SOP for environment monitoring by Drain point monitoring in pharmaceutical

1.0 Purpose:

To lay down the procedure for drain point monitoring.

2.0 Scope:

This procedure is applicable for sampling and monitoring of drain point.

3.0 Responsibility:

Microbiologist / Designee:
• To prepare the swab sampling sticks as per procedure.

• To perform sampling of drain point as per procedure.

• To perform testing of swab sample as per testing procedure.

• To observe the plate as per incubation procedure.

Head/ Designee - QC :
• To review, implement and ensure compliance of SOP.

Head/ Designee - QA :
• To approve, implement and ensure compliance of SOP.

4.0 Procedure:

4.1 Carryout the microbiological testing of the drain as per procedure mentioned below.

4.2 Prepare the swab sticks with 10 ml of BSCP or 0.9% saline and sterilize it as per SOP of Media preparation and it's sterilization. After sterilization mark the swab sticks with
location point no. and date of sampling.

4.3 Take the swabs at the respective area and withdraw the sample from critical point of drain.

4.4 Vortex the tubes along with swab and use the suspension for further analysis.

4.5 Total Aerobic Bacterial Count by Pour Plate Method:

4.5.1 This method is to be used for estimation of total aerobic bacterial counts.

4.5.2 Inoculate 1ml of sample in duplicate, in pre-sterilized petriplates.

4.5.3 Pour pre-sterilized soyabean casein digest agar (about 40–45°C) into these plates.
Swirl gently to mix uniformly. Keep inoculated and uninoculated medium plates as positive and negative controls respectively.

4.5.4 Incubate the plates at 30-35°C for 3 to 5 days.

4.5.5 Count the number of colonies on each plate and take the average of the two plates in calculation and express the result in cfu/swab.

4.6 Total Fungal Count:

4.6.1 This method is to be used for estimation of total fungal counts.

4.6.2 Inoculate 1ml of sample in duplicate, in pre-sterilized petriplates.

4.6.3 Pour pre-sterilized sabouraud dextrose agar medium (about 40-45°C) into these
plates. Swirl gently to mix uniformly. Keep inoculated and un-inoculated medium plates as positive and negative controls respectively.

4.6.4 Incubate the plates at 20-25°C for 5 days.

4.6.5 Count the number of colonies on each plate and express average for the two plates in terms of cfu/swab.

4.6.6 Record the observation in formats.

4.7 Pathogens:

4.7.1 Testing procedure for detection of pathogens – Salmonella, E. coli, Staphylococcus aureus, and Pseudomonas aeruginosa.

4.7.2 Filter aseptically rest of sample solution through a suitable membrane filter (0.45 micron) mounted on a sterile filtration assembly.

4.7.3 Remove the membrane filter. Put into pre-sterilized 100ml soyabean casein digest medium flask.

4.7.4 Incubate the flask for 24 to 48 hours at 30-35°C.

4.7.5 Proceeds as follows if any kind of turbidity is observed in the soyabean casein digest medium flask.

4.7.6 Test for E. coli:

4.7.6.1 Inoculate aseptically the 100 ml MacConkey broth with 1.0 ml soyabean casein digest medium tube having growth.

4.7.6.2 Incubate the enrichment medium tube for 24 to 48 hours at 42-44°C in an incubator.

4.7.6.3 If turbidity is observed, streak a loop full of the broth suspension on preincubated MacConkey agar plates and incubate at 30-35°C for 18-72 hours.

4.7.6.4 No growth of colonies on MacConkey agar plate confirms the absence of E. coli where as growth of colony indicates the presence of E. coli.

4.7.6.5 Confirm the presence of E. coli with indole production test. Inoculate 10 ml of 0.1% of sterile peptone water with 1.0 ml of broth or a loopful of culture from MacConkey agar plate; incubate at 30-35° C for 48 hours followed by addition of Kovac’s reagent. A red ring formation confirms the production of indole i.e. E. coli, while no formation of such ring confirms the E. coli absence.

4.7.6.6 Note: When using any medium, positive inoculated control and negative control shall be employed to check fertility and sterility of the medium respectively.

4.7.8 Test For Staphylococcus aureus:

4.7.8.1 Streak a loop full of the enriched soyabean casein digest medium suspension on Mannitol salt agar plates and incubate at 30-35°C for 18-72 hours.

4.7.8.2 No growth of colonies confirms the absence of Staphylococcus aureus; whereas yellow or white colonies surrounded by yellow zone indicate the presence of Staphylococcus aureus. Confirm the presence with coagulase production test.

4.7.8.3 Inoculate suspected colony into 0.5 ml of mammalian plasma in test tube and incubate at 37°C for 03 to 24 hours. Formation of gel to any level confirms the presence of Staphylococcus aureus.

4.7.8.4 Note:When using any medium, positive inoculated control and negative control shall be employed to check fertility and sterility of the medium respectively.

4.7.9 Test for Pseudomonas aeruginosa:

4.7.9.1 Streak a loop full of the enriched soyabean casein digests medium suspension on Cetrimide agar plates and incubate at 30-35°C for 18-72 hours.

4.7.9.2 No growth of colonies confirms the absence of Pseudomonas aeruginosa while growth of colonies indicates the possible presence of Pseudomonas aeruginosa.

4.7.9.3 Confirm the presence with help of oxidase test. Transfer a suspected colony on oxidase disk impregnated with N-N-Dimethyl-Paraphenylene-diamine-dihydrochloride. Violet colour production in 5-10 seconds at 25-30°C confirms the presence of Pseudomonas aeruginosa. A delayed positive reaction appear in 10-60 seconds, while a reaction after 60 seconds or no change at all is considered negative reaction.

4.7.9.4 Note: When using any medium, positive inoculated control and negative control shall be employed to check fertility and sterility of the medium respectively

4.7.10 Test for Salmonella:

4.7.10.1 Inoculate the 10 ml Rappaport vassiliadis salmonella enrichment broth aseptically with 1.0 ml soyabean casein digest medium flask having growth. Incubate the enrichment medium flask for 18 to 24 hours at 30-35°C in an incubator.

4.7.10.2 If turbidity production is observed, streak a loop full of the broth suspension on Xylose Lysine Deoxycholate agar plates and incubate at 30-35°C for 18-48 hours.

4.7.10.3 The possible presence of Salmonella is indicated by growth of well developed, red colonies with or without black centers. However the absence of such kind of colonies confirms the absence of salmonella.

4.7.10.4 If Salmonella is present, confirm firstly by streaking the slant surface of Triple sugar Iron agar followed by stabbing slant, incubate for 24-48 hours. If examination discloses no evidence of tubes having alkaline (pink) slants and acids (yellow) butt (with or without concomitant blackening of the butt from hydrogen sulfide production), the specimen meets the requirement for the absence of genus Salmonella.

4.7.10.5 Note:When using any medium, positive inoculated control and negative control shall be employed to check fertility and sterility of the medium respectively.

4.7.10.6 Record the observation in drain point monitoring test report.

4.8 Acceptance Criteria:
1. Total Aerobic Bacterial Count-Not More Than 200 CFU/swab

2. Total Fungal Count-Not More Than 10 CFU/swab

3. E. coli-Should be absent

4. Salmonella-Should be absent

5. Pseudomonas-Should be absent

6. S. aureus-Should be absent

4.9 Frequency:
Once in a 3 month ±15 days.