Enterobacteriaceae

Expriment 8
Isolation and Identification of

Enterobacteriaceae
Question
 How to identify the following common species of
the family Enterobacteriaceae?
 Escherichia coli
 Salmonella typhi
 Shigella flexneri
 Proteus vulgaris
 Enterobacter aerogenes
Identification of enteric bacilli
Specimen
Specimen Cultivation
Stool; with selective and differential medium
anal swab
Suspected colonies
Biochemical tests To confirm genus
Serological tests To confirm species

Note: 5 students/group
Oganism Student in a group

E coli Student A
Proteus vulgaris Student B
S typhi Student C
S dysenteriae Student D
Enterobacter aerogen Student E
 Culture media and inoculation methods
Culture Medium Prior Color Inoculation Method

1.EMB agar Dark red Four-area streak inoculation
2.Indole broth Pale yellow Liquid inoculation
3.Citrate slant Green Streak slant surface
4.Urea slant Pale yellow Streak slant surface
5.Double-sugar Stab into it and streak slant

Red
iron slant surface
Notes:
1. Every student inoculate your bacterium on 5 different culture
media.
2. After each inoculation, label each media and put them in the
incubator.
Culture Medium used in this experiment
1 、 EMB agar (Eosin-Methylene Blue Agar)
 Combination selective-differential medium
 dark red at first
 Selects Gˉ bacteria and differentiates lactose
fermenters from lactose non-fermenters.
 Reagents: Eosin and Methylene-blue
Lactose: only carbohydrate source
 Principle ：
Lactose Acid
Purple to black
(greenish, metallic sheen)
E.coli P.vulgaris S.typhi S.dysenteriae E.aerogens
(+) (-) (-) (-) (+)

2 、 Idole Broth
•Purpose: Distinguish Enterobacteriaseae based on the
ability to produce indole from trytophan.
•Pale yellow at first.
•Principle:
trytophan tryptophanase Kovac’s reagent
Indole roseindole
•Interpretation:
Positive: a pink to red color ring at the interface of reagent
and broth
Idole Broth

(+) (+) (-) (+) (-)
Positive : Negative:
a pink to red color colorless to
ring at the interface of yellow
reagent and broth

3 、 Citrate Slant
•Purpose: Determine if organism can use citrate
as its sole source of carbon. Green at first.
•Reagents:
bromthymol blue: pH indicator
sodium citrate: the sole source of carbon
•Principle:
sodium citrate sodium carbonate, pH↑
bromthymol blue (indicator) green to blue
Citrate Slant

(-) (-) (-) (-) (+)
E.coli －
E.aerogens ＋
4 、 Urea slant
•Purpose: determine if organism can produce the
urease and break down urea or not .
•Reagents: urea, phenol red: pH indicator.
Pale yellow at first
•Principle:
Urea
Urease
ammonia
Phenol red
Pink
Urea slant

(-) (+) (-) (-) (+)
Positive: red
Negative: no color change
5 、 Double sugar iron agar slant
 Red at first
 Purpose ： carbohydrate fermentation test
 Two sugars
 Glucose (0.1%)
 Lactose (1%)
 Ferrous sulfate (FeSO4) : for the detection of H2S (hydrogen
sulfide) production
 Phenol red: pH indicator
Double sugar iron agar slant
 Principle Glucose (0.1%)
 Carbohydrate fermentation test Lactose (1%)
Sugar Acid Yellow

Phenol red
Gas
 Hydrogen sulfide test

Ferrous sulfate
H2S Ferrous sulfide
Black deposit
Principle:
 Only Glucose fermenting: yellow ONLY in the bottom
because
 glucose: 0.1%
Slant: oxidized →neutral →red
Bottom: not oxidized →yellow
 Lactose fermenting: yellow both in the slant and bottom
because
 glucose: 1%
 Gas production: cracks
 H2S: black
Double-sugar iron slant
Yellow Slant, Yellow Bottom = lactose fermenters = A/A
Red Slant, Yellow Bottom = non-lactose fermenter but ferments
glucose = K/A
Red Slant, Red Bottom = no fermentation
Cracks = Gas production
Black = H2S
interpretation:
Bi-sugar iron slant
Results E.coli P.vulgaris S.typhi S.dysenteriae E.aerogens

Slant yellow red red red yellow
bottom yellow yellow yellow yellow yellow
Gas (+) (-) (-) (-) (+)
H2S (-) (+) (+) (-) (-)
LABORATORY INDICATIONS
lactose
Organisms EMB Urease Citrate Indole
fermentation
E. coli + +/Gas - - +
Enterobacter + +(Gas) -(+) + +
Shigella - - - - +
Salmonella - -(H2S) - - -
Proteus - -/H2S + - +
Serological test
 Slide
 Divide the slide into two equal parts.
(1) One drop of sera for shigella One drop of sera for salmonella
Serological test
 Slide test
(2) Two colonies of Bacteria , mix

Serological test
 Slide test
Positive Negative
(3) Gently shake the slide and observe the result

Experiment report :
Isolation and Identification of Enterobacteriaceae
Class Group name No. Date
1. results
Double sugar iron slant
EMB Urea Citrate Indole Glucose Lactose H2S
E.coli
Proteus
S.typhi
S.dysenteriae
Aerogen
2. discussion
Discussions focus on
1. Describe the actions of these 5 media.

2. How to differentiate among these 5 species
according to their biochemical reactions?
Four-area streak inoculation
1/10 II
I I 1/5
Rotate plate 90
Flame loop
Flame loop
Rotate 90
Rotate 90
III
1/4
IV
Liquid medium inoculation
Slant inoculation
Slant tube
Stabbing inoculation
Stabbing line Bi-sugar iron slant

Enterobacteriaceae

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Expriment 8

Isolation and Identification of

Biochemical tests To confirm genus

Serological tests To confirm species

Oganism Student in a group

Culture Medium Prior Color Inoculation Method

5.Double-sugar Stab into it and streak slant

(+) (-) (-) (-) (+)

E.coli P.vulgaris S.typhi S.dysenteriae E.aerogens

ring at the interface of yellow

reagent and broth

E.coli P.vulgaris S.typhi S.dysenteriae E.aerogens

E.coli P.vulgaris S.typhi S.dysenteriae E.aerogens

Sugar Acid Yellow

 Hydrogen sulfide test

Results E.coli P.vulgaris S.typhi S.dysenteriae E.aerogens

(2) Two colonies of Bacteria , mix

(3) Gently shake the slide and observe the result

1. Describe the actions of these 5 media.

Stabbing line Bi-sugar iron slant

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