lab. no. 7. ii. enterobacteriaceae it divided into two main groups: it divided into two main groups:...

Lab. No. 7

Bacteria Gram’s Stain

Gram’s +ve Gram’s -ve

Cocci Bacilli Cocci Rods

II. EnterobacteriaceaeII. Enterobacteriaceae It divided into two main groups:It divided into two main groups:According to their effect on lactoseAccording to their effect on lactose Lactose Fermenters:Lactose Fermenters: (coliforms) (coliforms)Examples: Examples: E.coli, KlebsiellaE.coli, Klebsiella.. Lactose Nonfermenters:Lactose Nonfermenters:Examples: Examples: Protus, SalmonellaProtus, Salmonella. . Late Lactose Fermenters:Late Lactose Fermenters:Example: Example: ShigellaShigella..

II. EnterobacteriaceaeII. Enterobacteriaceae Gram’s Stain:Gram’s Stain:Gram’s –ve rods , having single arrangement.Gram’s –ve rods , having single arrangement.

Macroscopical Examination:Macroscopical Examination: ProteusProteus showing a swarming growth. showing a swarming growth.

KlebsiellaKlebsiella has a mucoid colonies. has a mucoid colonies.

E.ColiE.Coli has a very bad odor. has a very bad odor.

II. EnterobacteriaceaeII. Enterobacteriaceae

II. EnterobacteriaceaeII. Enterobacteriaceae Oxidase test:Oxidase test:

Principle:Tetramethyl p-phenylene diamine

(oxidase reagent)colourless

Cytochrome oxidase enzyme

Indophenol(Purple colour)

II. EnterobacteriaceaeII. Enterobacteriaceae Oxidase test:Oxidase test:

Results:+ve Test: Appearance of purple colour within 1-2 min.

purple colour

+ve test

No colour

-ve test

Pseudomonas

Enterobacteriaceae

II. EnterobacteriaceaeII. Enterobacteriaceae Oxidation Fermentation (O/F) Test:Oxidation Fermentation (O/F) Test:

Principle:

Using sensitive O/F medium ( Hugh and Leifson Medium) .

All enterobacteriaceae are O+/F+ (Fermentative)

II. EnterobacteriaceaeII. Enterobacteriaceae Nitrate Test:Nitrate Test:

Principle:

All enterobacteriaceae reduce nitrate to nitrite

II. EnterobacteriaceaeII. Enterobacteriaceae Nitrate Test:Nitrate Test:

Procedure:

Nitrate broth

test m.o

Nit.A

Nit. B

Red colourIncubate at 35oC for 24

hrs

MacConkey’s agar is a selective medium for Gram’s –ve bacteria because it contains bile salt and crystal violet to inhibit the growth of Gram’s +ve bacteria.

Definitive tests to differentiate betweenDefinitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters

Growth on MacConkey’s agar:Growth on MacConkey’s agar:

Principle:

It also contains lactose and nutral red indicator

Definitive tests to differentiate betweenDefinitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters

Growth on MacConkey’s agar:Growth on MacConkey’s agar:Principle:

LactoseLactose fermenter

acidNutral red

Pink colonies

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

Definitive tests to differentiate betweenDefinitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters

Growth on MacConkey’s agar:Growth on MacConkey’s agar:

Results:

Pink colonies

Definitive tests to differentiate betweenDefinitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters

Growth on MacConkey’s agar:Growth on MacConkey’s agar:

Lactose fermenter

Pale coloniesLactose nonfermenter

Definitive tests to differentiate betweenDefinitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters

Growth on Triple Sugar Iron (TSI) agar:Growth on Triple Sugar Iron (TSI) agar:

Principle:

This medium contains three types of sugars (lactose, sucrose and dextrose).

The conc. of lactose and sucrose is 10 times that of dextrose.

It also contains phenol red indicator.

Definitive tests to differentiate betweenDefinitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters

Growth on Triple Sugar Iron (TSI) agar:Growth on Triple Sugar Iron (TSI) agar:

Principle:

butt slant

Definitive tests to differentiate betweenDefinitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters

Growth on Triple Sugar Iron (TSI) agar:Growth on Triple Sugar Iron (TSI) agar:

Principle:1. No Fermentation:1. No Fermentation:

Butt: alkaline (red) Slant: alkaline (red)

Definitive tests to differentiate betweenDefinitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters

Growth on Triple Sugar Iron (TSI) agar:Growth on Triple Sugar Iron (TSI) agar:

Principle:2. Dextrose Fermentation:2. Dextrose Fermentation:

a) Initial reaction:

Dextrose

acid

(after 10 – 12 hrs)

Butt: acidic (yellow) Slant: acidic (yellow)

Growth on Triple Sugar Iron (TSI) agar:Growth on Triple Sugar Iron (TSI) agar:

Principle:2. Dextrose Fermentation:2. Dextrose Fermentation:

b) Delayed reaction:

Definitive tests to differentiate betweenDefinitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters

(after 24 hrs)

Peptone O

2

Alkaline amines

Butt: acidic (yellow) Slant: alkaline (red)

Small amount of acid

Proteus, Salmonells & ShigellaProteus, Salmonells & Shigella

Definitive tests to differentiate betweenDefinitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters

Growth on Triple Sugar Iron (TSI) agar:Growth on Triple Sugar Iron (TSI) agar:

Principle:3. Lactose Fermentation:3. Lactose Fermentation:

Lactose

Large amount of acid

Peptone O

2

Alkaline amines

Butt: acidic (yellow) Slant: acidic (yellow)

E.Coli & KlebsiellaE.Coli & Klebsiella

Growth on Triple Sugar Iron (TSI) agar:Growth on Triple Sugar Iron (TSI) agar:

Principle:

Definitive tests to differentiate betweenDefinitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters

This medium contains also ferrous sulfate as an indicator for H2S production.

