Lab. No.5 (A)
StaphylococciStreptococciMicrococci
Neisseria
CorynbacteriumClostridumBacillus
Enterobacteriaceae Pseudomonas.
Bacteria Gram’s Stain
Gram’s +ve
Cocci Bacilli
Gram’s -ve
Cocci Bacilli
Enterobacteriaceae
enterobacteriaceaeGram-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(
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.
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:
Principle:
LactoseLactose fermenter
acidNeutral red
Pink colonies
Growth on MacConkey’s agar cont,.
Procedure:
1. Inoculate MacConkey’s agar plate with the test organism by streaking.
2. Incubate the plate at 35oC for 24 hrs.
Flame & Cool
Flame & Cool
Flame & Cool
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.
colour
-ve test
purple colour
+ve test
Pseudomonas Enterobacteriaceae
No
2 -Nitrate test:
Principle:
All enterobacteriaceae reduce nitrates to nitrites
Procedure:
Nitrate broth
test m.o
Nit.A
Nit. B
Red colourIncubate at
35oC for 24 hrs
Principle:
sensitive O/F medium is a specifically formulated medium to detect weak acids produced from saccharolytic Gram’s –ve bacteria.
sensitive O/F Test (Oxidation Fermentation Test)
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.
Black ppt. of ferrous sulfidH2S + FeSo4 FeS
Procedure:
Principle:
butt slant
Results:
Lactose Fermentation:
Lactose
Large amount of acid
Peptone O
2
Alkaline amines
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
Mixed acids pH less than
4.4MR indicator
Red colour
Acetyl methyl carbinolα-naphthol
KOH
Brick red colour
MR +ve VP +ve
partial glucoseFermentation
100% Glucose fermentation
Acidic pathway
3 -Citrate Utilization Test
Principle:
CitrateAs a sole source of carbon
Na2CO3
alkaline
Bromothymol blueindicator
Blue colour
+ve
Results:
+ve +ve -ve -ve -ve -ve +ve +ve
I M V C+ + - -
E.Coli
I M V C- - + +
Klebsiella
Indole
Results:
MR VP Citrate
IMVC
I MR VP 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:
black colonies with metallic sheen.
E.Coli
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
Lactose fermenter
Pink colonies on MacConkey & acidic butt and slant on TSI
IMVC test& EMB
IMVC++ - -
& black colonies with metalic shines on EMB
E.coli
IMVC- - ++
Klebsiell
a
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