In the name of God
Department
Of
Microbiology
Yasouj University of Medical Science
Enterobacteriacae identification
By: Dr. S. S. Khoramrooz, Ph.D.
Department of Microbiology, Faculty of Medicine,
Yasuj University of Medical Sciences, Yasuj, Iran
1
Characters of Enterobacteriaceae
• All Enterobacteriaceae
• Gram-negative rods
• Ferment glucose with acid production
• Reduce nitrates into nitrites
• Oxidase negative
• Facultative anaerobic
• Motile except Shigella and Klebsiella
• Non-capsulated except Klebsiella
• Non-fastidious
• Grow on bile containing media (MacConkey agar)
2
Classification of Enterobacteriaceae
Enterobacteriaceae
Lactose fermenters
E. coli, Citrobacter,
Klebsiella, Enterobacter
Non-lactose fermenter
Salmonella, Shigella
Proteus, Yersinia
There are several selective and differential media used to
isolate distinguishes between LF & LNF
The most important media are:
MacConkey agar
Eosin Methylene Blue (EMB) agar
Salmonella Shigella (SS) agar
In addition to Kiligler Iron agar (KIA)
3
Tests To Know
• Case Study Tests
• Indole
• Methyl Red/Voges Proskauer
• Citrate
• H2S production in SIM
• Urea hydrolysis
• Motility
• Lactose fermentation
• Glucose fermentation & gas production
• Decarboxylation of amino acis
• Fermentation of sugars
• Reaction on selective media
4
Growth of Enterobacteriaceae on
MacConkey agar
Colorless colonies
Pink colonies
Uninoculated plate Lactose non feremters
Lactose feremters
Salmonella, Shigella,
E. coli, Citrobacter
Proteus
Klebsiella, Enterobacter
5
Kligler Iron Agar
Lactose Fermentation
Glucose fermentation
Gas Production (H2 & CO2 )
H2S Production
6
Kligler Iron Agar (KIA)
glucose
lactose
Ferrous sulfate
pH indicator: phenol red
• Proteins
7
8
Red/Red
Alkaline /Alkaline K/K
Lactose -/Glucose –
Yellow/Yellow Acid/Acid
A/A Lactose +/Glucose +
Red/Yellow
K/A Lactose -/Glucose +
Alkaline/Acid
Gas Red/Yellow
Alkaline/Acid
K/A Lactose -/Glucose +
Gas +
Red/Yellow
Alkaline/Acid
Alkaline/Acid
H2S -
K/A Lactose -/Glucose +
Gas -
Red/Black
H2S -
H2S +
K/A Lactose -/Glucose +
Gas +
H2S +
9
Result
Reaction on KIA
Butt
color
Red
Slant
color
Red
Example
H2S
Negative
Negative
Yellow
Yellow
Yellow
Red
Red
Yellow
Positive
black in
butt
Negative
Result
Alk/Alk/(No action on sugars)
A/Alk/(Glucose fermented
without H2S)
A/Alk/+
(Glucose fermented
with H2S)
A/A/(three sugars are
fermented)
Non fermenter
e.g.
Pseudomonas
LNF
e.g. Shigella
LNF
e.g. Salmonella &
Proteus
LF
e.g. E. coli,
Klebsiella,
Enterobacter10
11
IMViC Test
• Indole, Methyl Red, Voges-Prosakaur, Citrate
(IMViC) Tests:
• The following four tests comprise a series of
important determinations that are collectively
called the IMViC series of reactions
• The IMViC series of reactions allows for the
differentiation of the various members of
Enterobacteriaceae.
12
IMViC: Indole test

Principle

Certain microorganisms can metabolize tryptophan
by tryptophanase

The enzymatic degradation leads to the formation of
pyruvic acid, indole and ammonia

The presence of indole is detected by addition of
Kovac's reagent.
Tryptophane
amino acids
Tryptophanase
Indole + Pyurvic acid + NH3
Kovac’s Reagent
Red color in upper organic layer`
13
IMViC: Indole test
 Result:

A bright pink color in the top
layer indicates the presence of
indole

The absence of color means that
indole was not produced i.e.
indole is negative
Negative test
e.g. Klebsiella
Positive test
e.g. E. coli
 Special Features:

