Lab. No. 7

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

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

Macroscopical Examination: Macroscopical Examination:  Proteus showing a swarming growth.  Klebsiella has a mucoid colonies.  E.Coli has a very bad odor. II. Enterobacteriaceae

Oxidase test: Oxidase test: Principle: Tetramethyl p-phenylene diamine (oxidase reagent) colourless Cytochrome oxidase enzyme Indophenol (Purple colour)

II. 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. 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. Enterobacteriaceae Nitrate Test: Nitrate Test: Principle:  All enterobacteriaceae reduce nitrate to nitrite

II. Enterobacteriaceae Nitrate Test: Nitrate Test: Procedure: Nitrate broth test m.o Nit.A Nit. B Red colour Incubate at 35 o C 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 between Definitive 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 between Definitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters Growth on MacConkey’s agar: Growth on MacConkey’s agar: Principle: Lactose Lactose fermenter acid Nutral red Pink colonies

Procedure: 1. Inoculate MacConkey’s agar plate with the test organism by streaking. 2. Incubate the plate at 35 o C for 24 hrs. Flame & Cool Definitive tests to differentiate between Definitive 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 between Definitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters Growth on MacConkey’s agar: Growth on MacConkey’s agar: Lactose fermenter Pale colonies Lactose nonfermenter

Definitive tests to differentiate between Definitive 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 between Definitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters Growth on Triple Sugar Iron (TSI) agar: Growth on Triple Sugar Iron (TSI) agar: Principle: buttslant

Definitive tests to differentiate between Definitive 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: Butt: alkaline (red)Slant: alkaline (red)

Definitive tests to differentiate between Definitive 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: 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: b) Delayed reaction: Definitive tests to differentiate between Definitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters (after 24 hrs) Peptone O2O2 Alkaline amines Butt: acidic (yellow)Slant: alkaline (red) Small amount of acid Proteus, Salmonells & Shigella

Definitive tests to differentiate between Definitive 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: Lactose Large amount of acid Peptone O2O2 Alkaline amines Butt: acidic (yellow)Slant: acidic (yellow) E.Coli & Klebsiella

Growth on Triple Sugar Iron (TSI) agar: Growth on Triple Sugar Iron (TSI) agar: Principle: Definitive tests to differentiate between Definitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters   This medium contains also ferrous sulfate as an indicator for H 2 S production. H 2 S + FeSo 4 FeS Black ppt. of ferrous sulfid Proteus & Salmonells

Growth on Triple Sugar Iron (TSI) agar: Growth on Triple Sugar Iron (TSI) agar: Procedure: Definitive tests to differentiate between Definitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters

Results: Butt: Slant: H 2 S Production: acidic (yellow) -ve E.Coli & Klebsiella acidic (yellow) alkaline (red) -ve Shigella acidic (yellow) alkaline (red) +ve Growth on Triple Sugar Iron (TSI) agar: Growth on Triple Sugar Iron (TSI) agar: Proteus & Salmonells

Definitive tests for Lactose fermenters E.coli & Klebsiella IMV i C Test: IMV i C Test: It is composed of a group of four tests: Indole production test. Indole production test. Methyl red (MR) test. Methyl red (MR) test. Voges-Proskauer (V-P) test. Voges-Proskauer (V-P) test. Citrate Utilization test. Citrate Utilization test.

IIIIndole Production Test Definitive tests for Lactose fermenters E.coli & Klebsiella IMV i C Test: IMV i C Test: Principle: Tryptophane Tryptophanase enzyme Indole Kovac’s reagent Purple red color in the upper organic layer

 Indole Production Test IMV i C Test: IMV i C Test: Results: Definitive tests for Lactose fermenters E.coli & Klebsiella +ve -ve

MMMMR-VP Test: IMV i C Test: IMV i C Test: Principle: Definitive tests for Lactose fermenters E.coli & Klebsiella Dextrose Acidic pathway OR Neutral pathway Mixed acids  pH less than 4.4 MR indicator Red colour Acetyl methyl carbinol α-naphthol KOH Brick red colour MR +ve VP +ve

