Follow
Objective This procedure describes the process for routine environmental monitoring of clean room and other microbiologically controlled environments. Environmental monitoring is intended to provide information to ascertain that a controlled environment is operating within an adequate state of control.
Scope: This SOP is applicable for routine monitoring of Production area, Microbiology Laboratory, Sampling area, Sampling booth and Dispensing booth , Laminar air flow bench of (Company Name).
Responsibility
1. QC officer/ Microbiologist is responsible for carrying out the procedure.
2. Sr. Officer / Assistant Manager shall be responsible to ensure proper and timely execution of procedure.
3. Overall responsibility for training, implementation & follow-up with the QC Manager or his nominee.
Precautions: Wear hand gloves and then rinse the hands with 70%IPA before starting environmental monitoring activities
Procedure
1 The environmental monitoring shall be carried out as per the frequency mentioned in section 6.10 by active air sampling / settle plate. The locations of active air sampling/ settle plate are identified in the drawing no.: Track-II/QC-(01-10) & also mentioned in Annexure-VI & VII.
2 The number of plates to be exposed per location is stated in the Annexure-VI (for production floor) and Annexure VII (for Microbiology Laboratory). The number of plate (i.e. number of sampling) is calculated on the basis of area of the location (One plate per 10sq.m).
3 Expose the plate on the basis of criticality of the points and at a uniform distance to cover the location and when a single plate is used, place it at the centre of the location.
4 For the sampling booth and dispensing booth, place the plate at the centre of the booth.
5 Settle Plate
5.1 Prepare, sterilize and dispense the media into the Petri plates and pre-incubate the plates as per SOP: Preparation of microbiological media (SOP No. QC/101)
5.2 Aseptically check the pre-incubated plates for any evidence of microbiological contamination under LAF bench.
5.3 Discard the plates having microbiological growth.
5.4 Decontaminate the external surface of the Petri plates with the help of a sterile cloth / cotton soaked in a sanitizing agent (70 % IPA).
5.5 Place the required number of Petri plates in a sterilized / sanitized container; close the lid of the container.
5.6 Transfer the container to the area to be monitored.
5.7 Enter into the respective area as per the SOP of Entry and Exit procedure.
5.8 Mark the Petri plates with the following details,
• Location Number or Name of the location
• Plate No.
• Date of exposure
5.9 Place the Petri plates on the corresponding designated plate exposure area and remove the upper lid of the Petri plates. Note the beginning time of the exposure and write it on the plate.
5.10 Place the upper lid on edge of the Petri plates in a slanting position.
5.11 Expose the media plates at the locations for 4 hours.
5.12 After completion of 4 hours of exposure, close the Petri plates with lid.
5.13 Collect all Petri plates in the sanitized container and bring back the exposed plates to the Laboratory.
5.14 Incubate one plate at 30-35° C for 3 days for bacteria and another at 20 – 25°C for Yeasts and Molds for 5 days.
5.15 Incubate an unexposed media filled Petri plate along with the exposed plates and mark it as negative control and record the results of negative control in Annexure-I.
5.16 After incubation, count the number of Colony Forming Units (CFU) per plate per location with the help of colony counter.
5.17 Note down the observation in the format attached as Annexure –I.
5.18 Identification of pathogens is carried out during environmental monitoring of manufacturing area (Air sample or Settle plate sample), additionally in case there is any evidence of pathogenic growth in any environmental monitoring sample (Daily, weekly, monthly etc), then identification test of specific pathogens will be carried out.
5.19 For identification of pathogens inoculate the loop full suspected colony by sterile inoculating loop from the environmental monitoring plates in a bottle containing 100 ml of tryptic soy broth. Incubate the bottle at 30-35°C for 18 to 24 hours.
5.20 After incubation observe the bottle for growth and if growth is observed identify the concern pathogens by following the pathogen identification procedure described in GTP/007.
5.21 If the counts obtained are above the limits specified below, immediately inform it to QC manager to investigate the results and take necessary actions.
