1. Dr.T.V.Rao MD
Dr.T.V.Rao MD
2/20/2014

2. Dr.T.V.Rao MD
2/20/2014

3. Commonly
present in large intestine
Non sporing , Non Acid fast, Gram –
bacilli.
A complex family of organisms,
Some are non pathogenic
A few are highly Pathogenic,
Some commensals turn out to be
pathogenic. as in UTI after
catheterization.
Dr.T.V.Rao MD
2/20/2014

4. • Are facultative anaerobes
• If motile, motility is by peritrichous flagella
• Many are normal inhabitants of the
intestinal tract of man and other animals
• Some are enteric pathogens and others are
urinary or respiratory tract pathogens
• Differentiation is based on biochemical
reactions and differences in antigenic
structure

5. • Most grow well on a variety of lab media
including a lot of selective and differential
media originally developed for the
selective isolation of enteric pathogens.
 Most of this media is selective by
incorporation of dyes and bile salts that
inhibit G+ organisms and may suppress the
growth of nonpathogenic species of
Enterobacteriaceae.
 Many are differential on the basis of whether
or not the organisms ferment lactose and/or
produce H2S.

6.  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)
Dr.T.V.Rao MD 2/20/2014

7.  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)
Dr.T.V.Rao MD 2/20/2014

8. Gram
negative bacilli or coccobacilli
Non-spore forming
Colony morphology on BAP or CA of
little value, as they look the same,
except for Klebsiella
Selective and differential media are
used for initial colony evaluation
(ex. MacConkey, HE, XLD agars)

9.  Some
•
•
•
•
Enterobacteriaceae are true pathogens
Salmonella spp.
Shigella spp.
Yersinia spp.
Certain strains of E. coli (ETEC, EPEC, EIEC, EHEC)
 Most
members of the Enterobacteriaceae are
opportunistic or cause secondary infections of
wounds, the urinary and respiratory tracts, and
the circulatory system e.g. E. coli.
 Enterobacteriaceae divided into TWO main
groups according to action on LACTOSE
• Lactose Fermenters (LF)
 E. coli, Citrobacter, Klebsiella, Enterobacter
• Lactose Non-Fermenters (LNF)
 Salmonella, Shigella, Proteus, Yersinia
Dr.T.V.Rao MD
2/20/2014

10. Enterobacteriaceae
Lactose fermenters
E. coli, Citrobacter,
Klebsiella, Enterobacter
Non-lactose fermenter
Salmonell, 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
Dr.T.V.Rao MD
In addition to Triple Sugar 2/20/2014
Iron (TSI) agar

11. Oxidase Test
Negative
Positive
Enterobacteriaceae
Pseudomonas
MacConkey’s agar
& TSI
 O/F test: O+/F Nitrate test: +ve further
reduction to N2
Pink colonies on MacConkey colorless colonies on MacConkey
& acidic butt and slant on TSI & acidic butt alkaline slant onTSI
 Growth on cetrimide agar:
Lactose non-fermenter
Pale colonies with green
pigmentation
Lactose fermenter
IMViC test
& EMB
IMViC
++ - & black colonies
with metalic
shines on EMB
E.coli
No H2S production
(no blacking in TSI)
IMViC
- - ++
Shigella
Urease production
+ve
Motility
Not motile
H2S production
(blacking in TSI)
-ve
SS agar
Motile
colorless colonies with black centers
Proteus
Dr.T.V.Rao MD 2/20/2014
Salmonella

12.  MacConkey agar is selective & differential medium for Enterobacteriaceae
MacConkey Agar
Contains
Bile salts
Crystal violet
Inhibit growth of G+ve bacteria
Lactose
Cause of differential
Cause of selectivity
Lactose feremnters
Pink coloniesDr.T.V.Rao MD
Neutral red
pH indicator
Acidic: Pink
Lactose non feremnters
colorless colonies
2/20/2014

13. Enterobacteriaceae
Lactose Non-Fermenters
Lactose Fermenters
No acid
Acid
Neutral red
Colorless colonies
Pink colonies
Escherichia coli
Klebsiella spp
Enterobacter spp
Citrobacter spp
Dr.T.V.Rao MD
Salmonella spp
Schigella spp
Proteus spp
2/20/2014
Yersinina spp

14. 

