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Isolation and characterization of Pseudomonas

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  1. 1. Isolation and Characterization of Pseudomonas
  2. 2. Pseudomonas <ul><li>Ubiquitous antibiotic-resistant bacteria </li></ul><ul><li>Some strain can even grow in distilled water </li></ul><ul><li>Pseudomonas are of utmost importance to the formation of snow and rain around the world. </li></ul><ul><li>(Bacteria – The Main Ingredient in Snowflakes) </li></ul><ul><li> </li></ul><ul><li>Pigments secreted by psuedomonads </li></ul><ul><li>A) Pyocin:- blue pigment </li></ul><ul><li>B) Fluorescin:- yellow pigment </li></ul><ul><li>C) Pyorubin:- red-brown pigment </li></ul>
  3. 3. Type Species: Pseudomonas aeruginosa <ul><li>Pseudomonas aeruginosa is a versatile gram negative bacterium that grows in soil, water, plants, animal tissues and other places that contain moisture. </li></ul><ul><li>Unlike many environmental bacteria, Pseudomonas aeruginosa has a remarkable capacity to cause disease in susceptible hosts </li></ul><ul><li>It is an “Opportunistic Pathogen”. </li></ul>
  4. 4. Characteristics of Pseudomonas aeruginosa <ul><li>Gram-negative rods </li></ul><ul><li>Motile (by single or multiple polar flagella) </li></ul><ul><li>Obligate (strict) aerobes (most strains) </li></ul><ul><li>Most distinguishing feature: formation of the diffusible, chloroform-soluble, blue pigment-Pyocyanin. </li></ul><ul><li>Non–spore forming, Nonfermentative </li></ul><ul><li>Catalase positive, Oxidase positive </li></ul><ul><li>Resistant to a large range of antibiotics </li></ul>
  5. 5. Material & Methods <ul><li>Isolation </li></ul><ul><li>Gram Staining </li></ul><ul><li>Biochemical tests </li></ul>
  6. 6. Isolation <ul><li>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. </li></ul><ul><li>1.0gm of soil was weighed and subjected for serial dilutions upto 10- 5 by Serial Dilution method. </li></ul><ul><li>Media Used: Nutrient Agar Medium </li></ul><ul><li>Kings B Medium (King et al. 1954) </li></ul>
  7. 7. Serial Dilution Method /
  8. 8. Growth on Nutrient Agar Plate <ul><li>Growth on Nutrient Agar Medium Showing Fluorescence </li></ul>
  9. 9. Growth on Kings B Medium <ul><li>Pale white, translucent colonies </li></ul><ul><li>Circular, convex, and smooth in appearance </li></ul>
  10. 10. Growth on Agar Slants <ul><li>Showing Fluorescence </li></ul>
  11. 11. Gram Staining <ul><li>The Gram-positive bacterial cells showed violet but Gram-negative cells turned pink/red coloration (Vincent, 1970). </li></ul><ul><li>Result: Stained Pink color: Gram –ve </li></ul><ul><li>The Gram negative cell wall contains a thin Peptidoglycan layer and does not hold onto the crystal violet stain. </li></ul>
  12. 12. Gram Negative Cell Wall <ul><li> </li></ul>
  13. 13. Biochemical Tests <ul><li>If the gram-stain showed gram-negative bacilli, the following tests can be done </li></ul><ul><li>Oxidase Test </li></ul><ul><li>Catalase Test </li></ul><ul><li>Urease Test </li></ul><ul><li>SIM Test </li></ul><ul><li>Glucose Fermentation Broth & O/F Medium </li></ul><ul><li>Carbohydrate Fermentation </li></ul><ul><li>MRVP (Methyl red & Voges-Proskauer) Test </li></ul><ul><li>Citrate Test </li></ul><ul><li>Gelatin Hydrolysis </li></ul><ul><li>Siderophore Production </li></ul><ul><li>HCN Production </li></ul>
  14. 14. <ul><li>Oxidase Test </li></ul><ul><li>This laboratory test is based on detecting the production of the enzyme cytochrome oxidase by Gram-negative bacteria. </li></ul><ul><li>Appearance of Blue-Purple color: Positive </li></ul>.
  15. 15. 2. Catalse Test <ul><li>This test is used to detect the enzyme catalase. </li></ul><ul><li>Liberation of bubbles (O2) immediately: Positive </li></ul>
  16. 16. 3. Urease Test <ul><li>This test is used to detect the enzyme Urease, which breaks down urea into ammonia. </li></ul><ul><li>Lack of color change : negative </li></ul>
  17. 17. 4. SIM- Hydrogen Sulphide, Indole & Motility Test <ul><li>The absence of a black color indicates that H2S was not produced. </li></ul><ul><li>No conversion of Ferrous sulfate to ferrous sulfide: H2S Negative </li></ul><ul><li>Pseudomonas can not break Tryptophan into Amino group and Indole </li></ul><ul><li>No change in color after addition of Kovac’s Reagent: Negative </li></ul><ul><li>No growth seen around the line of inoculation. </li></ul><ul><li>Motility Negative (P. aeruginosa doesn’t grow well in anaerobic conditions). </li></ul>
  18. 18. 5. Glucose Fermentation Broth and O/F Medium <ul><li>Tube 1& 2: Glucose O/F Medium </li></ul><ul><li>Pseudomonas shows Respiration of Glucose- Acid seen in upper part of 1 st tube </li></ul><ul><li>Tube 3: Glucose Fermentation Broth </li></ul><ul><li>Pseudomonas shows no fermentation of Glucose </li></ul>1 2 3
  19. 19. 6. MRVP (Methyl red & Voges-Proskauer) Test <ul><li>Methyl Red: neutral acetoin is produced and color changes from Red to Yellow </li></ul><ul><li>Result: Negative </li></ul><ul><li>Voges-Proskauer Test: The medium turns light brown </li></ul><ul><li>Result: Negative </li></ul>
  20. 20. 7. Citrate Test <ul><li>Test for the ability of bacteria to convert citrate into oxaloacetate. </li></ul><ul><li>Media turned bright blue: Positive </li></ul>
  21. 21. 8. Gelatin Hydrolysis <ul><li>This test is used to detect the enzyme Gelatinase which digest the Gelatin present in the medium. </li></ul><ul><li>Medium liquefied even after refrigeration: Positive </li></ul>Negative Result Positive Result
  22. 22. 9. Siderophore Production <ul><li>Pseudomonas Agar F stimulates the production of fluorescein and reduces that of pyocyanin and/or Pyorubin. </li></ul>
  23. 23. 10. HCN Production <ul><li>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 colour change. </li></ul>