5.22 Environmental monitoring
5.22.1 If the counts are equal to or more than alert limit
5.22.1.1 Observe the next day’s plates for same locations/areas.
5.22.1.2 If the counts are satisfactory for next day, investigate for the probable cause of high count.
5.22.1.3 Check for any unauthorized entry in the area or any major maintenance work.
5.22.1.4 Check cleaning and disinfection record.
5.22.1.5 Check the temperature / humidity records and the differential pressure record.
5.22.1.6 Observe the general behaviour of operators in the core working area.
5.22.1.7 In case no improvement of the count observed, inform the production manager & ask to clean the area thoroughly after day’s work.
5.22.1.8 Monitor the environment & check the monitoring results for any improvement.
5.22.1.9 If improvement observed, the work will continue as usual.
5.22.2 If the counts are more than action limit
5.22.2.1 Carry out the investigation as above; inform the situation to production manager.
5.22.2.2 Disinfect the area thoroughly at the end of the day’s work.
5.22.2.3 Monitor the environment & check the monitoring results.
5.22.2.4 Check all the records pertaining to the sterilization of media and personnel in the production area.
5.22.2.5 Check the record for proper preparation and rotation of the disinfectants.
5.22.2.6 If improvement observed then, allow the work as usual.
5.22.2.7 In case no improvement of the count observed, then inform the situation to production manager & ask to thoroughly clean the area again & then monitor the environment till the situation improves.
5.22.2.8 Train the operators & staff.
5.22.2.9 Review the validation of HEPA filters.
5.22.2.10 If improvement observed, allow the work as usual.
5.22.3 Out of specification of environmental monitoring results (Out of Acceptance limit)
5.22.3.1 In case the environmental monitoring result is out of specification, inform the situation to production manager & immediately stop the production.
5.22.3.2 Investigate the situation (e.g. carry out personnel monitoring, check for any unauthorized staff entry in the area, cheek the SOP deviation, review the integrity of HEPA filters etc).
5.22.3.3 Ask the production manager to clean the area two times daily (Once in the morning & once in the afternoon).
5.22.3.4 Carry out the environmental monitoring.
5.22.3.5 If the situation does not improve continue the above steps till the situation improves.
5.22.3.6 If the situation improves then continue work as usual.
6 Active Air sampling
6.1 Carry out -the same procedure as described in 6.5.1 to 6.5.8
6.2 Decontaminate the internal and external surface of the head of air sampler wiping with sanitizing agent (i.e. 70% IPA) before & after every sampling.
6.3 Place a single Petri plate on each head of the air sampler.
6.4 Set the instrument for the desired sampling time and flow rate as per the SOP: Operation of Air sampler (SOP No.: QC126 & SOP No.:QC170).
6.5 Take the air sampler to the location where the air is to be sampled, hold it with the plate facing to the required direction at a height of 1 meter from the floor (height of the air sampler head) as per Annexure-XI and take air sample following the Operation of Air sampler (SOP No.: QC126 & SOP No.: QC170).
6.6 Collect the Petri plates in the sanitized container and bring back the exposed plates to the Lab.
6.7 For dual head, Incubate all exposed TSA Petri plates in inverted position in the laboratory incubator at 30- 35°C for bacteria for 3 days and all exposed SDA Petri plates in inverted position in the laboratory incubator at 20- 25°C for Yeasts and Molds for 5 days.
6.8 In case of single head air sampler, incubate all exposed TSA petriplates in inverted position in the laboratory incubator at 20- 25°C for Yeasts and Molds for 3 days followed by another 2 days at 30- 35°C for bacteria.
6.9 Incubate an unexposed media filled Petri plate along with the exposed plates and mark it as negative control and record the results of negative control in Annexure-I.
6.10 After incubation, count the number of Colony Forming Units (CFU) per plate per location with the help of colony counter.
6.11 Note down the observation in the format attached as Annexure –I.
6.12 Calculate the number of organisms per cubic meter of air.
6.13 Identification of pathogens will be carried out during environmental monitoring of manufacturing area (Air sample or Settle plate sample), additionally in case there is any evidence of pathogenic growth in any environmental monitoring sample (Daily, weekly, monthly etc), then identification test of specific pathogens will be carried out.
6.14 For identification of pathogens inoculate the loop full suspected colony by sterile inoculating loop from the environmental monitoring plates in a bottle containing 100 ml of tryptic soy broth. Incubate the bottle at 30-35°C for 18 to 24 hours.
