Isolation and identification of Enteric Bacteria

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Isolation and identification of Enteric Bacteria
Experiment five
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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.
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Dimidiation of the enterobacteria according to
the fermentation of lactose

• Lactase fermenters saprophytic and commensal
• Escherichia Klebsiella
  Citrobater
• Enterobacter
• Non lactase fermenters pathogens
• Salmonella Shigella some
  Citrobacter
• Proteus Serratia

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Isolation culture and identification procedure

Colonial characteristic observation Specimens
isolation Gram Staining
(SS/EMB plate)
Serological identification
TSI

Biochemical reaction

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Specimens

• Different specimens should be taken depending on
  the kind and the process of the disease.
• blood
• bone marrow
• Urine
• stool

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Isolation

• Culture medium S.S agar
• Method streak plate
• Result
• Non-pathogenetic colonies middle size, red
• Suspect colonies colorless, small,
  opaque

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Biochemical reactions of Salmonella, etc

•  
• Species bottom slope
  H2S motility
• E.coli AG
  AG -
• Salmonella A -
  /-
• Shigella A
  - /- -
• bottom ferment dextrose
  slope lactose
• Aacid AG acid and gas

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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
  

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EMB agar
negative

• E.coli black and typically have a metallic
  green sheen

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EMB agar
E.coli S.typhi S.dysenteriae
( ) ( - ) ( - )
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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

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S.S agar
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S.S agar
E.coli S.typhi S.dysenteriae
pink colorless colorless
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Items of the experiment

• (3) Indole Broth
• purpose Distinguish Enterobacteriaseae based on
  the ability to produce indole from tryptophan.
• principle
• Interpretation
• Positive Test - red ring
• Negative Test - no color development

Kovacs reagent
tryptophanase
indole
rosindole
tryptophan
(red)
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Indole Broth
-

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Indole Broth
E.coli S.typhi S.dysenteriae
( ) ( - ) ( - )
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(4) Double sugar iron slant
Items of the experiment

• main component
• Glucose 0.1
• Lactose 1
• Phenol red as an indicator
• Ferrous sulfate(FeSO4) FeSO4H2S
  ?FeS(black)H2SO4
• purpose It provides information about
  carbohydrate
• fermentation.
• Principle

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Only glucose fermenting
Slant oxidized ?neutral ?red Butt not oxidized
?yellow
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Lactose fermenting
yellow both on the slant and in the butt
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H2S production
FeSO4H2S FeS(black)H2SO4
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Gas production
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Double suger iron slant
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Double suger iron slant
E.coli S.typhi S.dysenteriae
Slant yellow red red
butt yellow yellow yellow
Gas ( ) ( - ) ( - )
H2S ( - ) ( ) ( - )