Final Practical 8 -1 (2) PDF

Practical No. 8 Pseudomonas and Vibrios  Pseudomonas: Microscopic Morphology Gram negative straight Gram Staining rods - scattered 100 x (Oil immersion lens) Pseudomonas: Culture Characteristics Growth on Nutrient agar and Pigment production Medium- large size...

Practical No. 8 Pseudomonas and Vibrios  Pseudomonas: Microscopic Morphology Gram negative straight Gram Staining rods - scattered 100 x (Oil immersion lens) Pseudomonas: Culture Characteristics Growth on Nutrient agar and Pigment production Medium- large size colonies Smooth Greenish pigment (pyoverdin) diffused into medium Characteristic odor (fruity smell)  Pseudomonas aeruginosa: Pigment production Pyocyanin pigment – Blue colour Pseudomonas: Culture Characteristics Growth on Blood agar Colonies are: Large Flat Often hemolytic Pseudomonas: Culture Characteristics Growth on MacConkey’s agar Colonies are: Colourless Non lactose fermenting Pseudomonas aeruginosa: Biochemical Characteristics KIA (Kligler Iron Agar) Oxidase Test O-F test(oxidative-fermentative ) API 20NE Identification  1. KIA INTERPRETATIO N Pseudomonas growing in KIA 1. Alkaline / Alkaline Slant 2. Acidic / Acidic 3. Gas Production 4. Alkaline / Acidic 5. H2S Production Red colour= Alkaline Red colour= Alkaline Gas production = No Butt H2S production = No  2. Oxidase test Pseudomonas aeruginosa: Oxidase +ve 3. O-F Test (oxidative-fermentative) The oxidative-fermentative test determines if a gram-negative organism metabolizes glucose by fermentation (anaerobic) or oxidation (aerobic ).  P.aeruginosa: O-F Test Acid production in the open tube (yellow) and not in the oil-covered tube indicates an Oxidative result. a) P. aeruginosa incubated for 24 hours. Oil-covered tube b) P. aeruginosa incubated for 48 hours. c) P. aeruginosa incubated for 5 days. Open tube  O-F Test for E.coli Acid production and color change (yellow), in both the oil-covered (anaerobic) tube and open (aerobic) tube indicates positive fermentative result. Oil-covered tube Open tube O-F test for Alcaligens faecalis, Moraxella No change in colour of the medium indicates this organism is non-saccharolytic and unable to use glucose either oxidatively or fermentatively. Oil-covered tube Open tube  4. API 20NE Identification API is Analytical Profile Index NE stands for Non-enteric It is composed of 20 biochemical tests One kit identifies an organism to species level  API 20NE RECORDING SLIP FOR API 20NE RESULTS  API 20NE After inoculation, strip is incubated at 37˚C for 18-24 hours. After incubation, results are recorded after adding reagents in appropriate tubes.  Identification of Pseudomonas by API 20NE NO3 Glucose +ve +ve Recorded results for API 20NE strip inoculated with Pseudomonas Vibrios  Important species Vibrio cholerae Vibrio parahemolyticus Vibrio alginolyticus Vibrio vulnificus  Vibrios: Microscopic Morphology Gram staining Gram negative Curved rods (comma- shaped) 100 x (Oil immersion lens) V.cholerae: Single flagellum at one end SINGLE FLAGELLUM  V. cholerae: Culture Characteristics Growth on Thiosulphate Citrate bile salt sucrose (TCBS) agar Colonies of V.cholerae on TCBS agar: TCBS agar contains indicator bromothymol blue which differentiates sucrose fermenting (yellow colonies) from non-sucrose fermenting Vibrios. ❑ V.cholerae serogroups: ✔ O1 ✔O139  Other Vibrios: Culture Characteristics Growth on TCBS agar V. parahaemolyticus, V.mimicus, V. vulnificus produce: Greenish–blue colonies Do not ferment sucrose  V.cholerae: Culture Characteristics Growth on MacConkey’s agar Colonies on MacConkey’s agar are: Small Colorless Non-lactose fermenting (NLF)  Vibrio cholerae: Growth in alkaline peptone water Vibrio cholerae Turbidity No grows in alkaline turbidity peptone water Turbidity: +ve for growth  V.cholerae: Culture Characteristics Growth on Blood agar (sub cultured from alkaline peptone water) ❑ V.cholerae O1 and O139 often produce β-hemolytic colonies on blood agar Vibrios: Biochemical Tests KIA (Kligler Iron Agar) Oxidase Test API 20NE identification  1. KIA INTERPRETATIO Vibrio cholerae growing in KIA N 1. Alkaline / Alkaline slant 2. Acidic / Acidic 3. Gas production 4. Alkaline / Acidic 5. H2S production Red colour= Alkaline yellow colour= Acidic Gas production = No Butt H2S production = No  2. Oxidase test Vibrio cholerae :Oxidase +ve  Identification of Vibrio cholerae by API 20NE Recorded results for API 20NE strip inoculated with Vibrio cholerae  3. API 20NE Identification API is Analytical Profile Index NE stands for Non-enteric It is composed of 20 biochemical tests One kit identifies an organism to species level  API 20NE RECORDING SLIP FOR API 20NE RESULTS  API 20NE After inoculation, strip is incubated at 37˚C for 18-24 hours. After incubation, results are recorded after adding reagents in appropriate tubes.  V.cholerae O1and O139: Serological identification using specific antiserum O1 O139 Antiserum  V.cholerae O1 serotypes: Serological identification using monovalent antiserum There are 3 Serotypes: V.cholerae ogawa V.cholerae inaba V.cholerae hikojima Separate antisera (monovalent) is required to identify these serotypes of V.cholerae. QUESTIONS???