6.15 After incubation observe the bottle for growth and if growth is observed identify the concern pathogens by following the pathogen identification procedure described in GTP/007.
6.16 If the counts obtained are above the limits specified below, immediately inform it to QC manager to investigate the results and take necessary actions.
6.17 Carry out -the same procedure as described in 6.5.22 to 6.5.22.3.6
7 Surface monitoring (By swab sampling)
7.1 Use pre sterilized swab or sterilize the swab in an autoclave.
7.2 Place the required no. of sterilized swab sticks with tube containing 10 ml of sterile saline solution in a Sterilized/Sanitized container and tightly secure the lid of the container.
7.3 Transfer the container to the area to be monitored.
7.4 Enter the respective area as per the SOP: Entry and exit procedure.
7.5 Select the surface of the location (floor or wall) or the equipment based on maximum possibility of contamination (worst case). Refer to Annexure – XII & XIII for sampling from the surface.
7.6 Sampling point on the surface of the wall should be selected at a height of around 1 meter from the floor (working height). The sampling point on the floor should be selected from a position where the exposure of human or product is likely to high.
7.7 Remove the swab sticks with tubes from the container and take it to the location to be monitored and Mark the tube with the following details:
• Location number, equipment no or name of the location
• Swab No.
• Date of sampling
7.8 Hold the swab stick from the bottom, squeeze the tip against inner surface of the tube to remove excess solvent.
7.9 Use one side of the moistened swab to wipe about 5 5 cm2 area with 10 firm horizontal strokes.
7.10 At the end of each stroke, lift the swab carefully.
7.11 Turn the swab over to its other side; wipe the same area with 10 firm vertical strokes.
5 cm 5 cm
5 cm 5 cm
7.12 Then deep the swab into the tube and tighten the cap, bring it back to the laboratory.
7.13 Gently vortex the swab sampling tube containing swab sticks and pours the contents in the filter holder funnel and filter it. Use the filtration system as per SOP: operation of filtration system (SOP No: 125)
7.14 Rinse the tube with 3 x 10 ml of sterile saline solution.
7.15 Filter the test sample under partial vacuum.
7.16 When the sample is filtered rinse funnel by filtering three 20-30 ml portions of sterile purified water. This flushes residue from the walls of the funnel and helps to secure a uniform distribution of colonies on the filter surface.
7.17 Upon completion of final rinse and filtration process, shut off the vacuum.
7.18 Immediately transfer the membrane filter with sterile smooth-tip forceps on to Microbial content test agar/soybean casein digest agar with lecithin and polysorbate 80 plates.
7.19 Place the filter with a rolling motion to avoid entrapment of air.
7.20 Keep a negative control by filtering 100 ml sterile purified water before filtering the actual test sample and record the results of negative control in Annexure-II for room & Annexure-III for equipments.
7.21 Pass 20 to 30 ml of sterile purified water through the funnel, between different test samples when same funnel is used for multiple samples.
7.22 Incubate all exposed Petri plates in inverted position in the laboratory Incubator at 20- 25°C for Yeasts and Molds for 3 days followed by another 2 days at 30-35°C for Bacteria.
7.23 After incubation, count the number of Colony Forming Units (CFU) per plate per location with the help of colony counter.
7.24 Note down the observation in the format attached as Annexure- II for room & Annexure-III for equipments.
7.25 For identification of pathogens of room surface deep a filter paper after filtering the sample in a bottle containing 100 ml of Tryptic Soya Broth. Incubate the bottle at 30-35°C for 18 to 24 hours.
7.26 After incubation observe the bottle for growth and if growth is observed identify the concern pathogens by following the pathogen identification procedure described in GTP/012.
7.27 Note down the observation in the format attached as Annexure II for room.
7.28 If the counts obtained are above the limits specified below investigate the result and take necessary actions.
7.29 Carry out -the same procedure as described in 6.5.22 to 6.5.22.3.6
8 Surface monitoring (By contact plate)
8.1 Do the same procedure as described in 6.5.1 to 6.5.8.
8.2 Remove the lid and press the medium surface to the smooth and flat surface of specific location of the rooms or personnel lab coat and gloves.
