IDENTIFICATION OF PSEUDOMONAS AERUGINOSA

OBJECTIVE
The test for Identification of Pseudomonas aeruginosa is provided to determine compliance with the requirements given in individual monograph / specifications.
PRINCIPLE
Identification of Pseudomonas aeruginosa is the identification or detection of a pathogenic bacteria which may cause infection to human body. It can be identified by using specific differential media which support only the growth of Pseudomonas aeruginosa.’
REAGENT AND MEDIA
A. 1 % w/v solution of N,NN’N’-Tetramethyl-p-phenylene diammonium dichloride (Reagent A)
B. Cetrimide Agar
Pancreatic Digest of Gelatin 20.0 g
Magnesium Chloride 1.4 g
Potassium Sulfate 10.0 g
Cetrimide 0.3 g
Agar 13.6 g
Glycerol 10.0 mL
Water 1000 mL
Heat to boiling for 1 minute with shaking. Adjust the pH 7.0 +0.2. Sterilize the media an autoclave at 121 OC for 15 min.
C. Cetrimide Broth
Prepare the broth with the addition of all the ingredients specified in Cetrimide agar except the agar.
D. Pseudomonas Agar Medium for detection of Hourescein
Pancreatic Digest of Casein 10.0 g
Peptic Digest of Animal Tissue 10.0 g
Anhydrous Dibasic Potassium Phosphate 1.5 g
Magnesium Sulfate (MgSO4. H2O) 1.5 g
Glycerin 10.0 mL
Agar 15.0 g
Water 1000 mL
pH after sterilization : 7.2 ± 0.2
Dissolve the solid components in the water before adding the Glycerin. Heat, with frequent stirring, and boil for 1 minute.
E. Pseudomonas Agar Medium for detection of Pyocyanin
Pancreatic Digest of Gelatin 20.0 g
Anhydrous Magnesium Chloride 1. 4 g
Anhydrous Potassium Sulfate 10.0 g
Agar 15.0 g
Glycerin 10.0 mL
Water 1000 mL
pH after sterilization : 7.2 ± 0.2
Dissolve the solid components in the water before adding the glycerin. Heat, with frequent stirring, and boil for 1 minute .
F. Fluid Soyabean Casein Digest Medium
Pancreatic Digest of Casein 17.0 g
Papacy Digest of Soybean Meal 3.0 g
Sodium Chloride 5.0 g
Dibasic Potassium Phosphate 2.5 g
Dextrose (C6H12O6. H2O) 2.5 g
Distilled Water 1000 mL
Final pH after Sterilization 7.3 ± 0.2
Dissolve the solids in the water, warming slightly to effect solution. Cool to room temperature and adjust the pH with 0.1 N Sodium Hydroxide or phosphoric acid and . Filter, if necessary. Distribute the media into suitable containers and sterilize in an autoclave at 121 OC for about 15 min.
Incubate all the above media for 24 h at 37 OC before use to check any external contamination.
Prepare media by using different ingredients as above or use readymade dehydrated media of Hi media / Difco make. Rehydrate the required quantity as per instructions on the bottle label and sterilize at 15 psi at 121 OC for 15 min.
PRE-TREATMENT OF THE PREPARATION BEING EXAMINED
Water-Soluble Products :
1. Dissolve or dilute 10 g or 10 mL of the preparation being examined, unless otherwise prescribed, in Lactose broth or another suitable medium shown not to have anti-microbial activity under the conditions of the test and adjust the volume to 100 mL with the same medium.
2. If necessary, adjust the pH to about 7.0
Non-Fatty Products Insoluble in Water :
1. Suspend 10 g or 10 mL of the preparation being examined, unless otherwise prescribed, in Lactose or another suitable medium shown not to have anti-microbial activity under the conditions of the test and dilute to 100 mL with the same medium.
2. If necessary divide the preparation being examined and homogenize the suspension mechanically.
3. A suitable surface-active agent such as 0.1 % Polysorbate 80 may be added to assist the suspension of poorly wettable substance.
4. If necessary, adjust the pH of the suspension to about 7.0
Fatty Products :
1. Homogenize 10 g or 10 mL of the preparation being examined, unless otherwise prescribed with 5 g of Polysorbate 20 or Polysorbate 80.
2. If necessary, heat to not more than 40 OC . Mix carefully while maintaining the temperature in a water bath or in an oven.
3. Add 85 mL of Lactose broth or another suitable medium shown not to have anti-microbial activity in the conditions of the test, heated to not more than 40 OC, if necessary.
4. Maintain this temperature for the shortest time necessary for formation of an emulsion and in any case for not more than 30 min.
