PHT 416 Lab 8

Steps Microscopic Morphology Growth Biochemical Tests Nutrient agar Blood agar Mannitol Salt Agar MacConkey’s agar

Bacteria Gram’s Stain Gram’s +ve Cocci Bacilli Gram’s -ve Cocci Bacilli Staphylococci Streptococci Neisseria Corynbacterium Clostridum Bacillus Enterobacteriaceae Pseudomonas

Gram –ve bacilli

Oxidase test Principle: Tetramethyl p-phenylene diamine (oxidase reagent colourless) Cytochrome oxidase enzyme Indophenol (Purple colour) -ve test Colourless +ve test Purple colour Pseudomonas Enterobacteriaceae

1- Growth on MacConkey’s agar Lactose fermenters Lactose non- fermenters Enterobacteriaceae Pink colonies Pale colonies Escherichia, Klebsiella Salmonella, Shigella, Proteus Lactose acid Neutral red Pink colonies fermenter

2- Triple sugar iron (TSI) agar Lactose fermentersLactose non-fermenters Enterobacteriaceae Escherichia, KlebsiellaSalmonella, Shigella, Proteus Principle:  it contains three types of sugars (lactose, sucrose and dextrose) (The conc. of lactose and sucrose is 10 times that of dextrose.)  It contains phenol red indicator.  It contains soft agar to enhance gas appearance.  It contains also ferrous sulfate as an indicator for H 2 S production. H 2 S + FeSo 4 FeS Black ppt. of ferrous sulfid

2- Triple sugar iron (TSI) agar Lactose fermentersLactose non-fermenters Enterobacteriaceae Escherichia, KlebsiellaSalmonella, Shigella, Proteus acidic (yellow) -ve E.Coli & Klebsiella acidic (yellow) alkaline (red) -ve Shigella acidic (yellow) alkaline (red) +ve Proteus & Salmonells

2- Triple sugar iron (TSI) agar Lactose fermentersLactose non-fermenters Enterobacteriaceae Escherichia, KlebsiellaSalmonella, Shigella, Proteus acidic (yellow) -ve E.Coli & Klebsiella Lactose Fermentation: Lactose Large amount of acid Butt: acidic (yellow)Slant: acidic (yellow)

Lactose Fermentation: Lactose Large amount of acid Butt: acidic (yellow) Slant: acidic (yellow) E.Coli & Klebsiella

2- Triple sugar iron (TSI) agar Lactose fermentersLactose non-fermenters Enterobacteriaceae Escherichia, KlebsiellaSalmonella, Shigella, Proteus acidic (yellow) -ve E.Coli & Klebsiella acidic (yellow) alkaline (red) -ve Shigella acidic (yellow) alkaline (red) +ve Proteus & Salmonells

1. Dextrose Fermentation: a) Initial reaction: Dextrose acid (after 10 – 12 hrs) Butt: acidic (yellow)Slant: acidic (yellow) Proteus, Salmonells & Shigella

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

3- Nitrate Test Lactose fermentersLactose non-fermenters Enterobacteriaceae Escherichia, KlebsiellaSalmonella, Shigella, Proteus Procedure: Nitrate broth Test m.o Red colour Incubate at 35 o C for 24 hrs All enterobacteriaceae reduce nitrates to nitrites Nit. A Nit. B

4- Oxidation Fermentation (O/F) Test Lactose fermentersLactose non-fermenters Enterobacteriaceae Escherichia, KlebsiellaSalmonella, Shigella, Proteus Principle:  Using sensitive O/F medium (Hugh and Leifson Medium)  All enterobacteriaceae are O + /F + (Fermentative)

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 (Hugh and Leifson Medium) 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. To be more sensitive this medium contains:

Growth on MacConkey’s agar Lactose fermenters Lactose non-fermenters Enterobacteriaceae Pink colonies Escherichia, Klebsiella Salmonella, Shigella, Proteus Triple sugar iron (TSI) agar acidic (yellow) -ve

1- Growth on MacConkey’s agar E. coliKlebsiella Enterobacteriaceae Lactose Fermenters Colonies are mucoid 2- Growth on Eosin Methylene Blue Agar black colonies with metallic sheen Purple colonies Pink Colonies

3- IMVC Test They are group of four tests: Indole production test. Methyl red (MR) test. Voges-Proskauer (V-P) test. Citrate Utilization test.

Tryptophane Tryptophanase enzyme Indole Kovac’s reagent Purple red color in the upper layer Indole Production Test MR-VP Test Glucose Acidic pathway OR MR +ve/Red colourVP +ve/Eosin-pink colour partial glucose fermentation 100% Glucose fermentation Acidic pathway Citrate Utilization Test Citrate As a source of carbon Na 2 CO 3 alkaline Bromothymol blue indicator Blue colour +ve- ve

+ve -ve +ve I M V C E.Coli I M V C Klebsiella IMVC

Growth on MacConkey’s agar Lactose fermentersLactose non-fermenters Enterobacteriaceae Escherichia, KlebsiellaSalmonella, Shigella, Proteus Triple sugar iron (TSI) agar Pale colonies acidic (yellow) alkaline (red) -ve Shigella acidic (yellow) alkaline (red) +ve Proteus & Salmonells

ShigellaSalmonella, Proteus Enterobacteriaceae Lactose non-fermenters 1- Growth on Salmonella-Shigella (S-S) agar. Pale colonies with black centersPale colonies 2- Citrate Utilization Test +ve -ve ? Citrate As a source of carbon Na 2 CO 3 Bromothymol blue indicator  It is a 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.

Culture Characteristics Facultative anaerobes, grow on simple media. Proteus shows a swarming growth

Proteus Definitive Test to Differintate between Proteus & Salmonella 1- Urease Production Test Salmonella Urea Urease enzyme NH 3 Phenol red Pink colour ( alkaline) +ve-ve 2- Gelatin Liquefaction Test Gelatin Gelatinase enzyme Peptides gel liquid Positive test: Gelatin medium remain liquid even after refrigeration. Gelatinase +veGelatinase -ve

Oxidase test Principle: Tetramethyl p-phenylene diamine (oxidase reagent colourless) Cytochrome oxidase enzyme Indophenol (Purple colour) -ve test Colourless +ve test Purple colour Pseudomonas Enterobacteriaceae

Nutrient Agar Its growth on showing greenish coloration due to exopigment production. Pseudomonas Growth on Cetrimide Agar: Only Pseudomonas species can grow on cetrimide agar showing growth of pale colonies with diffusion of green pigmentation. Cetrimide agar is a highly selective medium for Pseudomonas species due to presences of cetrimide which inhibits the growth of other bacteria. It contains also MgCl 2 & K 2 So 4 to facilitate production of the charactaristic green pigmentation of Pseudomonas.

Pseudomonas 1- Oxidation Fermentation (O/F) Test O - /F - O + /F + O + /F - Results: Fermentative Enterobacteriaceae Oxidative Pseudomonas Non Saccharolytic

2- Nitrate Test Principle: Nitrate reductase nitrite (nit. A) (nit. B) Red diazonium salt Enterobacteriaceae If no red color Further reduction Nitrogen (N 2 ) Add zinc dust (reducing agent) Pseudomonas

Procedure: Nitrate broth test m.o Nit. A Nit. B Red color No red colour Add zinc dust Incubate at 35 o C for 24 hrs 2- Nitrate Test Red colour after addition of nit.A & nit.B Reduction of Nitrate to nitrite Enterobacteriaceae Red colour after addition of zinc dust -ve reduction No red colour after addition of zinc dust Further reduction to Nitrogen pseudomonas Results: