procedure for environmental monitoring of bio load in controlled areas by using settle plates air sampling and contact plate

1.0    Purpose:               

To lay down the procedure for environmental monitoring of bio-load in controlled areas by using settle plates, air sampling and contact plate.

2.0   Scope:

This procedure applicable to controlled areas like pharma areas at different locations where crystallization, centrifuging, vacuum drying, sifting, blending, micronisation, compaction and packing of finished product are carried out and Microbiology testing area.

3.0   Responsibility:           

The responsibility of performing environmental monitoring in controlled areas lies with Microbiologist/Analyst.

4.0  Definitions / Abbreviations:

4.1Pharma area / Common packing area: Area to isolate and handle finished products where air-handling system are provided to prevent cross contamination           

4.2Microbiology testing area: Area to isolate and handle the testing of water samples/ finished products/positive cultures where air handling systems are provided to prevent contamination.

4.3Pre-incubation: Plates prepared before 18 to 48 hours of use and incubated.

5.0  Procedure:  

5.1Environmental monitoring by Settle Plates:

5.1.1Prepare and sterilize Soyabean-Casein Digest agar as per requirement and pour 15-20 mL in  sterile 90 mm petri dishes aseptically  and incubate for 18 to 48 hours at 30-35°C for settle plates and air sampling. Decontaminate the pre-incubated Soya bean-Casein Digest agar plates by spraying with 0.2 µ filtered 70% Isopropyl alcohol (IPA) and duly labeled indicating the location, keep them in SS box / SS can and close the box / can with the cover.

5.1.2Pharma area/Common packing area: Transfer SS can into the pharma areas/common packing area and expose one petri plate at each location near the return air duct for two hour as per schematic layout of sampling points in pharma area for environmental monitoring as shown in Annexure – 1,2,3,4 & 6, by opening the lid of each plate and allow to expose for two hour. After the stipulated period of exposure close the lid and keep the plate in SS can. Repeat the same procedure at different locations in the area.  Follow this procedure in uni-direction.     

5.1.3Microbiology testing area: Transfer SS tray containing Soyabean-Casein Digest agar plate to microbiology testing area by UV passbox (UV exposure time should be minimum 5 minutes) and expose them for two hour as per the schematic layout of sampling points in microbiology testing area for environmental monitoring as             shown in Annexure-5, by removing the lid of each plate and keep the lid aside.  Follow this procedure in uni-direction i.e. from LAF bench, testing room, change room and buffer room.

5.1.4At the same time keep one plate without exposure as negative control for pharma area and microbiology testing area and simultaneously check the media GPT (Positive control).

5.1.5The exposed plates along with negative control shall be shifted to microbiology lab by closing in a SS box/SS can and incubate them in inverted position for first 72 hours(3 days) at 20-25°C and remaining 48 hours(2 days) at 30-35°C.

5.1.6Following the incubation, examine the plates for growth and count the number of Colony Forming Unit (CFU) and record the results in their respective formats and report the results.

5.1.7Acceptance Criteria:

5.2Environmental monitoring by Air Sampling:

5.2.1Autoclave the S.S cone / Sampling header of air sampler at 15 lbs / 121° C for 30 minutes.

5.2.2Decontaminate the air samplers by spraying with 0.2 µ filtered 70% IPA and transfer into sampling location.

5.2.3Transfer the autoclaved S.S cone / Sampling header to the location of air sampling and unwrap the parchment / butter paper and place S.S cone / Sampling header inside the air sampler.

5.2.4Place the agar plate in position without lid and place the S.S feeder cone circular assembly/ Sampling header in position.

5.2.5Pharma area/Common packing area: Transfer SS can into the pharma areas/common packing area and set the air sampler to pass air of 1000 liters per 10 minutes over pre-incubated Soya-bean-Casein Digest agar plates,   inserted into the air sampler and sample the air at specified location as per the sampling points in Pharma area for environmental monitoring as shown in Annexure – 1, 2, 3, 4 & 6. Spray 70% IPA solution on sampling header before next sampling points and wait for evaporation of IPA.