H2S + FeSo4 FeS

Black ppt. of ferrous sulfid

Proteus & SalmonellsProteus & Salmonells

Growth on Triple Sugar Iron (TSI) agar:Growth on Triple Sugar Iron (TSI) agar:

Procedure:

Definitive tests to differentiate betweenDefinitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters

Definitive tests to differentiate betweenDefinitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters

Results:

Butt:

Slant:

H2S Production:

acidic (yellow)

acidic (yellow)

-ve

E.Coli & KlebsiellaE.Coli & Klebsiella

acidic (yellow)

alkaline (red)

-ve

ShigellaShigella

acidic (yellow)

alkaline (red)

+ve

Growth on Triple Sugar Iron (TSI) agar:Growth on Triple Sugar Iron (TSI) agar:

Proteus & SalmonellsProteus & Salmonells

Definitive tests Definitive tests for Lactose fermentersfor Lactose fermentersE.coli & KlebsiellaE.coli & Klebsiella

IMVIMViiC Test:C Test:

It is composed of a group of four tests:It is composed of a group of four tests: IIndole production test.ndole production test. MMethyl red (MR) test.ethyl red (MR) test. VVoges-Proskauer (V-P) test.oges-Proskauer (V-P) test. CCitrate Utilization test.itrate Utilization test.

Indole Production TestIndole Production Test

Definitive tests Definitive tests for Lactose fermentersfor Lactose fermentersE.coli & KlebsiellaE.coli & Klebsiella

IMVIMViiC Test:C Test:

Principle:Tryptophane Tryptophanase

enzymeIndole

Kovac’s reagent

Purple red color in the

upper organic layer

Indole Production TestIndole Production Test

IMVIMViiC Test:C Test:

Results:

Definitive tests Definitive tests for Lactose fermentersfor Lactose fermentersE.coli & KlebsiellaE.coli & Klebsiella

+ve -ve

MR-VP Test:MR-VP Test:

IMVIMViiC Test:C Test:

Principle:

Definitive tests Definitive tests for Lactose fermentersfor Lactose fermentersE.coli & KlebsiellaE.coli & Klebsiella

Dextrose

Acidic pathway OROR Neutral pathway

Mixed acids pH less than

4.4MR indicator

Red colour

Acetyl methyl carbinol

α-naphtholKOH

Brick red colour

MR +ve VP +ve

Citrate Utilization TestCitrate Utilization Test

IMVIMViiC Test:C Test:

Definitive tests Definitive tests for Lactose fermentersfor Lactose fermentersE.coli & KlebsiellaE.coli & Klebsiella

Principle:

CitrateAs a sole source of carbon

Na2CO3

alkaline

Bromothymol blueindicator

Blue colour

+ve

IndoleIndole

IMVIMViiC Test:C Test:

Results:

Definitive tests Definitive tests for Lactose fermentersfor Lactose fermentersE.coli & KlebsiellaE.coli & Klebsiella

MRMR VPVP CitrateCitrate

IMVIMViiCC

I MR VP C

IMVIMViiC Test:C Test:

Results:

Definitive tests Definitive tests for Lactose fermentersfor Lactose fermentersE.coli & KlebsiellaE.coli & Klebsiella

+ve +ve -ve -ve -ve -ve +ve +ve

I M Vi C+ + - -

E.Coli

I M Vi C- - + +

Klebsiella

Growth on Eosin Methylene Blue (EMB)Agar:Growth on Eosin Methylene Blue (EMB)Agar:

Definitive tests Definitive tests for Lactose fermentersfor Lactose fermentersE.coli & KlebsiellaE.coli & Klebsiella

Principle:

This medium contains lactose, eosin and methylene blue.

It is differential medium for E.coli which produce a characteristics black colonies with metalic sheen.

Growth on Eosin Methylene Blue (EMB)Agar:Growth on Eosin Methylene Blue (EMB)Agar:

Results:

Definitive tests Definitive tests for Lactose fermentersfor Lactose fermentersE.coli & KlebsiellaE.coli & Klebsiella

black colonies with metalic sheen.E.Coli

Klebsiella Purple colonies.