Used in the differentiation of genera
and species. e.g. E. coli (+) from
Klebsiella (-).
14
IMViC test
Methyl Red-Voges Proskauer (MR-VP) Tests
Principle
Acidic pathway
Mixed acids
 pH less than
4.4
Methyl Red
indicator
Red color
Glucose
Or
Neutral pathway
Acety methyl carbinol
(ACETOIN)
solution A
solution B
MR positive VP positive
E. coli
Klebsiella
15
Pink color
Butylene Glycol Pathway of Glucose
Fermentation
• In the butylene glycol pathway
• pyruvic acid to acetoin and butylene glycol.
• Acetoin and butylene glycol are detected by
oxidation to diacteyl at an alkaline pH.
• and the addition of -naphthol which forms a
red-colored complex with diacetyl.
• Important biochemical property used for the
identification of Klebsiella, Enterobacter, and
Serratia.
16
Voges-Proskauer Reaction
• Acetoin and butylene glycol are detected by oxidation to
diacteyl at an alkaline pH, and the addition of -naphthol
which forms a red-colored complex with diacetyl.
• The production of acetoin and butylene glycol by glucose
fermentation is an important biochemical property used
for the identification of Klebsiella, Enterobacter, and
Serratia.
17
IMViC test: MRVP test
Method
 Inoculate the tested organism into MRVP broth
 Incubate the tubes at 37°C for 24 hours
• For methyl red: Add 6-8 drops of methyl
red reagent.
• For Voges-Proskauer: Add 12 drops of
solution A (-naphthol), mix, 4 drops of
Solution B (40% KOH), mix
18
IMViC test: MR/VP test
Results
Methyl Red test
Voges-Proskauer test
Pink: Positive VP (Klebsiella)
Red: Positive MR (E. coli)
Yellow or orange: Negative MR (Klebsiella)
No pink: Negative VP (E. coli)
19
Citrate Utilization Test
Principle:
Citrate
Pyruvate
Na2CO3
CO2 + Na + H2O
Alkaline,↑pH
Simmone’s Citrate media
Contains Citrate as a sole of C source
Bromothymol blue
Positive test
Blue colour
Positive test: Klebsiella, Enterobacter, Citrobacter
Negative test: E. coli
20
Citrate Utilization Test
Method
 Streak a Simmon's Citrate agar slant with
the organism

Incubate at 37°C for 24 hours.
21
Citrate Utilization Test
Result
 Examine for growth (+)
 Growth on the medium is
accompanied by a rise in pH to
change the medium from its
initial green color to deep blue
Positive
Klebsiella, Enterobacter
Negative
E. coli 22
Principle
Urease Test

Urea agar contains urea and phenol red

Urease is an enzyme that catalyzes the conversion of urea to CO2
and NH3

Ammonia combines with water to produce ammonium hydroxide,
a strong base which ↑ pH of the medium.

