PHT 416 Lab 8
Steps
• Microscopic
Morphology
Nutrient agar
• Growth
Blood agar
Mannitol Salt Agar
MacConkey’s agar
• Biochemical Tests
Bacteria Gram’s Stain
Gram’s +ve
Cocci
Staphylococci
Streptococci
Bacilli
Corynbacterium
Clostridum
Bacillus
Gram’s -ve
Cocci
Neisseria
Bacilli
Enterobacteriaceae
Pseudomonas
Gram –ve bacilli
Oxidase test
Principle:
Tetramethyl p-phenylene diamine
(oxidase reagent colourless)
Cytochrome oxidase enzyme
Indophenol
(Purple colour)
+ve test
Purple colour
-ve test
Colourless
Pseudomonas
Enterobacteriaceae
Enterobacteriaceae
Lactose
fermenters
Lactose nonfermenters
Salmonella, Shigella, Proteus
Escherichia, Klebsiella
1- Growth on MacConkey’s agar
Lactose
Lactose
fermenter
Pink colonies
acid
Neutral red
Pink colonies
Pale colonies
Enterobacteriaceae
Lactose fermenters
Lactose non-fermenters
Escherichia, Klebsiella
Salmonella, Shigella, Proteus
2- Triple sugar iron (TSI) agar
Principle:
 it contains three types of sugars (lactose, sucrose and
dextrose)
(The conc. of lactose and sucrose is 10 times that of
dextrose.)
 It contains phenol red indicator.
 It contains soft agar to enhance gas appearance.
 It contains also ferrous sulfate as an indicator for H2S
production. H S + FeSo
FeS
2
4
Black ppt. of ferrous sulfid
Enterobacteriaceae
Lactose fermenters
Lactose non-fermenters
Escherichia, Klebsiella
Salmonella, Shigella, Proteus
2- Triple sugar iron (TSI) agar
Lactose Fermentation:
Lactose
Large amount of acid
acidic (yellow)
acidic (yellow)
-ve
E.Coli & Klebsiella
Butt: acidic (yellow)
Slant: acidic (yellow)
Enterobacteriaceae
Lactose fermenters
Lactose non-fermenters
Escherichia, Klebsiella
Salmonella, Shigella, Proteus
2- Triple sugar iron (TSI) agar
acidic (yellow)
acidic (yellow)
acidic (yellow)
acidic (yellow)
alkaline (red)
alkaline (red)
-ve
-ve
+ve
E.Coli & Klebsiella
Shigella
Proteus & Salmonells
Proteus, Salmonells & Shigella
1. Dextrose Fermentation:
a) Initial reaction:
(after 10 – 12 hrs)
Dextrose
acid
Butt: acidic (yellow)
Slant: acidic (yellow)
Proteus, Salmonells & Shigella
Dextrose Fermentation:
b) Delayed reaction:
(after 24 hrs)
Small amount of acid
Butt: acidic (yellow)
Slant: alkaline (red)
Enterobacteriaceae
Lactose fermenters
Lactose non-fermenters
Escherichia, Klebsiella
Salmonella, Shigella, Proteus
3- Nitrate Test
All enterobacteriaceae reduce nitrates to
nitrites
Procedure:
Test m.o
Incubate at 35oC
for 24 hrs
Nitrate broth
Nit. A
Nit. B
Red colour
Enterobacteriaceae
Lactose fermenters
Lactose non-fermenters
Escherichia, Klebsiella
Salmonella, Shigella, Proteus
4- Oxidation Fermentation (O/F) Test
Principle:
 Using sensitive O/F medium (Hugh and Leifson Medium)
 All enterobacteriaceae are O+/F+ (Fermentative)
Sensitive O/F Test (Hugh and Leifson Medium)
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.
Enterobacteriaceae
Lactose fermenters
Lactose non-fermenters
Escherichia, Klebsiella
Salmonella, Shigella, Proteus
Growth on MacConkey’s agar
Triple sugar iron (TSI) agar
Pink colonies
acidic (yellow)
acidic (yellow)
-ve
E. coli
Enterobacteriaceae
Lactose Fermenters
Klebsiella
1- Growth on MacConkey’s agar
Pink Colonies
Colonies are mucoid
2- Growth on Eosin Methylene Blue Agar
black colonies with metallic sheen
Purple colonies
3- IMVC Test
They are group of four tests:
Indole production test.
