1. APPARATUS
1.1. Autoclave
1.2. BOD - Incubator
1.3. Bacteriological Incubator
1.4. pH – meter
1.5. Hot air Oven
1.6. Laminar Air flow
1.7. Analytical Balance
1.8. Spirit Lamp / Bunsen Burner
1.9. Micropipette
1.10. Petri dishes
1.11. Test tubes
1.12. Media bottles
2. REAGENTS AND MEDIAS
2.1 1 % w/v solution of N,N N’N’-Tetramethyl-p-phenylene diammonium dichloride
(Reagent A)
2.2 Cetrimide Agar
2.3 Prepare media as per the current version of Respective GTP.
If the dehydrated medium is not available then prepare the medium as follows.
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 min. with shaking. Adjust the pH so that after sterilization it is 7.0 to 7.4. Sterilize by heating in an autoclave at 121–124 OC for about 30 min.
2.4 Pseudomonas Agar Medium for detection of Fluorescin
2.5 Prepare media as per the current version of Respective GTP.
If the dehydrated medium is not available then prepare the medium as follows
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
Dissolve the solid components in the water before adding the Glycerin. Heat, with frequent agitation and boil for 1 minute to effect solution.
Heat to boiling for 1 min. with shaking. Adjust the pH so that after sterilization it is 7.2 ± 0.2. Sterilize by heating in an autoclave at 121–124O C for about 30 min.
2.6 Pseudomonas Agar Medium for detection of Pyocyanin
2.7 Prepare media as per the current version of Respective GTP.
If the dehydrated medium is not available then prepare the medium as follows.
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
Dissolve the solid components in the water before adding the glycerin. Heat, with frequent agitation, and boil for 1 minute to effect solution.
Heat to boiling for 1 min. with shaking. Adjust the pH so that after sterilization it is 7.2 ± 0.2. Sterilize by heating in an autoclave at 121–124O C for about 30 min.
2.8 Soyabean Casein Digest Medium
2.9 Prepare media as per the current version of Respective GTP.
If the dehydrated medium is not available then prepare the medium as follows.
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 add, if necessary, sufficient 0.1 N Sodium Hydroxide to give a final pH after Sterilization between 7.1 and 7.5. Filter, if necessary, to clarify, distribute into suitable containers and sterilize in an autoclave at 121–124O C for about 30 min.
All the above media should be incubated for 48 h at 32.50C?2.50C before use. Any contaminated media should be discarded.
3. PROCEDURE
3.1. PRE-TREATMENT OF THE PREPARATION BEING EXAMINED
3.1.1. Water - Soluble Products :
3.1.1.1. Dissolve or dilute 10 g or 10 ml of the preparation being examined, unless otherwise prescribed, in Soyabean Casein digest medium 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
3.1.1.2. If necessary, adjust the pH to about 7.0 ± 0.2.
3.2. Enrichment
3.2.1. 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 300 C to 350C for 18 to 48 hours.
3.2.2. Examine the medium for growth, and if growth is present, mix by gently shaking.
3.3. Primary Test
3.3.1. 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.
3.3.2. Cover and invert the dishes and incubate at 300C to 350C for 18 to 72 hours.
Grams stain the suspected colony as per Respective GTP.
3.3.3. 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.’
3.3.4. 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
Characteristic Colonial Morphology
Gram stain
Generally greenish
Negative rods
3.4. Secondary test
3.4.1. Transfer some material of morphologically different , isolated colonies to Soyabean casein digest medium and incubate at 410 to 430C for 18 to 24 hours.
3.4.2. The product passes the test if no growth occurs.
3.5. Note :
3.5.1. 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.
3.5.2. The test is invalid if the results do not indicate that the control contains Pseudomonas aeruginosa.
autoclave, bod - incubator, bacteriological incubator, general testing procedure