5.2.6Microbiology testing area: Transfer SS tray containing Soyabean-Casein Digest agar plate to microbiology testing area by UV passbox (UV exposure time should be minimum 5 minutes). Place the agar plate in position without lid and place the S.S feeder cone circular assembly/ Sampling header in position. Set the air sampler to pass the air of 1000 liters per 10 minutes over pre-incubated Soya-bean-Casein Digest agar plates, inserted into the air sampler and sample the air at specified location as per the schematic layout of  sampling points in microbiology testing area for environmental monitoring as shown in Annexure – 5. Spray 70% IPA solution on sampling header before next sampling points and wait for evaporation of IPA

5.2.7After sampling, remove the petri plate, cover with lid and put it in a SS tray (microbiology testing area) / SS can (pharma area). Repeat the same procedure at different locations.

5.2.8At the same time keep one plate with out exposure as negative control and simultaneously check the media GPT (Positive control).

5.2.9The sampled plates along with negative control shall be shifted to microbiology lab by closing in a SS can/SS tray and incubate them in inverted position for first 72 hours (3 days) at 20-25°C and remaining 48 hours (2 days) at 30-35°C.

5.2.10Record the results with positive hole conversion table in their respective formats.

Pr = N [1/N + 1/N-1 + 1/N-2 + 1/n-r+1]

Pr = Potable statistical total                N = Number of holes

r = Number of colony forming units counted on 90mm petri dish.

5.2.11Acceptance Criteria:

5.3Environmental monitoring by Contact Plate :

5.3.1Use Rodac plate or prepare required amount of Soyabean Casein Digest Agar as per manufacturer instruction and pour approx. 15-20 ml of sterilize Soyabean Casein Digest agar into 55 mm diameter sterile plates.

5.3.2Incubate plates at 30 to 35°C for not less than 18 to 48 hours.

5.3.3Decontaminate the pre-incubated agar plates by spraying with 0.2 µ filtered 70%IPA and duly

labeled indicating the location, keep them in SS box / SS can and    close the box / can with the cover.

5.3.4Pharma area/Common packing area: Transfer SS can / box into the Pharma areas/common packing area and touch the media portion onto the surface of the equipment  in each pharma area room, If pharma room contains no equipment wall / floor will be considered as of sampling points in Pharma area for environmental monitoring as shown in Annexure – 1,2,3 4 & 6.

5.3.5Microbiology testing area: Transfer SS can / box contains Soyabean-Casein Digest agar plate to microbiology testing area by UV passbox (UV exposure time should be minimum 5 minutes) and touch the media portion to the surface of sampling point as per schematic layout of sampling points in microbiology testing area for environmental monitoring as shown in Annexure – 5. Follow this procedure in uni-direction i.e. from testing room, LAF bench, change room and buffer room.

5.3.6Wipe the sampling location with 70% IPA after sampling.

5.3.7The contact plates along with negative control shall be shifted to microbiology lab by closing in a SS box/SS can and incubate them in inverted position for first 72 hours(3 days) at 20-25°C and remaining 48 hours(2 days) at 30-35°C and simultaneously check the media GPT(Positive control).

5.3.8Acceptance Criteria:

5.4Frequency:

5.4.1Pharma area / Common packing area: Settle plates, air sampling, and contact plate shall be performed once in 3 months ± 7 days.

5.4.2Microbiology testing area: Settle plate and air sampling shall be performed monthly and contact plate shall be performed once in 3 months ± 7 days.

5.5Trend charts for environmental monitoring data shall be prepared annually.

5.6Procedure in case of non – compliance:

5.6.1Pharma Area / Common packing area: In case microbial levels exceed the alert limit inform production to improve the cleaning. Monitor till compliance.

5.6.2In case microbial level exceeds the action limit stop the activity immediately take corrective action. Monitor till compliance.

5.6.3In case microbial levels exceed the standard limit stop the activity immediately and inform to QCD Head Monitor till compliance.

5.6.4Microbiology testing area: If the counts are beyond the limits clean the area as per cleaning procedure.

5.6.5Fumigate the area with double the quantity of Stericare/Virosil/Hydrogen peroxide.

5.6.6Personnel hygiene of the people working shall be checked.

5.6.7AHU condition and physical monitoring of the area shall be checked.

5.6.8Monitor the plate counts for three consecutive days. If the CFU are found within the limits microbiology testing shall be initiated. Repeat the step 5.6.1 to 5.6.7 if the CFU are not found with in the limits.

6.0Related Documents:

                Procedure for operation, calibration and maintenance of air sampler.

Settle plates, contact plate, environmental, procedure, microbiology