INTRODUCTION:
Pseudomonas aeruginosa is a Gram-negative, aerobic, rod-shaped bacterium with unipolar motility. An opportunistic human pathogen, P. aeruginosa is also an opportunistic pathogen of plants. It secretes a variety of pigments, including pyocyanin (blue-green), fluorescein (yellow-green and fluorescent, now also known as pyoverdin), and pyorubin (red-brown).
It is oftenpreliminarily identified by its pearle scent appearance and grape-like odor in vitro. Definitive clinical identification of P. aeruginosa often includes identifying the production of pyocyanin and fluorescein as well as its ability to grow at 42°C. It is capable of growth in diesel and jet fuel, where it is known as a hydrocarbon utilizing microorganism (or "HUM bug"), causing microbial corrosion. It creates dark gellish mats sometimes improperly called "algae" because of their appearance.
PREPARATION OF CULTURE MEDIA:
Sodium hydroxide 1N: Dissolve 4.0 g of sodium hydroxide in water to make 100 mL.
Hydrochloric Acid 1N: Dissolve 8.5ml of Hydrochloric Acid in water to make 100 mL.
Soyabean Casein Digest broth Medium (SCDM)/ Cetrimide Agar (CA): Reconstitute dehydrated media as directed by the manufacturer and sterilize in an autoclave at 121°C for 20 minutes.
PROCEDURE:
Sample preparation and Pre Enrichment:
Aseptically add 1g of specimen if it’s a solid or 1ml accurately measured, if the specimen is a liquid to make 100ml of Soyabean Casein Digest broth medium or transfer 10 ml (Quantity corresponding to not less than 1 g or 1 mL specimen) of sample from the Total aerobic microbial count sample preparation to make 100ml Soyabean Casein Digest broth medium unless otherwise described in the specific STP.
Negative Control: Take 100 mL of sterile Soyabean-Casein Digest broth Medium without any inoculation.
Positive Control: Add 1mL
Incubate the sample, negative control and positive control 30-35°C for 18 - 24 hours.
Examine the medium for growth.
Interpretation:
Negative control flasks should not show any growth.
Growth should be present in positive control.
If growth is present (turbidity observed) in the sample flask, or the media difficult to observe the growth with sample (Likely some powders and capsules can alter the color and the clearness of the media) mix by gentle shaking and proceed to test for Pseudomonas aeruginosa. If there is no growth in the sample flask, that indicates, sample meets the requirement for absence of Pseudomonas aeruginosa.
Note: Incase of water sample add 1 ml sample in 9 ml Soyabean Casein Digest broth medium for test and negative control with out adding anything and positive control by adding Pseudomonas aeruginosa culture in 10 ml media.
Selective Enrichment and conformation test:
Add 15 to 20 mL of Cetrimide Agar into each petriplate.
Streak the surface of Cetrimide agar medium by taking loopful of inoculum from sample.
Steak the surface of Cetrimide agar with standard culture of Pseudomonas aeruginosa.
Maintain a negative control by the omitting the sample and standard culture in respective plates.
Cover and invert the dishes and incubate at 30 to 35°C for 18 - 72 hours.
After incubating, examine the plates.
Interpretation:
Negative control should not show any colonies.
If the colonies on the sample plate do not match as per characteristics given in table-1, which indicates that the sample meets the requirements for the absence of Pseudomonas aeruginosa. If the colonies on the sample plate match to the characteristics given table-1, which indicates that the sample does not meets the requirements for the absence of Pseudomonas aeruginosa. If require perform identification test.
Table1. Morphologic Characteristic of Pseudomonas aeruginosa on
Selective Agar Medium
|
Selective Medium |
Characteristic Colonial Morphology |
|
Cetrimide Agar Medium |
Generally Greenish |
General testing procedure, pseudomonas, opportunistic pathogen