5. If necessary, adjust the pH of the emulsion to about 7.0
For a Fluid Specimen in Aerosol Form :
1. Chill the container in an Alcohol-dry Ice mixture for approx. 1 h, cut open the container, allow it to reach room temperature, permit the propellant to escape, or warm to drive off the propellant if feasible and transfer the quantity of test material required for the procedures specified in one of the two proceeding paragraphs, as appropriate.
2. Where 10 g or 10 mL of the specimen, whichever is applicable,. cannot be obtained from 10 containers in aerosol form, transfer the entire contents from 10 chilled containers to the culture medium, permit the propellant to escape, and proceed with the test on the residues.
As per IP
Primary Test
1. Place the prescribed quantity in a sterile screw-capped jar containing 100 mL of Fluid Soyabean Casein Digest Medium and incubate at 35 - 37 OC for 18 - 24 h.
2. Subculture on a plate containing a layer of Cetrimide agar and incubate at 35 -37 OC for 18 - 24 h.
3. Examine the resulting growth by Gram’s stain and apply the oxidase test; Gram-negative bacilli giving oxidase test indicate the presence of Pseudomonas.
Confirmatory Tests
Oxidase Test: Place 2 to 3 drops of a freshly prepared 1 % w/v solution of reagent A on a piece of filter paper (Whatman No.1 is suitable) and smear with the suspect colony. if a purple color is produced within 5 to 10 seconds, the test is positive.
Pigments Test: please refer Page 7 of this SOP from point1 - 5 for pigment test.
As per BP
Primary Test
1. Pre - treat the preparation being examined as described above but using buffered Sodium Chloride - Peptone solution pH 7.0, or another suitable medium shown not to have anti-microbial activity under the conditions of the test, in place of Lactose Broth.
2. Inoculate 100 mL of Casein soybean digest broth with a quantity of the solution, suspension or emulsion thus obtained containing 1 g or 1 mL of the preparation being examined.
3. Mix and incubate at 35 - 37 OC for 24 - 48 h.
4. Subculture on a plate of Cetrimide agar and incubate at 35 - 37 OC for 24 - 48 h.
5. If no growth of micro-organisms is detected, the preparation being examined passes the test.
Confirmatory Test
1. If growth of colonies of Gram-negative rods, usually with a greenish fluorescence occurs, apply an oxidase test and test the growth in casein soybean digest broth at 42 OC.
2. Place 2 or 3 drops of a freshly prepared 1 % w/v solution of Reagent A on filter paper (Whatman No. 1 is suitable) and smear with the suspect colony; if a purple color is produced within 5 - 10 seconds., the test is positive.
3. The preparation being examined passes the test if cultures of the type described do not appear or if the confirmatory biochemical test is negative.
As per USP
Primary Test
1. To the specimen add Fluid Soybean-Casein digest medium to make 100 mL, mix, and incubate.
2. Examine the medium for growth, and if growth is present, use an inoculating loop to streak a portion of the medium on the surface of cetrimide agar plated on petridishes. Cover and invert the dishes, and incubate.
3. If upon examination, none of the plates contains colonies having the characteristics listed in Table for the media used, the test specimen meets the requirements for freedom from Pseudomonas aeruginosa.’
Confirmatory Test
Oxidase and Pigment Tests :
1. With the aid of an inoculating loop, streak representative suspect colonies from the agar surface of Cetrimide agar medium on the agar surfaces of Pseudomonas agar medium for detection of Fluorescent and pseudomonas agar medium for detection of pyocyanin contained in petridishes.
2. If numerous colonies are to be transferred, divide the surface of each plate into quadrants, each of which may be inoculated from a separate colony.
3. Cover and invert the inoculator media, and incubate at 35 ±2 OC for not less than 3 days.
4. Examine the streaked surface under ultraviolet light.
5. Examine the plates to determine whether colonies having the characteristics, listed in Table are present.
6. Confirm any suspect colonial growth n one or more of the media as pseudomonas aeruginosa by means of the oxidase test.
7. Upon the colonial growth place or transfer colonies to strips or disks of filter paper that previously has been impregnated with reagent A; if there is no development of a pink color, changing to purple, the specimen meets the requirements of the test for absence of pseudomonas aeruginosa.
8. The presence of pseudomonas aeruginosa maybe confirmed by other suitable cultural and biochemical tests, if necessary.
Morphologic Characteristics of Pseudomonas aeruginosa on Selective And Diagnostic Agar Media

NOTE:
1. Carry out a control test by repeating the test adding the prescribed quantity and a volume of broth containing 10 to 50 pseudomonas aeruginosa (NCTC 6750), prepared from a 24 h. culture in Nutrient broth, to a sterile, screw-capped jar containing 100 ml of Cetrimide broth or Fluid Soyabean Casein Digest Medium.
2. The test is invalid if the results do not indicate that the control contains Pseudomonas.