Lab. No. 4 (A)

StaphylococciStreptococciMicrococci NeisseriaCorynbacterium Clostridum Bacillus Enterobacteriaceae Pseudomonas. Bacteria Gram’s Stain Gram’s +ve Cocci Bacilli Gram’s -ve Cocci Bacilli

Enterobacteriaceae

enterobacteriaceae Gram-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. 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. 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: Lactose Nonfermenters: e.g., protus, salmonella,shigella. e.g., protus, salmonella,shigella.

1-morphology: 1-morphology: Gram’s –ve bacilli, having single arrangement.

2-cultural characteristic: 2-cultural characteristic: - Facultative anaerobes, - 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:

Lactose Lactose fermenter acid Neutral 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 35 o C for 24 hrs. 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 colour Incubate at 35 o C 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: 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: 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- 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 H 2 S production. Black ppt. of ferrous sulfid H 2 S + FeSo 4 FeS

Procedure:

Principle: buttslant

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

Test: 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 4.4 MR indicator Red colour Acetyl methyl carbinol α-naphthol KOH Brick red colour MR +ve VP +ve partial glucose Fermentation 100% Glucose fermentation Acidic pathway

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

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

 Indole Results: MMMMR VVV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 → 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., 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).

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 Klebsiella