↑ in the pH causes phenol red r to turn a deep pink. This is
indicative of a positive reaction for urease
Urea
Urease
CO2 + NH3
H2O
NH4 OH
↑ in pH
Phenol Red
Method
 Streak a urea agar tube with the organism
 incubate at 37°C for 24 h
Pink
Positive test
23
Urease Test
Result
•
If color of medium turns from
yellow to pink indicates positive
test.
•
Proteus give positive reaction after
4 h while Kelebsiella and
Enterobacter gave positive results
after 24 h
Positive test
Negative test
24
From
Motility
+
left to right:
–
+
25
SIM
Sulfide, Indole, Motility
3/15/2016
S. S. Khoramrooz
26
Phenylalanine Deaminase Reaction
• Enterobacteriaceae utilize amino acids in a variety of ways
including deamination.
• Phenylalanine is an amino acid that forms the keto acid
phenylpyruvic acid when deaminated.
• Phenylpyruvic acid is detected by addition of ferric
chloride that forms an intensely dark olive-green colored
complex when binding to phenylpyruvic acid.
• The deamination of phenylalanine is an important
biochemical property of Proteus, Morganella, and
Providencia.
28
Amino Acid Decarboxylation
• Enterobacteriaceae contain decarboxylases with substrate
specificity for amino acids, and are detected using Moeller
decarboxylase broth overlayed with mineral oil for
anaerobiosis.
• Moeller broth contains glucose for fermentation, peptone
and beef extract, an amino acid, pyridoxal, and the pH
indicator bromcresol purple.
29
Amino Acid Decarboxylation
• If an Enterobacteriaceae contains amino acid
decarboxylase, amines produced by decarboxylase action
cause an alkaline pH, and bromcresol purple turns purple.
• Lysine, ornithine, and arginine are utilized.
•
A base broth without amino acid is included in which
glucose fermentation acidifies the broth, turning the
bromcresol purple yellow.
30
Amino Acid Decarboxylation1
Lysine → Cadaverine
Ornithine → Putrescine
Arginine → Citrulline → Ornithine → Putrescine
1Conversion
of arginine to citrulline is a dihydrolase reaction
31
Amino Acid Decarboxylation
• Decarboxylation patterns are essential for the genus
identification of Klebsiella, Enterobacter, Escherichia, and
Salmonella.
• Decarboxylation patterns are also essential for the species
identification of Enterobacter aerogenes, Enterobacter
cloacae, Proteus mirabilis, and Shigella sonnei.
32
Amino Acid Decarboxylation
Lys
Orn
Arg
Klebsiella
+
–
–
Enterobacter
+/–
+
+/–
Escherichia
+
+/–
–/+
Salmonella
+
+
+
33
Amino Acid Decarboxylation
Lys
Orn
Arg
E. aerogenes
+
+
–
E. cloacae
–
+
+
P. mirabilis
–
+
–
P. vulgaris
–
–
–
Shigella D
–
+
–
Shigella A-C
–
–
–
34
IPViC Reactions for Initial Grouping of
the Enterobacteriaceae
• Indole
• Phenylalanine deaminase
• Voges-Proskauer
• Citrate
35
Initial Grouping of the Enterobacteriaceae
(VP=Voges Proskauer, PDA=Phenylalanine
Deaminase)
GENERA
VP
PDA
Klebsiella
POSITIVE
NEGATIVE
Enterobacter POSITIVE
NEGATIVE
Serratia
POSITIVE
NEGATIVE
Hafnia
POSITIVE
NEGATIVE
Pantoea
POSITIVE
NEGATIVE
36
Initial Grouping of the
Enterobacteriaceae
GENERA
VP
PDA
NEGATIVE
POSITIVE
Morganella
NEGATIVE
POSITIVE
Providencia
NEGATIVE
POSITIVE
Proteus
1
1
Proteus mirabilis: 50% of strains VP positive 37
Initial Grouping of the
Enterobacteriaceae
GENERA
Escherichia
Shigella
Edwardsiella
Salmonella
Citrobacter
Yersinia
VP
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
PDA
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
NEGATIVE
38
Initial Grouping of the
Enterobacteriaceae1
GENERA
INDOLE
CITRATE
Escherichia
POSITIVE
NEGATIVE
Shigella
POSITIVE2
Yersinia
POSITIVE3
Edwardsiella POSTIVE
NEGATIVE
NEGATIVE
NEGATIVE
1
VP negative, PDA negative
2
Shigella groups A, B, and C variably positive
for indole production (25-50%), group D
Shigella negative.
3
Yersinia enterocolitica 50% positive
39
Initial Grouping of the
Enterobacteriaceae1
GENERA
Salmonella
Citrobacter
INDOLE
NEGATIVE
NEGATIVE
CITRATE
2
POSITIVE
POSITIVE
1
VP negative, PDA negative
2
Salmonella serotype Paratyphi A and Typhi
negative
40
Key Characteristics of the
Enterobacteriaceae
TSI
E
A/A
coli
Shi
AC
Shi
D
Ed
Sal
Cit
Ak/
A
Ak/
A
Ak/
A
Ak/
A
A/A
Ak/
A
Yer A/A
ON
GAS H2S VP
IND CIT PDA UR
MO LYS OR
AR
/
+ +   +    + +
+
/
    +       
+/






+




+
+
+   
+/

 
+


+

+
+
+

+
+
+
(1) RT=room temperature
    + 
  + + + 
+/
+
+
+
 

+/
+/
/
  +  + 
+/

RT
(1)
 + 41
Key Characteristics of the
Enterobacteriaceae
Kle
pne
Kle
oxy
En
aer
En
cloa
Serr
(1)
Haf
TSI
ON
GAS H2S VP
A/A
+
+
+
+
+
+
+
+
+
+
+
+
+
A/A
A/A
A/A
A/A
Ak/
A
Pan A/A
Alk/
A
/+







+
+
+
+
+
+
IND CIT PDA UR

+




+
+
+
+
+







+/ /+ +/ /+
MO
LYS OR
AR
+  +  
+  +  
 + + + 
+/ +  + +
 + + + 
 + + + 
/+    
(1) Produces DNase, lipase, and gelatinase
42
Key Characteristics of the
Enterobacteriaceae
Prot
mir
a
Prot
vulg
Mor
Pro
v
GAS H2S VP
IND CIT PDA UR
MO LYS OR
AR
TSI
ON
Ak/
A
 + +
A/A
 +/ +  + /+ + + +s   
 +   +  + + +  +
    + + + + +   
Ak/
A
Ak/
A
+/

