Detection of Pseudomonas aeruginosa

1.    APPARATUS

1. Autoclave
2. BOD - Incubator
3. Bacteriological Incubator
4. pH – meter
5. Hot air Oven
6. Laminar Air flow
7. Analytical Balance
8. Spirit Lamp / Bunsen Burner
9. Micropipette
10. Petri dishes
11. Test tubes
12. Media bottles   

2.  REAGENTS   AND  MEDIAS

1.  Oxidase disc (N,N N’N’-Tetramethyl-p-phenylene diammonium dichloride)       
2. Cetrimide Agar
3. Pseudomonas Agar Medium for detection of Fluorescin 
4. Pseudomonas Agar Medium for detection of Pyocyanin
5. Soyabean Casein Digest Medium

Prepare media (2.2- 2.5) as per the current version of Respective GTP.

All the above media should be incubated for 48 h at 32.5⁰C±2.5⁰C before use.  Any contaminated media should be discarded.

1. PROCEDURE    
   1. Pre-Treatment Of The Preparation Being Examined
      1. 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 ± 0.2.
   2.  Enrichment
      1. Take 10 ml or 10 g, the quantity corresponding to 1.0g or 1.0ml (as per the specification)  of the pretreated sample in a suitable vessel, add a volume of Fluid Soyabean-Casein digest medium to make 100 ml and incubate at 32.5⁰C±2.5⁰C for 24 to 48 hours.
      2. Examine the medium for growth, and if growth is present, mix by gently shaking.
      3. By means of an inoculating loop, streak a portion from the Fluid Soyabean-Casein digest Medium on the surface of Cetrimide agar plated on petridishes.
      4. Cover and invert the dishes and incubate at 32.5⁰C ± 2.5⁰C for 24 to 48 hours.
      5. Grams stain the suspected colony as per Respective GTP.
      6. 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.’
      7. If colonies matching the description in Table –1  given below are found, proceed with further identification.

Table –1

            Morphological Characteristics of Pseudomonas aeruginosa

On Cetrimide Agar Medium

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 inoculated media, and incubate at 32.5 ±2.5 ^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 on one or more of the media as pseudomonas aeruginosa by means of the oxidase test.
7. Upon the colonial growth place or transfer colonies on oxidase disc; 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.

Table –2

Morphologic Characteristics of Pseudomonas aeruginosa on                 

Selective And Diagnostic Agar Media

1. Note :
   1.  Carry out a control test by repeating the test, adding the prescribed quantity  and a volume of broth containing 10 to 50 cells / colonies of Pseudomonas aeruginosa (ATCC), prepared from a 24 h. culture in fluid soybean-casein digest medium, to a sterile 100 ml of fluid soybean-casein digest medium.
   2. The test is invalid if the results do not indicate that the control contains Pseudomonas aeruginosa.         

Pseudomonas aeruginosa, general testing procedure, tetramethyl-p-phenylene