Pseudomonas aeruginosa: Isolation and identification

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Pseudomonas aeruginosa: Isolation and identification

Pseudomonas aeruginosa is a gram-negative, motile rod belonging to the family Pseudomonadaceae. These bacteria are commonly found in soil and water. Pseudomonas aeruginosa can resist variety of physical conditions such as dyes, weak antiseptics, commonly used antibiotics and tolerate high salt concentration. Pseudomonas aeruginosa has minimal nutritional and can grow in distilled water. Most pseudomonads are opportunistic pathogen known to cause disease in humans. Pseudomonas aeruginosa and Pseudomonas maltophila are mostly responsible for disease conditions. Pseudomonas aeruginosa produces a number of water-soluble pigments, including the yellow-green or yellow-brown fluorescent pigment pyoverdin (fluorescein).When pyoverdin combines with the blue water-soluble pigment pyocyanin, the bright green color characteristic of Pseudomonas aeruginosa is created.

Characteristics of Pseudomonas aeruginosa
• Motile (by single or multiple polar flagella)
• gram-negative rods
• Obligate (strict) aerobes (most strains)
• Oxidase (usually) and catalase positive
• Nonfermentative
• chemoheterotrophic
• Many organic compounds used as C and N sources, but only a few carbohydrates by oxidative metabolism
• Glucose used oxidatively
• On Nutrient agar→ Colonies are surrounded by bluish-green coloration
• hemolytic colonies are observed on blood agar
• On MacConkey agar- pale yellow colonies i.e. non-lactose fermenters
• On selective media “Cetermide” → pigments are more obvious
• Pseudomonas aeruginosa able to grow at temperatures as high 42 degrees.

Application to Biotechnology
P. aeruginosa, as well as many other Pseudomonas spp., has the ability to degrade aromatic hydrocarbons such as methylbenzenes. Methylbenzenes, which are the by-products of petroleum industries are considered as an environmental pollutant that are present in the atmosphere, soils, and in surface water. Methylbenzenes are used as solvents for enamels and paints as well as in the production of drugs and chemicals. P. aeruginosa can break down toluene, the simplest form of methylbenzene through the oxidation of the methyl group to aldehyde, alcohol, and an acid, which is then converted to catechol. Hence, P. aeruginosa can be used in pollution control
Identification and Isolation of P. aeruginosa

Cetrimide Agar

Pseudosel Agar or Cetrimide Agar is used for the selective isolation of Pseudomonas aeruginosa from clinical and non-clinical specimens. The medium was first developed by Lowburry and is a modification of Tech Agar. Cetrimide is a cationic detergent which selectively inhibits the growth of variety of microorganisms including Pseudomonas species other than Pseudomonas aeruginosa. Cetrimide, when comes in contact with bacterial cell, causes the release of nitrogen and phosphorous which denatures membrane proteins of bacterial cell

Composition of the Cetrimide Agar media
Formula / Liter
Enzymatic Digest of Gelatin                                    20 g
Magnesium Chloride                                              1.4 g
Potassium Chloride                                                 10 g
Cetrimide (Cetyltrimethylammonium Bromide)      0.3 g
Glycerol                                                                   10 mL
Agar                                                                         13.6 g
Final pH: 7.2 ± 0.2 at 250C

• Glycerol acts as the source of carbon
• Enzymatic Digest of Gelatin supplies essential nutrients for P. aeruginosa such as nitrogen, vitamins, and carbon.
• Magnesium chloride and Potassium chloride stimulates the production of pyocyanin and fluorescein.
• Agar is the solidifying agent.
• Cetrimide is the selective agent. It is a toxic substance that inhibits the growth of many bacteria. Cetrimide enhances the production of both pyocyanin and fluorescein pigment.

Procedure of preparation of media
1. Suspend 45.3 g of the medium and 10 ml of glycerol in 1000ml of distilled water.
2. Boil for one minute to completely dissolve the medium.
3. Autoclave the medium at 121°C for 15 minutes.
4. Cool the medium and pour into sterile Petri plates.

Expected Results
1. Positive: Presence of growth
2. Negative: No growth

Typical colony characteristics
After 18 – 48 h of incubation, colonies may be identified as of P. aeruginosa when it exhibits a blue-green to green pigment and fluoresce under short wavelength (254 nm) UV light.

Uses of Cetrimide Agar
• Cetramide agar is used for the selective isolation and identification of P. aeruginosa from clinical and nonclinical samples.
• It is also used for determining the ability of an organism to produce pigments (fluorescein and pyocyanin)

Storage and shelf life of Cetrimide agar
• store plated media away from direct light at 2-8ºC.
• Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed.
• Product is temperature sensitive; protect from light, excessive heat, moisture, and freezing.

Limitation of Cetrimide Agar
• Some enterics show a slight yellowing of the medium; however, this coloration is easily distinguished from fluorescein production because this yellowing does not fluoresce.
• Some non-fermenters and some aerobic spores formers may exhibit a water-soluble tan to brown pigmentation on this medium. Serratia strains may exhibit a pink pigmentation.
• Studies of Lowbury and Collins showed P. aeruginosa can lose its fluorescence under UV if the cultures are left at room temperature for a short time. Fluorescence reappears when plates are re-incubated.
• Further testing is recommended for complete identification of P. aeruginosa.

By |2018-04-09T12:05:39+00:00April 9th, 2018|Fermentation, Food Microbiology|Comments Off on Pseudomonas aeruginosa: Isolation and identification

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