Diagnostic Microbiology Lab Manual-1 PDF

Procedure of Biochemical Tests 1. Motility test: Motility media are SIM media and MIO media. 2. IMVIC Reaction: Stands for: ▪ Indole test ▪ Methyl red test ▪ voges-Proskauer test ▪ Citrate test INDOLE TEST Principle: To test the ability of an organisms to split indole from the Tryptopha...

Procedure of Biochemical Tests 1. Motility test: Motility media are SIM media and MIO media. 2. IMVIC Reaction: Stands for: ▪ Indole test ▪ Methyl red test ▪ voges-Proskauer test ▪ Citrate test INDOLE TEST Principle: To test the ability of an organisms to split indole from the Tryptophane molecule. (Media: Tryptophane broth or peptone broth, or SIM media or MI0, Reagent: Kovac's) Procedure: 1. Inoculate the test tubes f Tryptophane broth with a loopful of bacterial growth. 2. Incubate the tubes at 35-37° C for 24 hours. 3. After incubation period, open the test tube add 5 drops of Kovac’s reagent. 4. Read the result immediately Positive----- Pink or red ring. Negative --- yellow ring. METHYL RED TEST Principle: To determine the ability of an organisms to produce acid as final products from glucose fermentation. (Media: MR/VP broth, Reagent: Methyl red Procedure: Inoculate the test tubes of MR/VP broth with a loopful of bacterialgrowth. 1. Incubate the tubes at 35-37 °C for 48 hours. 2. After incubation period, add 4 drops of Methyl red reagent. 3. Read the result immediately Positive --- Red Negative --- Yellow VOGES PROSKAUER TEST Principle: To determine the ability of an organisms to produce acetoin as final products from glucose fermentation. (Media: MR/VP broth, Reagent: 1- Alpha naphthol, 2- 40% KOH) Procedure: 1. Inoculate the test tubes of MR/VP broth with a loopful of bacterialgrowth. 2. Incubate the tubes at 35-37C for 48 hours. 3. After-incubation period, add to the tube ( 1ml of bacterial broth), 6drops of Alpha naphthol and 2 drops of 40% KOH reagents. 4. Loosely the cap of tube and place it in slant position for 15 to 45 minutes. 5. After 15 minutes read the result if it is positive report it and if it is negative keep it for another 30 minutes. Red --- Positive Yellow --- Negative CITRATE TEST Principle: To determine if the organism is capable to utilize citrate as the only sole source of carbon. (Media: Simmon's Citrate agar, Indicator: Bromothymol blue). Procedure: 1. Inoculate the slant of test tubes with an organism as fish-tail. 2. Incubate the tubes for 1 to 4 days at 35-37 °C with loosely cap 3. After incubation period read the result immediately. Deep blue ----- Positive No Change (Green) ----Negative KLIGLER IRON AGAR (KIA) Principle: To determine the ability of microorganism to ferment carbohydrate (glucose & lactose), determine gas production (CO2) andHydrogen sulfide gas production. Formula: Glucose 0.1% Lactose 1% peptone Phenol red Sodium Thiosultefate Ferric Ammonium Citrate (Media: KIA, Indicator Phenol Red) H2S (colorless gas) reacts with IRON in the medium (ferric ammonium sulfate as H2S indicator) to produce a BLACK PRECIPITATE (ferrous sulfide FeS. H2S+ ferrous sulfate or ferric ammonium citrate ferrous sulfide Procedure: 1. Inoculate the test tube of KIA with an organism: Stabbing in the butt then Fish tail on the slant. 2. Incubate the tubes at 35-37°C for 24 hours (Time is Critical). 3. Read the result immediately after the incubation period. UREASE TEST Principle: To determine the presence of Urease enzyme. (Media: Urea broth media or Urea agar/ slant, Indicator Phenol Red). Procedure: 1.The surface of slant agar media is streaked with the test organisms asfish-tail (Urea agar/ slant) 2. Incubate the tubes at 35-37°C for 24 up to 72 hours. 3. After incubation period read the result: Red to pink color --- Positive No change (yellow) --- Negative OXIDASE TEST Principle: To determine the presence of Cytochrome oxidase enzyme Procedure: pick up one or two isolated colonies by a wooden applicator stick and rub it directly on filter paper impregnated with N. N. N. N. Tetramethyl paraphenylen diamine dihydrochloride (TPD) as reagent. Read the result within 10 second (Do not use wire loop or needle cause a false positiveresult). Dark purple or blue color --- Positive No change --- Negative Enterobacteriaceae This family divided into two groups according to their ability to ferment lactose: 1. Lactose fermenter groups. »Escherichia coli »Klebsiella sp. »Enterobacter sp. »Citrobacter sp. 2. None lactose fermenter groups. »Proteus sp Salmonella sp Shigella sp »Morganella Morgani »Providencia sp »Seratia sp »Yersinia pestis Characteristics of Enterobacteriaceae family: - Gram