+/
+ + +s  + 
s = swarming motility
43
Biochemical Characteristics of
Escherichia coli and Shiglla
TSI
Lactose
ONPG
Sorbitol
Indole
Methyl red
VP
Citrate
Lysine
Motility
1Shigella
E. coli
A/Ag
+
+
+
+
+
–
–
+
+
E. coli O157:H7
A/Ag
+
+
–
+
+
–
–
+
+
Shigella
Alk/A
–
–/+1
+/–
+/–
+
–
–
–
–
sonnei (group D) ONPG +
44
Biochemical Characteristics of
Salmonella
TSI
H2S (TSI)
Citrate
Lysine
Ornithine
Dulcitol
Rhamnose
Indole
Methyl red
VP
Most Serotypes
Alk/A
+
+
+
+
+
+
–
+
–
Typhi
Alk/A
+ (weak)
–
+
–
–
–
–
+
–
Paratyphi A
Alk/A
–
–
–
+
+
+
–
+
–
45
Xylose Lysine Deoxycholate (XLD)
Agar: Composition
•
•
•
•
•
•
•
•
•
•
•
•
Xylose
Lysine
Lactose
Sucrose
Sodium chloride
Yeast extract
Sodium deoxycholate
Sodium thiosulfate
Ferric ammonium citrate
Agar
Phenol red
pH = 7.4
0.35%
0.5%
0.75%
0.75%
0.5%
0.3%
0.25%
1.35%
XLD Agar: Growth of Salmonella
• Salmonella selective due to bile salt.
• Xylose fermentation (except Salmonella serotype
Paratyphi A) acidifies agar activating lysine
decarboxylase.
– With xylose depletion fermentation ceases, and colonies of
Salmonella (except S. Paratyphi A) alkalinize the agar due to
amines from lysine decarboxylation.
• Xylose fermentation provides H+ for H2S production
(except S. Paratyphi A).
XLD Agar: Appearance of Salmonella
• Ferric ammonium citrate present in XLD agar reacts
with H2S gas and forms black precipitates within
colonies of Salmonella.
• Agar becomes red-purple due to alkaline pH produced
by amines.
• Back colonies growing on red-purple agar-presumptive
for Salmonella.
XLD Agar: Growth of Escherichia coli
and Klebsiella pneumoniae
Escherichia coli and Klebsiella pneumoniae are
lysine-positive coliforms that are also lactose and
sucrose fermenters.
The high lactose and sucrose concentrations result in
strong acid production, which quenches amines
roduced by lysine decarboxylation.
Colonies and agar appear bright yellow. Neither
Escherichia coli nor Klebsiella pneumoniae produce
H2S.
XLD Agar: Growth of Shigella and
Proteus
Shigella species do not ferment xylose, lactose, and
sucrose, do not decarboxylate lysine, and do not
produce H2S. Colonies appear colorless.
Proteus mirabilis ferments xylose, and thereby provides
H+ for H2S production.
Colonies appear black on an agar unchanged in color
(Proteus deaminates rather than decarboxylates amino
acids).
Proteus vulgaris ferments sucrose, and colonies appear black
on a yellow agar.
Hektoen Enteric (HE) Agar:
Composition
•
•
•
•
•
•
•
•
•
•
•
•
Peptone
Yeast extract
Bile salts
Lactose
Sucrose
Salicin
Sodium chloride
Ferric ammonium citrate
Acid fuchsin
Thymol blue
Agar
pH = 7.6
1.2%
0.3%
0.9%
1.2%
1.2%
0.2%
0.5%
1.4%
HE Agar: Growth of Enteric
Pathogens and Commensals
• High bile salt concentration inhibits growth of grampositive and gram-negative intestinal commensals, and
thereby selects for pathogenic Salmonella (bile-resistant
growth) present in fecal specimens.
• Salmonella species as non-lactose and non-sucrose
fermenters that produce H2S form colorless colonies
with black centers.
• Shigella species (non-lactose and non-sucrose
fermenters, no H2S production) form colorless colonies.
• Lactose and sucrose fermenters (E. coli, K.
pneumoniae) form orange to yellow colonies due to acid
production.
58
Pseudomonas aeruginosa

Some strains appear mucoid

Particularly common in patients with cystic fibrosis

Some strains produce diffusible pigments

Pyocyanin [blue]

Fluorescein [yellow]

Pyorubin [redbrown]
59
Laboratory Diagnosis
Culture
Grow easily on common isolation media such as blood agar and MacConkey
37-42C
Identification
The colonial morphology (e.g., colony size, hemolytic activity, pigmentation,
odor)
+
Biochemical tests (e.g., positive oxidase reaction)
60
61
62
The End
63