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