Negative slightly short or slightly longer rods, single - Some of genera are motile due to presence of peritrichous flagella and other genera are not motile. - Aerobic or facultative anaerobic. - All genera of this family ferment glucose. - Oxidase: Negative. - Reduce nitrate to nitrite. - Catalase: Positive. - Colonies: Large, gray and smooth with beta or gamma hemolysis onBAP and some genera give mucoid colonies. Identification of Enterobacteriaceae members: - Growth of organisms on Blood and Chocolate agar mostly Look the same. Gram stains... Look the same (Gram-Negative rods). Oxidase Test MacConkey Agar Kligler Iron Agar (KIA). Nitrate reduction test. IMVIC Reaction Motility Test Urease Test Identification of Enterobacteriaceae mnembers 1- E. coli -EPEC (Enteropathogenic ) -ETEC (Enterotoxigenic ) -EHEC (0157) H7 (Enterohemorrhagic ) -EIEC (Enteroinvasive ) -EAEC (Enteroaggregative) - Gram Negative bacilli single - Sometime give beta hemolysis in blood gar On MacConkey agar: E. coli can be, Lactose fermenter or non- Lactosefermenter Colonies appear smooth and large, On Eosin Methylene Blue agar media(EMB). It gives green metallic sheen colonies (greenish to silver color). IMVIC: + + - -- KIA: A/A,G A/A,G, H2S K/A,G, & K/A Motility: Motile Urease: Negative H2S: Mostly Negative 2- Enterobacter aerogenes: Gram-Negative Bacilli, single n MacConkey agar: colonies appear mucoid pink-red IMVIC: - - ++ KIA: A/A,G, KIA,G Motility: Motile Urease: Negative except Enterobacter cloacae H2S: Negative 3- Klebsiella sp Gram-Negative Bacilli, single On MacConkey agar: colonies appear large slimy viscous (gummy) or mucoid with pink-red color. IMVIC: - - + + (K. pneumonia e) + - + + (K. oxytoca) KIA: A/A,G Motility: Not Motile Urease: Positive H2S: Negative 4- Citrobacter sp Gram-Negative Bacilli, single On MacConkey agar: colonies appear pink-red mucoid. Ferment lactose weakly. IMViC: - + - + Citrobacter freundii  + + - + Citrobacter sp KIA: K/A, H2S, A/A,H2S Citrobacter freundii A/A,G Citrobacter sp Motility: Motile Urease: Positive  Gram Negative Bacilli Enterobacteriaceae None Lactose fermenter group 5- Proteus sp. - Gram Negative Bacilli - single On MacConkey agar: colonies appear colorless with foul smell. - On blood and chocolate agar:- it gives swarming (like the wave of the sea) To prevent swarming: - Cystine Lactose Electrolyte Deficlent (CLED). It Is used for differentiation of microorganisms in urine - Phenyl Ethyl Alcohol PEA - Duplicate The concentration of Agar in The Media IMVIC: ++-- P. vulgaris - + (-/+) (-/+) P. mirabilis KIA: K/A,G,H2S A/A,G,H2S Motility: Motile Urease: Positive H2S: Positive 6- Marganella morgana - Gram Negative Bacilli, single - It have all Proteus character except it doesn't produce H2S IMVIC: + + - - KIA: K/A,G Motility: Motile Urease: Positive H2S: Negative 7- Salmonella sp. Gram Negative Bacilli, single On MacConkey agar: colonies appear colorless On Salmonella Shigella agar medium, colonies appear colorless with black center (H2S). IMVIC: -+-- S. typhi& S. paratyphi A -+-+ Other type of Salmonella KIA: K/A,H2S S. typhi K/A, G S. paratyphi A K/A, G,H2S Other type of Salmonella Motility: Motile Urease: Negative H2S: Positive except S. paratyphi A (10%: +) 8- Shigella sp. Gram-Negative Bacilli, single On MacConkey agar: colonies appear colorless and flat. On Salmonella Shigella agar medium, colonies appear colorless. IMVIC:(-/+) + - - ( serogroups A,B,C) -+-- S. sonnei (group D) KIA: K/A Motility: Not Motile Urease: Negative H2S: Negative 9- Providencia sp. Gram Negative Bacilli, single -On MacConkey agar: colonies appear colorless Similar to Proteus (without swarming in BAM) IMVIC: KIA: K/A,G K/A Motility : Motile Urease: Negative except P. rettgeri H2S: Negative 10- Serratia marcescens Gram Negative Bacilli, single On MacConkey agar: colonies appear none lactose fermenter with Briek red pigment. On blood agar and nutrient agar media colonies appear with brick red pigment. IMVIC: KIA: K/A,G K/A Motility: Motile Urease: Negative H2S: Negative 11- Yersinia pestis Gram-Negative Bacilli, single On MacConkey agar: colonies appear colorless IMVIC: - + - - KIA: K/A Motility: Not Motile Urease: Negative H2S: Negative Salmonella Shigella agar medium SS agar is a selective medium for isolation of Salmonella and Shigella from stool and food Product. Inhibitor factors: Brilliant green Bile salt Sodium thiosulfate Sodium citrate PH indicator: Neutral red Source of Carbohydrate: Lactose Examine the media for non-fermenting colonies (colorless), with or without H2S. Colonies with black center confirm H2S production.