Method:
• MacConkey agar is inoculated with tested organism
using streak plate technique
• Incubate the plate in incubator at 37 C/24 hrs
Results:
• LF organism appears as pink colonies (e.g. E. coli)
• NLF organism appears as colorless colonies (e.g.
Shigella)
Flame & Cool
2
1
3
4
5
Flame & Cool
Flame & Cool
Dr.T.V.Rao MD
2/20/2014

15. Colorless colonies
Uninoculated plate
Lactose non feremters
Salmonella, Shigella,
Proteus
Dr.T.V.Rao MD
Pink colonies
Lactose feremters
E. coli, Citrobacter
Klebsiella, Enterobacter
2/20/2014

16. Growth of Enterobacteriaceae on SS agar
Both are lactose fermenters
Both Salmonella sp. & Proteus product H2S
Pseudomonas colonies are nearly colorless

17. Coli-type colonies are very dark,
almost black e.g. E. coli

18. Principle
Acidic pathway
Glucose
Or
Acety methyl carbinol
(ACETOIN)
Mixed acids
 pH less than 4.4
Barrit’s A
Barrit;s B
Methyl Red
indicator
Red color
Neutral pathway
VP positive
MR positive
Klebsiella
Dr.T.V.Rao MD 2/20/2014
E. coli
Pink color

19. Indole,
Methyl Red, VogesProsakaur, 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.
Dr.T.V.Rao MD
2/20/2014

20. 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 Dr.T.V.Rao MD organic layer`
in upper 2/20/2014

21.  Method:
 Inoculate tryptone water with the
tested microorganism
 Incubate at 37 C for 24 hours
 After incubation interval, add 1 ml
Kovacs reagent, shake the tube
gently and read immediately
Dr.T.V.Rao MD
2/20/2014

22. Negative test
e.g. Klebsiella

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

Special Features:

Used in the differentiation of
genera and species. e.g. E. coli
(+) from Klebsiella (-).
Dr.T.V.Rao MD
2/20/2014
Positive test
e.g. E. coli

23. 
Inoculate the tested organism into One tube of MRVP broth

Incubate the tubes at 37 C for 24 hours

AFTER INCUBATION: Pour 1/3 of the suspension into a clean
nonsterile tube:

Run the MR test in the tube with 2/3, and the VP test in the open tube with
1/3.

For methyl red: Add 6-8 drops of methyl red reagent.

For Voges-Proskauer: Add 12 drops of Barritt's A (-naphthol),
mix, 4 drops of Barritt's B (40% KOH), mix

Let sit, undisturbed, for at least 1hour
Dr.T.V.Rao MD
2/20/2014

24. Results
Voges-Proskauer test
Methyl Red test
Pink: Positive VP (Klebsiella)
Red: Positive MR (E. coli)
Yellow or orange: Negative MR (Klebsiella)
No pink: Negative VP (E. coli)
Dr.T.V.Rao MD
2/20/2014

25. Principle:
Citrate
Citrate Utilization Test
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
Dr.T.V.Rao MD
2/20/2014

26. Method
 Streak a Simmon's Citrate agar slant with
the organism

Incubate at 37 C for 24 hours.
Dr.T.V.Rao MD
2/20/2014

27. 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 2/20/2014
Dr.T.V.Rao MD
Negative
E. coli

28. Principle




Urea
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
Urease
H2O
CO2 + NH3
NH4 OH
↑ in pH
Phenol Red
Method
 Streak a urea agar tube with the organism
 incubate at 37 C for 24 h
Dr.T.V.Rao MD
2/20/2014
Pink
Positive test