Definitive tests Definitive tests for Lactose for Lactose nonfermentersnonfermenters

Proteus & SalmonellaProteus & Salmonella Urease Production Test:Urease Production Test:

Principle:

UreaUrease enzyme

NH3 ( alkaline)

Phenol red indicator

Pink colour

Definitive tests Definitive tests for Lactose for Lactose nonfermentersnonfermenters

Proteus & SalmonellaProteus & Salmonella Urease Production Test:Urease Production Test:

Results:

+ve -ve

Proteus Salmonella

Definitive tests Definitive tests for Lactose for Lactose nonfermentersnonfermenters

Proteus & SalmonellaProteus & Salmonella Gelatin Liquefaction Test:Gelatin Liquefaction Test:

Principle:

Gelatin Gelatinase enzyme

Peptides

gel liquid

Definitive tests Definitive tests for Lactose for Lactose nonfermentersnonfermenters

Proteus & SalmonellaProteus & Salmonella Gelatin Liquefaction Test:Gelatin Liquefaction Test:

Results:Positive test: Gelatin medium remain liquid even after refrigeration.

Gelatinase +ve Proteus

SalmonellaGelatinase -ve

It is selective medium for Salmonella It is selective medium for Salmonella and Shigella.and Shigella.

It contains sodium thiosulfate and It contains sodium thiosulfate and ferric citrate as an indicator for Hferric citrate as an indicator for H22S.S.

Definitive tests for Definitive tests for Salmonella & ShigellaSalmonella & Shigella

Growth on Salmonella-Shigella (S-S) Agar:Growth on Salmonella-Shigella (S-S) Agar:

Principle:

Definitive tests for Definitive tests for Salmonella & ShigellaSalmonella & Shigella

Growth on Salmonella-Shigella (S-S) Agar:Growth on Salmonella-Shigella (S-S) Agar:

Results:

Salmonella Pale colonies with black centers

Shigella Pink colonies

Flame & Cool

Flame & Cool

Flame & Cool

Practicle WorkPracticle Work Oxidase test.Oxidase test. O/F test.O/F test. Nitrate broth.Nitrate broth. Indole broth.Indole broth. MR-VP broth.MR-VP broth. Citrate slant.Citrate slant. Urea Slant.Urea Slant. Gelatin test. Gelatin test. Growth on MacConkey’s agar.Growth on MacConkey’s agar. Growth on EMB agar.Growth on EMB agar. Growth on S-S agar.Growth on S-S agar.

ResultsResults Nitrate Test:Nitrate Test:

Nit.A

Nit. B

Red colour

No red colour

Add zinc dust

ResultsResults Indole Production TestIndole Production Test

Indole

Kovac’s reagent

Purple red color in the

upper organic layer

I

ResultsResults MR-VP Test:MR-VP Test:

MR-VPbroth

MR

VP

MR indicator

Red colour

MR +ve

α-naphtholKOH

Brick red colour

VP +ve

Oxidase TestOxidase Test

+ve-ve

Pseudomonas Enterobacteriaceae

O/F test: O+/F-

Nitrate test: +ve further reduction to N2

Growth on cetrimide agar: Pale colonies with green pigmentation

MacConkey’s agar& TSI

Lactose fermenter

Pink colonies on MacConkey& acidic butt and slant onTSI

Pale colonies on MacConkey& acidic butt alkaline slant onTSI

Lactose non-fermenter

IMViC test& EMB

IMViC++ - -& black colonies with metalic shines on EMB E.coli

IMViC- - ++

Klebsiella

No H2S production(no blacking in TSI)

H2S production(blacking in TSI)

ShigellaUrease production

& gelatin liquefaction

+ve

Proteus

-ve

S-S agar

Pale colonies with black centers

Salmonella

Pseudomonas

E.coli Klebsiella

shigella

Proteus Salmonella

ResultsResults

Gram’s Stain Gram’s –ve rods, single arrangement

O/F testO+/F- O+/F+ O+/F+ O+/F+ O+/F+O+/F+

Nitrate test

No red colour after addition of zinc dust+ve further reduction to N2

+ve reduction from nitrate into nitrite

Oxidase test +ve -ve -ve -ve -ve-ve

Cetrimide agar

Pale colonies with green pigmentation

- - -- -

E.coli Klebsiella shigella Proteus Salmonella

ResultsResults

MacConkey’s agar

Pink colonies Pink mucoid colonies

Pale colonies Pale coloniesPale colonies

TSI agarButt:Slant:H2S:

Acidic (yellow)Acidic (yellow)-ve

Acidic (yellow)Acidic (yellow)-ve

Acidic (yellow)Alkaline (red)-ve

Acidic (yellow)Alkaline (red)+ve

Acidic (yellow)Alkaline (red)+ve

IMViC test IMViC++ - -

IMViC- - ++

IMViC- + - -

IMViC- - - +

IMViC- + - +

EMB agar Black colonies with metalic sheen

Pink colonies Pale coloniesPale colonies Pale colonies

ResultsResultsE.coli Klebsiella shigella Proteus Salmonell

a

Urease production test

Gelatin liquefaction test

+ve (pink)

-ve-ve +ve -ve

+ve (liquid)

-ve-ve -ve -ve

S – S agar Pink coloniesPink coloniesPale coloniesPale colonies Pale colonies with black centers

With my Best Wishes,,,Manal Abu El-Khair