Pseudomonas

Isolation and Characterization of 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) Pyocin:- blue pigment
B) Fluorescin:- yellow pigment
C) Pyorubin:- red-brown pigment

Type Species: Pseudomonas aeruginosa
Pseudomonas aeruginosa is a versatile 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
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-aeruginos

Material & Methods
Isolation
Gram Staining
Biochemical tests

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 http://sciencefair.math.iit.edu/techniques/SerialDilution /

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

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

Growth on Agar Slants
Showing Fluorescence

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

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

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

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

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

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 colour change.

Pseudomonas

by Sandeep Sandhu on Feb 06, 2010

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