29. 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
Dr.T.V.Rao MD
2/20/2014
Negative test

30. TSI contains

•
Three different types of sugars


Lactose (10 part)

•
Glucose (1 part)
Sucrose (10 part)
Phenol red (acidic: Yellow)
TSI dispensed in tubes with equal butt & slant
Principle


•
To determine the ability of an organism to attack a specific
carbohydrate incorporated into a basal growth medium, with or
without the production of gas, along with the determination of
possible hydrogen sulphide production.
Dr.T.V.Rao MD
2/20/2014

31. Method:

•
Inoculate TSI medium with an
organism by inoculating needle by
stabbing the butt and streaking the
slant
• Incubate at 37 C for 24 hours
Dr.T.V.Rao MD
2/20/2014

32. Reaction on TSI
Butt color
Slant color
Yellow
Red
Yellow
Red
Yellow
Yellow
Red
Red
H2 S
Negative
Positive
black in butt
Negative
Negative
Result
s Result
Example
A/Alk/(Glucose fermented)
LNF
e.g. Shigella
A/Alk/+
(Glucose fermented with H2S)
LNF
e.g. Salmonella &
Proteus
A/A/(three sugars are fermented)
LF
e.g. E. coli, Klebsiella,
Enterobacter
Alk/Alk/(No action on sugars)
Non fermenter e.g.
Pseudomonas

33. Results
Example
Reaction on TSI
Butt
color
Red
Slant
color
Red
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)
Non fermenter
e.g.
Pseudomonas
LNF
e.g. Shigella
LNF
e.g. Salmonella &
Proteus
LF
e.g. E. coli,
Klebsiella,
Dr.T.V.Rao MDEnterobacter
2/20/2014
A/A/(three sugars are
fermented)

34. Summary of morphology, cultural characteristics,
and biochemical reactions of Enterobacteriaceae
Gram
stain
Oxidase
Nitrate
reductase
O/F
MacCon
key
SS
EMB
E. coli
-ve rod
-ve
+ve
O+/F+
LF
LF
Metallic
sheen
Citrobacter
-ve rods
-ve
+ve
O+/F+
LF
LF
Dark
Klebsiella
-ve rods
-ve
+ve
O+/F+
LF
LF
Dark
Enterobacter
-ve rods
-ve
+ve
O+/F+
LF
LF
Dark
Salmonella
-ve rods
-ve
+ve
O+/F+
NLF
NLF/
H2S
Colorless
Shigella
-ve rods
-ve
+ve
O+/F+
NLF
NLF
Colorless
Proteus
-ve rods
-ve
+ve
O+/F+
NLF
NLF/
H2S
Colorless
Dr.T.V.Rao MD
2/20/2014

35. Summary of morphology, cultural characteristics,
and biochemical reactions of Enterobacteriaceae
TSI
Indole
MR
VP
Citrate
Urease
Motility
E. coli
A/A/-
+ve
+ve
-ve
-ve
-ve
Motile
Citrobacter
freundii
A/A/-
+ve
+ve
-ve
+ve
-ve
Motile
Klebsiella
pneumoniae
A/A/-
-ve
-ve
+ve
+ve
+ve
Non
motile
Enterobacter
cloacae
A/A/-
-ve
-ve
+ve
+ve
+ve
Motile
Salmonella
typhi
A/Alk/+ -ve
+ve
-ve
+ve
-ve
Motile
Shigella
boydii
A/Alk/-
-ve
+ve
-ve
-ve
-ve
Non
motile
Proteus
mirabilis
A/Alk/+ -ve
+ve
-ve
+ve
+ve
Motile
Swarwing
Dr.T.V.Rao MD
2/20/2014

36. Programme
Created from
Web Sources for skills in
Identification of
Enterobacteriaceae
email
 doctortvrao@gmail.com
Dr.T.V.Rao MD
2/20/2014