CCCCitrate Utilization Test IMV i C Test: IMV i C Test: Definitive tests for Lactose fermenters E.coli & Klebsiella Principle: Citrate As a sole source of carbon Na 2 CO 3 alkaline Bromothymol blue indicator Blue colour +ve

 Indole IMV i C Test: IMV i C Test: Results: Definitive tests for Lactose fermenters E.coli & Klebsiella MMMMR VVVVP  Citrate IMV i C I MR VP C

IMV i C Test: IMV i C Test: Results: Definitive tests for Lactose fermenters E.coli & Klebsiella +ve -ve +ve I M V i C E.Coli I M V i C Klebsiella

Growth on Eosin Methylene Blue (EMB)Agar: Growth on Eosin Methylene Blue (EMB)Agar: Definitive tests for Lactose fermenters E.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 for Lactose fermenters E.coli & Klebsiella black colonies with metalic sheen. E.Coli Klebsiella Purple colonies.

Definitive tests for Lactose nonfermenters Proteus & Salmonella Urease Production Test: Urease Production Test: Principle: Urea Urease enzyme NH 3 ( alkaline) Phenol red indicator Pink colour

Definitive tests for Lactose nonfermenters Proteus & Salmonella Urease Production Test: Urease Production Test: Results: +ve -ve Proteus Salmonella

Definitive tests for Lactose nonfermenters Proteus & Salmonella Gelatin Liquefaction Test: Gelatin Liquefaction Test: Principle: Gelatin Gelatinase enzyme Peptides gel liquid

Definitive tests for Lactose nonfermenters Proteus & Salmonella Gelatin Liquefaction Test: Gelatin Liquefaction Test: Results: Positive test: Gelatin medium remain liquid even after refrigeration. Gelatinase +ve Proteus Salmonella Gelatinase -ve

 It is selective medium for Salmonella and Shigella.  It contains sodium thiosulfate and ferric citrate as an indicator for H 2 S. Definitive tests for Salmonella & Shigella Growth on Salmonella-Shigella (S-S) Agar: Growth on Salmonella-Shigella (S-S) Agar: Principle:

Definitive tests for Salmonella & 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 Practicle 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.

Results Nitrate Test: Nitrate Test: Nit.A Nit. B Red colour No red colour Add zinc dust

Results IIIIndole Production Test Indole Kovac’s reagent Purple red color in the upper organic layer I

Results MMMMR-VP Test: MR-VP broth MR VP MR indicator Red colour MR +ve α-naphthol KOH Brick red colour VP +ve

Oxidase Test +ve -ve Pseudomonas Enterobacteriaceae O/F test: O + /F - Nitrate test: +ve further reduction to N 2 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 IMV i C test & EMB IMV i C & black colonies with metalic shines on EMB E.coli IMV i C Klebsiella No H 2 S production (no blacking in TSI) H 2 S production (blacking in TSI) Shigella Urease production & gelatin liquefaction +ve Proteus -ve S-S agar Pale colonies with black centers Salmonella

PseudomonasE.coliKlebsiellashigellaProteusSalmonella Results Gram’s Stain Gram’s –ve rods, single arrangement O/F test O + /F - O + /F + Nitrate test No red colour after addition of zinc dust +ve further reduction to N 2 +ve reduction from nitrate into nitrite Oxidase test +ve -ve Cetrimide agar Pale colonies with green pigmentation

E.coliKlebsiellashigellaProteusSalmonella Results MacConkey’s agar Pink colonies Pink mucoid colonies Pale colonies TSI agar Butt: Slant: H 2 S: Acidic (yellow) -ve Acidic (yellow) -ve Acidic (yellow) Alkaline (red) -ve Acidic (yellow) Alkaline (red) +ve Acidic (yellow) Alkaline (red) +ve IMV i C test IMV i C IMV i C IMV i C IMV i C IMV i C - + EMB agar Black colonies with metalic sheen Pink coloniesPale colonies

Results E.coliKlebsiellashigellaProteusSalmonella Urease production test Gelatin liquefaction test +ve (pink) -ve +ve-ve +ve (liquid) -ve S – S agar Pink colonies Pale colonies Pale colonies with black centers

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