Download lab 6 PHT313

Bacteria Gram’s Stain
Gram’s +ve
Cocci
Staphylococci
Streptococci
Micrococci
Bacilli
Corynbacterium
Clostridum
Bacillus
Gram’s -ve
Cocci
Neisseria
Bacilli
Enterobacteriaceae
Pseudomonas.



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.
Classification of enterobacteriaceae




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 and shigella.
Natural Habitat
Normal commensals in the colon of both man and
animal e.g. Coliform group.
Some are saprophytes in water, soil and plants e.g.
Proteus species.
Few are primarily intestinal pathogens e.g.
Salmonella, Shigella and Yersinia.
Medical importance of the family



Indicator of fecal pollution of water.
Responsible for 30% of cases of hospital acquired
infections (nosocomial infection).
Difficult to treat with antibiotics (resistant to many
antibiotics).
1- Morphology:
Gram’s –ve bacilli ,
having single arrangement.
2-cultural characteristic:
Facultative anaerobes,
grow on simple media

Proteus showing a swarming growth.
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 non intestinal
bacteria).
Test sugar: lactose.
pH indicator: neutral red ( yellow in alkaline,
pink in acidic pH).
Principle:
Lactose
Lactose fermenter
Neutral red
acid
Pink colonies
Results:
Pink colonies
Lactose
fermenter
Pale colonies
Lactose
Non fermenter
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
pseudomonas
enterobacteriaceae
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
3- Oxidation Fermentation (O/F) Test:
Principle:
 Using sensitive O/F medium ( Hugh and Leifson
Medium) .
 All enterobacteriaceae are O+/F+
(Fermentative)
1- TSI test.
2- Growth on S-S agar.
3-Citrate utilization test.
4- Urease test.
5-Gelatin liquefaction test.
1-Triple Sugar Iron (TSI) agar:
Results:
1. Dextrose Fermentation:
a) Initial reaction:
(after 10 – 12 hrs)
Dextrose
acid
Butt: acidic (yellow)
Slant: acidic (yellow)
Dextrose Fermentation:
b) Delayed reaction:
(after 24 hrs)
Small amount of acid
Butt: acidic (yellow)
Slant: alkaline (red)
Proteus, Salmonella & Shigella
Principle:

This medium contains ferrous sulfate as an
indicator for H2S production.
H2S + FeSo4
FeS
Black ppt. of ferrous sulfid
Proteus & Salmonella
Results:
Butt:
acidic (yellow)
acidic (yellow)
acidic (yellow)
Slant:
acidic (yellow)
alkaline (red)
alkaline (red)
-ve
+ve
H2S Production:
-ve
E.Coli & Klebsiella
Shigella
Proteus & Salmonella
Principle:



It is selective and differential medium for
enteric pathogens especially Salmonella.
It contains the sugar lactose and neutral red as
an indicator
It also contains sodium thiosulfate and ferric
citrate as an indicator for H2S→ colonies with
black centers.
Results:
Salmonella & proteus
Pale colonies with black
centers
Shigella
Pale colonies
3-Citrate Utilization Test
Principle:
Citrate
As a sole source of carbon
Na2CO3
alkaline
Bromothymol blue
Blue colour
indicator
+ve

Results:-
Proteus &
Salmonella
+ve
Shigella
-ve
4- Urease Production Test:
Principle:
Urease enzyme
Urea
NH3 ( alkaline)
Phenol red indicator
Pink colour
Results:
+ve
-ve
Proteus
Salmonella
Definitive tests to differentiate between
Proteus & Salmonella
5- Gelatin Liquefaction Test:
Principle:
Gelatin
gel
Gelatinase
enzyme
Peptides
liquid
Results:
Positive test: Gelatin medium remain liquid even after refrigeration.
Gelatinase +ve
Proteus
Gelatinase -ve
Salmonella
Disease caused by shigella:
Bacillary Dysenteriae
Disease caused by salmonella:
Typhoid Fever
Diseases caused by proteus:
urinary tract infections ,bacteremia and pneumonia,
Proteus species produce infections in humans only
when the bacteria leave the intestinal tract.
Oxidase Test
-ve
+ve
Enterobacteriaceae
Pseudomonas
MacConkey’s agar
& TSI
 O/F test: O+/F-
Pink colonies on MacConkey Pale colonies on MacConkey
& acidic butt and slant onTSI & 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
 Nitrate test: +ve further
reduction to N2
No H2S production
(no blacking in TSI)
H2S production
(blacking in TSI)
Shigella
Urease production
& gelatin liquefaction
IMViC
- - ++
Klebsiella
+ve
Proteus
-ve
Salmonella
Results
Pseudomonas E.coli
Gram’s
Stain
Oxidase
test
O/F test
Nitrate
test
Klebsiella shigella Proteus
Salmonella
Gram’s –ve rods, single arrangement
+ve
O+/FNo red colour
after addition
of zinc dust
+ve further
reduction to N2
Cetrimid Pale colonies
with green
e agar
pigmentation
-ve
-ve
-ve
-ve
-ve
O+/F+ O+/F+ O+/F+ O+/F+ O+/F+
+ve reduction from nitrate into nitrite
-
-
-
-
-
Results
E.coli
Klebsiella
MacConkey’ Pink colonies Pink mucoid
s agar
colonies
TSI agar
Butt:
Slant:
H2S:
IMViC test
EMB agar
shigella
Pale colonies
Proteus
Salmonella
Pale colonies Pale colonies
Acidic (yellow)Acidic (yellow)Acidic (yellow) Acidic (yellow)Acidic (yellow)
Acidic (yellow)Acidic (yellow)Alkaline (red) Alkaline (red) Alkaline (red)
-ve
-ve
+ve
+ve
-ve
IMViC
++ - -
IMViC
- - ++
Black colonies Pink colonies
with metalic
sheen
IMViC
-+--
IMViC
---+
Pale colonies Pale colonies
IMViC
-+-+
Pale colonies
Results
E.coli
Urease
production
test
Gelatin
liquefaction
test
S – S agar
-ve
-ve
Klebsiella
+ve
-ve
shigella
Proteus
Salmonella
-ve
+ve
(pink)
-ve
-ve
+ve
(liquid)
-ve
Pink colonies Pink colonies Pale colonies
Pale
colonies
with black
centers
Pale
colonies
with black
centers