Lab. No. 4 (A)
Bacteria Gram’s Stain
Gram’s +ve
Cocci
Staphylococci
Streptococci
Micrococci
Bacilli
Corynbacterium
Clostridum
Bacillus
Gram’s -ve
Cocci
Neisseria
Bacilli
Enterobacteriaceae
Pseudomonas.
Enterobacteriaceae
enterobacteriaceae
Gram-negative, non-spore-forming bacilli.
Their natural habitat is the intestinal tract of
humans and animals.
Characters of enterobacteriaceae:
1- Facultative anaerobes that grow on
simple laboratory media.
2- Ferment glucose ± other
carbohydrates.
3-Oxidase negative.
4- Reduce nitrates to nitrites.
This family is classified on the basis of
biochemical reactions, especially
fermentation of carbohydrates.
It can be divided according to their effect on
lactose into:
Lactose Fermenters: (coliforms)
e.g., E.coli, klebsiella.
Lactose Nonfermenters:
e.g., protus, salmonella,shigella.
1-morphology:
Gram’s –ve bacilli ,
having single arrangement.
2-cultural characteristic:
- Facultative anaerobes,
- grow on simple media
Klebsiella has a mucoid colonies.
E.Coli has a very bad odor.
Growth on MacConkey’s agar:
Principle:
MacConkey’s agar is a selective and
differential medium
selective medium for gram –ve bacteria (bile
salt inhibit the growth of other bacteria).
Test sugar: lactose.
pH indicator: neutral red ( yellow in alkaline,
pink in acidic pH).
Growth on MacConkey’s agar cont.,
Lactose
Lactose fermenter
acid
Neutral red
Pink colonies
Procedure:
1. Inoculate MacConkey’s agar plate with
the test organism by streaking.
Flame & Cool
Flame & Cool
2. Incubate the plate at
Flame & Cool
35oC for 24
hrs.
Results:
Pink colonies
Lactose
fermenter
Pale colonies
Lactose
Non fermenter
3-biochemical reaction:
1- Oxidase test.
2- Nitrate test.
3- O/F test.
1- Oxidase test:
Principle:
Tetramethyl p-phenylene diamine
(oxidase reagent)
colourless
Cytochrome oxidase enzyme
Indophenol
(Purple colour)
Results:
+ve Test: Appearance of purple colour within 1-2 min.
purple colour
No colour
+ve test
-ve test
Enterobacteriaceae
Pseudomonas
2- Nitrate test:
Principle:
All enterobacteriaceae reduce nitrates to
nitrites

Procedure:
Nit.A
test m.o
Incubate
at 35oC
for 24
hrs
Nitrate broth
Nit. B
Red colour
sensitive O/F Test (Oxidation
Fermentation Test)
Principle:
sensitive O/F medium is a specifically
formulated medium to detect weak acids
produced from saccharolytic Gram’s –ve
bacteria.
To be more sensitive this medium
contains:
 Higher conc. Of sugar to increase amount of acid
produced.
 Lower amount of peptone to reduce formation of
alkaline amines which neutralize weak acids formed.
 Lower conc. Of agar making the medium semisolid
to facilitate diffusion of acid throughout the medium.
3- Oxidation Fermentation (O/F) Test:
Principle:
 Using sensitive O/F medium ( Hugh and Leifson
Medium) .
 All enterobacteriaceae are O+/F+
(Fermentative)
E coli and Klebsiella on
MacConkey’s media
E coli
Klebsiella
Identification of lactose
fermenters:
1- TSI test.
2- IMVC
It is composed of a group of four tests:
Indole production test.
Methyl red (MR) test.
Voges-Proskauer (V-P) test.
Citrate Utilization test.
3- Growth on Eosin- Methylene blue agar.
1-Triple sugar iron (TSI) agar:
Principle:
1-it contains three types of sugars (lactose, sucrose and
dextrose).
(The conc. of lactose and sucrose is 10 times that of dextrose.)
2-it contains phenol red indicator (PH adjusted to the alkaline side).
3-contains soft agar for enhancement gas appearance.
4- contains also ferrous sulfate as an indicator for H2S production.
H2S + FeSo4
FeS
Black ppt. of ferrous sulfid
Procedure:
Principle:
butt
slant
Results:
Lactose Fermentation:
Lactose
Large amount of acid
Butt: acidic (yellow)
Slant: acidic (yellow)
E.Coli & Klebsiella
2-IMVC Test:
They are group of four tests:
Indole production test.
Methyl red (MR) test.
Voges-Proskauer (V-P) test.
Citrate Utilization test.
1- Indole Production Test:
Principle:
inoculate the organism in peptone water for 24h.
Tryptophane
Tryptophanase
enzyme
Indole
Kovac’s reagent
Purple red color
in the upper
organic layer
Results:
+ve
-ve
2- MR-VP Test:
Principle:
inoculate the organism in glucose phosphate peptone for 48h,
one of 2 forms will be produced, either complete acidic or partial acidic
pathway:
glucose
Acidic pathway
OR
Acidic pathway
100% Glucose
fermentation
partial glucose
Fermentation
Mixed acids
 pH less than 4.4
Acetyl methyl
carbinol
α-naphthol
KOH
MR indicator
Red colour
Brick red colour
MR +ve
VP +ve
3- Citrate Utilization Test
Principle:
Citrate
As a sole source of carbon
Na2CO3
alkaline
Bromothymol blue
Blue colour
indicator
+ve
Results:
+ve
+ve
-ve
-ve
-ve
-ve
+ve
I M VC
++ - -
I M V C
- - + +
E.Coli
Klebsiella
+ve
Results:
IMVC
Indole
I
MR
MR
VP
VP
Citrate
C
3- Growth on Eosin Methylene Blue (EMB)
Agar:
Principle:
It is a selective and differential medium used for
isolation of fecal coliforms
eosin and methylene blue are both:
selective substances→ inhibit the growth of most
gram-positive organisms
pH indicators→ combine to form a dark purple
precipitate at acidic pH.
Test sugars: Sucrose& lactose.
Vigorous fermenters of lactose or sucrose →
dark purple dye complex→ dark purple to
black colonies.
Non-lactose/sucrose fermenters →
normally-colored or colorless colonies.
E.coli, often produces a green metallic
sheen due to precipitation of M.B. in the
medium from the very high amounts of
acid produced.
Klebsiella, produces colonies with dark
center surrounded by light colored- mucoid
rim (fish-eye appearance).
Results:
E.Coli
black colonies
with metallic
sheen.
Klebsiella
Purple colonies.
Diseases
E.Coli:
1- urinary tract infections.
2- neonatal meningitis .
3-gastroenteritis(rarely).
Klebsiella:
1-urinary tract infection.
2-lower respiratory tract infection
(Klebsiella pneumonia ).
Oxidase Test
-ve
Enterobacteriaceae
MacConkey’s agar
& TSI
Pink colonies on MacConkey & acidic butt and slant on
TSI
Lactose fermenter
IMVC test
& EMB
IMVC
++ - & black colonies with metalic
shines on EMB
E.coli
IMVC
- - ++
Klebsiella