Gas production
Double suger iron slant
Double suger iron slant
E.coli
S.typhi
S.dysenteriae
Slant
yellow
red
red
butt
yellow
yellow
yellow
Gas
(+)
(-)
(-)
H2 S
(-)
(+)
(-)