Lab. No. 5

Gram-negative, non-spore-forming bacilli. Gram-negative, non-spore-forming bacilli. Their natural habitat is the intestinal tract of humans and animals. Their natural habitat is the intestinal tract of humans and animals. Characters of enterobacteriaceae: Characters of enterobacteriaceae: 1- Facultative anaerobes that grow on simple laboratory media. 1- Facultative anaerobes that grow on simple laboratory media. 2- Ferment glucose ± other carbohydrates. 2- Ferment glucose ± other carbohydrates. 3-Oxidase negative. 3-Oxidase negative. 4- Reduce nitrates to nitrites. 4- Reduce nitrates to nitrites.

This family is classified on the basis of biochemical reactions, especially fermentation of carbohydrates. 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: It can be divided according to their effect on lactose into:  Lactose Fermenters: (coliforms) e.g., E.coli, klebsiella. e.g., E.coli, klebsiella.  Lactose Nonfermenters: e.g., protus, salmonella. e.g., protus, salmonella.  Late Lactose Fermenters: e.g., shigella. e.g., shigella.

Gram’s –ve bacilli, having single arrangement.

Facultative anaerobes, grow on simple media Facultative anaerobes, grow on simple media  Proteus showing a swarming growth.  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 & crystal violet inhibit the growth of gram +ve bacteria).  Test sugar: lactose.  pH indicator: neutral red ( yellow in alkaline, pink in acidic pH). 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. Flam & Cool

Results: Pink colonies Lactose fermenter Pale colonies Lactose nonfermenter

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 +ve test No colour -ve test Pseudomonas Enterobacteriaceae

2- Nitrate test: Principle:  All enterobacteriaceae reduce nitrates to nitrites

Procedure: Nitrate broth test m.o Nit.A Nit. B Red colour Incubate at 35 o C for 24 hrs

3- Oxidation Fermentation (O/F) Test: 3- Oxidation Fermentation (O/F) Test: Principle:  Using sensitive O/F medium ( Hugh and Leifson Medium).  All enterobacteriaceae are O + /F + (Fermentative)

Identification of lactose fermenters: 1- TSI test. 2- IMViC It is composed of a group of four tests: 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. 3- Growth on Eosin- Methylene blue agar. 4-urease production test.

1-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.

Principle: buttslant

Procedure:

Results: Lactose Fermentation: Lactose Large amount of acid Peptone O2O2 Alkaline amines Butt: acidic (yellow)Slant: acidic (yellow) E.Coli & Klebsiella

Test: 2-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.

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, 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

3- Citrate Utilization Test Principle: Citrate As a sole source of carbon Na 2 CO 3 alkaline Bromothymol blue indicator Blue colour +ve

 Indole Results: MMMMR VVVVP  Citrate IMV i C I MR VP C

Results: +ve -ve +ve I M V i C E.Coli I M V i C Klebsiella

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 → combine to form a dark purple precipitate at acidic pH  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. Vigorous fermenters of lactose or sucrose → dark purple dye complex → dark purple to black colonies. Non-lactose/sucrose fermenters → normally-colored or colorless colonies. Non-lactose/sucrose fermenters → normally-colored or colorless colonies., often produces a green metallic sheen due to precipitation of M.B. in the medium from the very high amounts of acid produced. 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). Klebsiella, produces colonies with dark center surrounded by light colored- mucoid rim (fish-eye appearance).

Results: black colonies with metalic sheen. E.Coli Klebsiella Purple colonies.

4-Urease production test:- Principle : Urea Urease enzyme NH3 ( alkaline) Phenol red indicator Pink colour

Results: +ve -ve Klebsiella E-Coli

1- TSI test. 2- Growth on S-S agr. 3-Citrate utilization test. 4- Urease test. 5-Gelatin liquefaction test. Identification of lactose- nonfermenters:

Results: 1. Dextrose Fermentation: a) Initial reaction: Dextrose acid (after 10 – 12 hrs) Butt: acidic (yellow)Slant: acidic (yellow) 1-Triple Sugar Iron (TSI) agar:

Dextrose Fermentation: b) Delayed reaction: (after 24 hrs) Peptone O2O2 Alkaline amines Butt: acidic (yellow)Slant: alkaline (red) Small amount of acid Proteus, Salmonells & Shigella

Principle:   This medium contains ferrous sulfate as an indicator for H 2 S production. H 2 S + FeSo 4 FeS Black ppt. of ferrous sulfid Proteus & Salmonells

Results: Butt: Slant: H 2 S Production: acidic (yellow) -ve E.Coli & Klebsiella acidic (yellow) alkaline (red) -ve Shigella acidic (yellow) alkaline (red) +ve Proteus & Salmonells

 It is selective and differential medium for enteric pathogens especially Salmonella.  It contains lactose and neutral red indicator  It also contains sodium thiosulfate and ferric citrate as an indicator for H 2 S → colonies with black centers. 2- Growth on Salmonella-Shigella (S-S) Agar: Principle:

Results: Salmonella & proteus Pale colonies with black centers Shigella Pale colonies

3-Citrate Utilization Test Principle: Citrate As a sole source of carbon Na 2 CO 3 alkaline Bromothymol blue indicator Blue colour +ve

Results:- Results:- Proteus & Salmonella Salmonella Shigella +ve -ve

Definitive tests to differentiate between Proteus & Salmonella 4- Urease Production Test: Principle: Urea Urease enzyme NH 3 ( alkaline) Phenol red indicator Pink colour

Results: +ve -ve Proteus Salmonella

Definitive tests to differentiate between Proteus & Salmonella 5- Gelatin Liquefaction Test: Principle: Gelatin Gelatinase enzyme Peptides gel liquid

Results: Positive test: Gelatin medium remain liquid even after refrigeration. Gelatinase +ve Proteus Salmonella Gelatinase -ve

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

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 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 coloniesPale colonies with black centers