Methyl red (MR) test.
Voges-Proskauer (V-P) test.
Citrate Utilization test.
Indole Production Test
Tryptophane
Purple red
color in
the upper
layer
Kovac’s reagent
Tryptophanase
enzyme
Indole
MR-VP Test
Glucose
Acidic pathway
Acidic pathway
OR
100% Glucose fermentation
MR +ve/Red colour
partial glucose fermentation
VP +ve/Eosin-pink colour
Citrate Utilization Test
Citrate
As a source of carbon
- ve
Na2CO3 Bromothymol blue
indicator
alkaline
Blue colour
+ve
IMVC
+ve
+ve
-ve
-ve
-ve
-ve
+ve
I M VC
++ - -
I M V C
- - + +
E.Coli
Klebsiella
+ve
Enterobacteriaceae
Lactose fermenters
Lactose non-fermenters
Escherichia, Klebsiella
Salmonella, Shigella, Proteus
Triple sugar iron (TSI) agar
Growth on MacConkey’s agar
Pale colonies
acidic (yellow)
acidic (yellow)
alkaline (red)
alkaline (red)
-ve
Shigella
+ve
Proteus & Salmonells
Enterobacteriaceae
Lactose non-fermenters
Shigella
?
Salmonella, Proteus
1- Growth on Salmonella-Shigella (S-S) agar.
Pale
colonies
Pale colonies
black centers
 It
is a
selective and differential medium
for with
enteric
pathogens especially Salmonella.
 It contains lactose and neutral red indicator
 It also contains sodium thiosulfate and ferric citrate as
an indicator for H2S→ colonies with black centers.
2- Citrate Utilization Test
Bromothymol blue
indicator
Citrate
As a source of
carbon
-ve
Na2CO3
+ve
Culture Characteristics
•Facultative
anaerobes,
grow on simple
media.
•Proteus shows
a swarming
growth
Definitive Test to Differintate between Proteus & Salmonella
Proteus
Salmonella
1- Urease Production Test
Urea
Urease
enzyme
NH3
( alkaline)
Phenol
red
Pink colour
+ve
-ve
2- Gelatin Liquefaction Test
Gelatin
gel
Gelatinase
enzyme
Peptides
liquid
Positive test: Gelatin medium remain liquid even after refrigeration.
Gelatinase +ve
Gelatinase -ve
Oxidase test
Principle:
Tetramethyl p-phenylene diamine
(oxidase reagent colourless)
Cytochrome oxidase enzyme
Indophenol
(Purple colour)
+ve test
Purple colour
-ve test
Colourless
Pseudomonas
Enterobacteriaceae
Pseudomonas
Growth
on Cetrimide
• Nutrient
CetrimideAgar
agar is a highly
selective
medium for
Agar:
Pseudomonas
to presences of
Its
growth on species due
cetrimide
which inhibitsOnly
the growth
of otherspecies
showing
greenish
Pseudomonas
bacteria. due to
coloration
can grow on cetrimide agar
of pale
• exopigment
It contains also MgCl2 &showing
K2So4 togrowth
facilitate
production.
coloniesgreen
with diffusion
of
production of the charactaristic
pigmentation
green pigmentation.
of Pseudomonas.




Pseudomonas
1- Oxidation Fermentation (O/F) Test
Results:
Positive
PositiveTest:
Test:
O-/FNon Saccharolytic
O+/F+
O+/F-
Fermentative
Oxidative
Enterobacteriaceae
Pseudomonas
Pseudomonas
2- Nitrate Test
Principle:
Nitrate
Nitrate
reductase
Further reduction
nitrite
(nit. A)
(nit. B)
Red diazonium salt
Enterobacteriaceae
If no red color
Add zinc dust (reducing agent)
Nitrogen (N2)
2- Nitrate Test
Procedure:
Red color
test m.o
Nitrate
broth
Incubate
at 35oC
for 24 hrs
Nit. A
Nit. B
No red colour
Add zinc dust
Results:
Red colour after
addition of nit.A & nit.B
Reduction of Nitrate to
nitrite
Enterobacteriaceae
Red colour after
addition of zinc dust
No red colour after
addition of zinc dust
-ve reduction
Further reduction to
Nitrogen
pseudomonas