Pseudomonas

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Isolation and Characterization of Pseudomonas

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Slide 1: 

Isolation and Characterization of Pseudomonas

Pseudomonas : 

Pseudomonas http://www.bazenyservis.cz/hygiena/hygiena.html Ubiquitous antibiotic-resistant bacteria Some strain can even grow in distilled water Pseudomonas are of utmost importance to the formation of snow and rain around the world. (Bacteria – The Main Ingredient in Snowflakes) http://en.wikipedia.org/wiki/Pseudomonas Pigments secreted by psuedomonads A) Pyocyanin:- blue pigment B) Pyoverdin:- yellow pigment C) Pyorubin:- red-brown pigment

Type Species: Pseudomonas aeruginosa : 

Type Species: Pseudomonas aeruginosa Pseudomonas aeruginosa is a gram negative bacterium that grows in soil, water, plants, animal tissues and other places that contain moisture. Unlike many environmental bacteria, Pseudomonas aeruginosa has a remarkable capacity to cause disease in susceptible hosts It is an “Opportunistic Pathogen”. http://commons.wikimedia.org/wiki/File:Pseudomonas_aeruginosa_Gram.jpg

Characteristics of Pseudomonas aeruginosa : 

Characteristics of Pseudomonas aeruginosa Gram-negative rods Motile (by single or multiple polar flagella) Obligate (strict) aerobes (most strains) Most distinguishing feature: formation of the diffusible, chloroform-soluble, blue pigment-Pyocyanin. Non–spore forming, Nonfermentative Catalase positive, Oxidase positive Resistant to a large range of antibiotics www.buzzle.com/articles/pseudomonas-aeruginosa

Material & Methods : 

Material & Methods Isolation Gram Staining Biochemical tests

Isolation : 

Isolation Soil samples were taken from different locations in various agricultural areas of Dehradun during March 2008. Soil samples from each field were collected to a depth of 30 cm. 1.0gm of soil was weighed and subjected for serial dilutions upto 10-5 by Serial Dilution method. Media Used: Nutrient Agar Medium Kings B Medium (King et al. 1954)

Serial Dilution Method : 

Serial Dilution Method http://sciencefair.math.iit.edu/techniques/SerialDilution/

Pure Culture on Nutrient Agar Plate : 

Pure Culture on Nutrient Agar Plate Growth on Nutrient Agar Medium Showing Fluorescence

Pure Culture on Kings B Medium : 

Pure Culture on Kings B Medium Pale white, translucent colonies Circular, convex, and smooth in appearance

Growth on Agar Slants : 

Growth on Agar Slants Showing Fluorescence

Gram Staining : 

Gram Staining The Gram-positive bacterial cells showed violet but Gram-negative cells turned pink/red coloration (Vincent, 1970). Result: Stained Pink color: Gram –ve The Gram negative cell wall contains a thin Peptidoglycan layer and does not hold onto the crystal violet stain.

Gram Negative Cell Wall : 

Gram Negative Cell Wall http://authors.ck12.org/wiki/images/8/85/BioII-1901-06a.jpg

Biochemical Tests : 

Biochemical Tests If the gram-stain showed gram-negative bacilli, the following tests can be done Oxidase Test Catalase Test Urease Test SIM Test Glucose Fermentation Broth & O/F Medium Carbohydrate Fermentation MRVP (Methyl red & Voges-Proskauer) Test Citrate Test Gelatin Hydrolysis Siderophore Production HCN Production

Oxidase Test : 

. Oxidase Test This laboratory test is based on detecting the production of the enzyme cytochrome oxidase by Gram-negative bacteria. Appearance of Blue-Purple color: Positive

2. Catalse Test : 

2. Catalse Test This test is used to detect the enzyme catalase. Liberation of bubbles (O2) immediately: Positive

3. Urease Test : 

3. Urease Test This test is used to detect the enzyme Urease, which breaks down urea into ammonia. Lack of color change : negative

4. SIM- Hydrogen Sulphide, Indole & Motility Test : 

4. SIM- Hydrogen Sulphide, Indole & Motility Test The absence of a black color indicates that H2S was not produced. No conversion of Ferrous sulfate to ferrous sulfide: H2S Negative Pseudomonas can not break Tryptophan into Amino group and Indole No change in color after addition of Kovac’s Reagent: Negative No growth seen around the line of inoculation. Motility Negative (P. aeruginosa doesn’t grow well in anaerobic conditions).

5. Glucose Fermentation Broth and O/F Medium : 

5. Glucose Fermentation Broth and O/F Medium Tube 1& 2: Glucose O/F Medium Pseudomonas shows Respiration of Glucose- Acid seen in upper part of 1st tube Tube 3: Glucose Fermentation Broth Pseudomonas shows no fermentation of Glucose 1 2 3

6. MRVP (Methyl red & Voges-Proskauer) Test : 

6. MRVP (Methyl red & Voges-Proskauer) Test Methyl Red: neutral acetoin is produced and color changes from Red to Yellow Result: Negative Voges-Proskauer Test: The medium turns light brown Result: Negative

7. Citrate Test : 

7. Citrate Test Test for the ability of bacteria to convert citrate into oxaloacetate. Media turned bright blue: Positive

8. Gelatin Hydrolysis : 

8. Gelatin Hydrolysis This test is used to detect the enzyme Gelatinase which digest the Gelatin present in the medium. Medium liquefied even after refrigeration: Positive Negative Result Positive Result

9. Siderophore Production : 

9. Siderophore Production Pseudomonas Agar F stimulates the production of fluorescein and reduces that of pyocyanin and/or Pyorubin.

10. HCN Production : 

10. HCN Production The isolate was grown on King's B agar supplemented with Glycine (5.8 mM) in Petri plates with lids fitted with a Whatman filter paper previously soaked in saturated picric acid solution. The change in the color of the filter paper from yellow to dark brown was assessed visually depending on the intensity of the color change.

Acknowledgement : 

Acknowledgement Dr. Vishal Kumar Deshwal Miss Kavita Vig Prof. Dr. D.K Maheshwari Prof. Dr. R.C Dubey