8.3 Ensure that entire agar surface contact the area to be sampled without producing any scratch on the media.
8.4 Cover the plate with the lid and bring back to lab keeping into the container.
8.5 After sampling, sanitize the sampled area with 70% IPA and make the surface dry with a clean cloth.
8.6 Incubate all exposed petriplates in inverted position in the laboratory incubator at 20- 25°C for Yeasts and Molds for 3 days followed by another 2days at 30-35°C for Bacteria.
8.7 Incubate an unexposed plate as negative control at the same time Incubate an unexposed plate as negative control at the same time and record the results of negative control in Annexure-IV for hand gloves, Annexure-V for personnel clothing & Annexure-II for room surface.
8.8 After incubation note down the observation in the format attached as Annexure-IV for hand gloves in Annexure-V for personnel clothing & Annexure-II for room surface.
8.9 If the counts obtained are above the limits specified below investigate the results and take necessary actions.
8.10 Carry out -the same procedure as described in 6.5.22 to 6.5.22.3.6
9 Equipment surface monitoring (By rinse sample)
9.1 Use pre-sterilized conical flask for sampling.
9.2 Place the required no. of sterilized conical flasks in a Sterilized /Sanitized container and tightly secure the lid of the container.
9.3 Transfer the SS container to the area to be monitored.
9.4 Enter into the respective area as per the SOP: Entry and exit procedure.
9.5 Clean the equipment as per the SOPs.
9.6 Remove the conical flasks from the container and take it to the location to be monitored and Mark the tube with the following details:
• Location of the equipment no or Name of the location.
• Date of sampling
9.7 Collect the rinse sample of the equipment into sterilized conical flask individually various parts as per the sampling plan.
9.8 Then tighten the cap of the conical flasks, bring it to back to laboratory.
9.9 Gently vortex the conical flasks containing rinse sample and pour the 10 ml contents in the filter holder funnel and filter it. Use the filtration system as per SOP: operation of filtration system (SOPNo:125).
9.10 Rinse the funnel with 3 x 10 ml of sterile purified water.
9.11 Filter the test sample under partial vacuum.
9.12 When the sample is filtered rinse funnel by filtering three 20-30 ml portions of sterile purified water. This flushes residue from the walls of the funnel and helps to secure a uniform distribution of colonies on the filter surface.
9.13 Upon completion of final rinse and filtration process, shut off the vacuum.
9.14 Immediately transfer the membrane filter with sterile smooth-tip forceps on to Microbial content test agar/soybean casein digest agar with lecithin and polysorbate 80 plate.
9.15 Place the filter with a rolling motion to avoid entrapment of air.
9.16 Perform duplicate control plate by filtering 10 ml purified water collecting from the purified water point location used for cleaning the equipment and keep a negative control by filtering 10 ml sterile water before filtering the actual test sample and record the results of negative control in Annexure-IX.
9.17 Pass 20 to 30 ml of sterile purified water through the funnel, between different tests samples when same funnel is used for multiple samples.
9.18 Incubate all exposed Petri plates in inverted position in the laboratory incubator at 20- 25°C for Yeasts and Molds for 3 days followed by another 2 days at 30-35°C for Bacteria.
9.19 After incubation, count the number of Colony Forming Units (CFU) per plate with the help of colony counter and deducting the number of colonies found in control plates from the counts of colonies of actual tests sample plates
9.20 Note down the observation in the format attached as Annexure- IX for the equipments.
9.21 For identification of pathogens immerge a filter paper after filtering the 100 ml sample in a bottle containing 100 ml of tryptic soy broth. Incubate the bottle at 30-35°C for 18 to 24 hours.
9.22 After incubation observe the bottle for growth and if growth is observed identify the concern pathogens by following the pathogen identification procedure described in GTP/012.
9.23 Note down the observation in the format attached as Annexure- IX for equipments.
9.24 If the counts obtained are above the limits specified below investigate the result and take necessary actions.
9.25 Carry out -the same procedure as described in 6.5.22 to 6.5.22.3.6
10 Acceptance criteria and frequency
10.1 Based on the acceptance criteria and the data obtained during the validation of facility the following alert and action limits are set for different methods and different locations:
