SOP for Monitoring of Microbiology Laboratory : Pharmaceutical Guidelines

SOP for Monitoring of Microbiology Laboratory

Standard operating procedure to monitor the microbiology laboratory for microbial load and non viable particle count.

1.0 PURPOSE

To lay down the procedure for monitoring of microbiology lab to determine nature and extent of microbiological load and non-viable particles.

2.0 SCOPE

It is applicable to the Microbiological lab.

3.0 RESPONSIBILITY

Microbiologist

4.0 ACCOUNTABILITY

Head of Department

5.0 PROCEDURE

5.1 Pre-operational checks for area monitoring

5.1.1 Two days pre-incubated plates shall be used.
5.1.2 Check the plates for any microbial contamination and physical integrity of solid media.
5.1.3 Outer surface of the plates should be sanitized with a filtered sporicidal agent.
5.1.4 Check the expiry / use before date of the media.
5.1.5 Check the cleaning status of the area where the plate has to be exposed.
5.1.6 Ensure the temperature, humidity and differential pressure of the area within the limit or not.
5.2 Precaution to be taken during area monitoring.
5.2.1 Expose and collect the plate in such a way to avoid personnel interference.
5.2.2 Open the lid in such a way that finger does not touch the surface of the media
5.2.3 Do not lean on plate while exposing and collecting the plate.
Note: Monitoring of microbiology lab is carried out by the following methods.

5.3 Passive air sampling (Settle plate method)

5.3.1 Prepare the Soyabean casein digest agar plates as per SOP for storage and preparation of microbial media. Use the prepared plates after 48 hours of pre-incubation.
5.3.2 Transfer the required number of pre-incubated Petri plate into pass box (LAL test room to Incubator).
5.3.3 Enter the respective area as per the SOP
5.3.4 Aseptically check the pre-incubated plates for any evidence of microbiological contamination.
5.3.5 Discard the plates having microbiological growth.
5.3.6 Decontaminate the external surface of the Petri plates with the help of a sterile mop soaked in a filtered sporicidal agent.
5.3.7 Mark the Petri plates at the base with the following details with a marker,
For negative control plate
For all the other plates
Media lot No
Name of the location
Date of use
Date of Sampling
Used by
Sampled by
5.3.8 Place the Petri plates on the plate exposure stands and remove the upper lid of the Petri plate and keep it in an inverted position on the resting stand. (Note: Inclined Petri plate stand in front of the riser and straight Petri plates stand at all other location if there is no stand available expose the plates on the floor at the specified location)
5.3.9 Expose the media plates at all the locations for 4 hours at the locations.
5.3.10 After 4 hours of exposure, close the Petri plate with lid.
Note: collect the plates in the same sequence in which the plates were exposed.
5.3.11 Collect all petriplates and transfer the plates into the incubator.
5.3.12 Incubate all exposed Petri plates in an inverted position in the incubator at 22.5 ± 2.5°C for 3 days and 32.5 ± 2.5°C for 2 days. (Keep each set not more than 10 plates in Stack)
5.3.13 Once in a month (preferably first week of the month) expose the separate set of plates inside the cRABS and incubate anaerobically at 32.5 ± 2.5°C for 3 days, follow the SOP for operating anaerobic jar.
Note: For anaerobic incubation the plates shall be pre-incubated under anaerobic condition for 2 days
5.3.14 After incubation observe and count the number of colonies on the colony counter or in the light source with the help of a marker.
5.3.15 Note down the observation in the format.
5.3.16 If the counts obtained are above the limits specified below investigate the results and take necessary actions as per SOP
5.3.17 Frequency of Monitoring
Class A: Once in a day whenever there is activity.
Class B: Once in a day
Class C: Once in a day
Class D: Twice in a week
Note: Whenever a sterilitytest is to be performed in a day monitoring shall be done at the same time.
5.3.18 Acceptance criteria
Alert limit:
Action limit:
Class A: 1 CFU / plate
Class A: 1 CFU / plate
Class B: 3 CFU / plate
Class B: 5 CFU / plate
Class C: 35 CFU / plate
Class C: 50 CFU / plate
Class D: 75 CFU / plate
Class D: 100 CFU / plate
5.3.19 Note: For transferring the plates into sterility testing area place the plates in pass box (Buffer room to LAL test room)

5.4 Active air sampling

5.4.1 Prepare the Soybean casein digest agar plates as per SOP “Procedure for Storage and Preparation of Microbiological Culture Media” SOP Use the prepared plates after 48 hours of pre-incubation.
5.4.2 Transfer the required number of media cassette, plate, and accessories into pass box (LAL test room to Incubator).
5.4.3 Enter the respective area as per the SOP
5.4.4 Aseptically check the pre-incubated plates for any evidence of microbiological contamination
5.4.5 Discard the plates having microbiological growth.
5.4.6 Sanitize the external surface of the media cassette/ plate with the filtered sporicidal agent.
5.4.7 Mark the petriplates at the base with the following details with a marker,
For negative control plate
For all the other plates
Media lot No
Name of the location
Date of use
Date of Sampling
Used by
Sampled by
5.4.8 Perform the air sampling as mentioned.
5.4.9 Place the media cassette / Plate on the Air sampler and operate the instrument as per SOP for air sampler.
5.4.10 Take the instrument to the locations, where the air is to be sampled, hold it in such a way plate facing the airflow at the required working height.
5.4.11 After completion of sampling collect all the plates and incubate in an inverted position in the incubator at 22.5 ± 2.5°C for 3 days and 32.5 ± 2.5°C for 2 days.
5.4.12 After incubation observe and count the number of colonies on the colony counter or in the light source with the help of a marker.
5.4.13 Note down the observation in the format.
5.4.14 If the counts obtained are above the limits specified below investigate the results and take necessary actions as per SOP.
5.4.14 Frequency of Monitoring
Class A: Once in a day
Class B: Once in a day
Class C: Once in a day
5.4.15 Acceptance criteria
Alert limit
Action limit
Class A: 1 CFU / plate
Class A: 1 CFU / plate
Class B: 7 CFU / plate
Class B: 10 CFU / plate
Class C: 75 CFU / plate
Class C: 100 CFU / plate
Note: Perform the Air sampling after completion of the activity.
5.4.16 Note: For transferring the plates into Sterility area place the plates in pass box
5.5 Study the contaminants as per SOP.
5.6 Non – Viable sampling
5.6.1 Perform the non-viable sampling.
5.6.2 Operate the sampler according to SOP
5.6.3 The sampling should be carried out as mentioned in the table.
5.6.4 Record the details of the count.
5.6.5 Perform the non-viable count at a specified location for 1 minute per run.
S.no
Room No/
Equipment ID
Class
No. of Locations
Acceptance criteria (particles per cubic feet)
Frequency
0.5 m / ft3
5 m / ft3
1
1
A
2
100
≥ 1
Once in fifteen day
2
2
A
3
100
≥ 1
Daily
3
3
A
2
100
≥ 1
Once in fifteen day
4
4
B
2
10,000
57
Once in fifteen day
5
5
B
2
10,000
57
Once in fifteen day
6
6
B
4
10,000
57
Once in fifteen day
7
7
B
3
10,000
57
Once in fifteen day
8
8
C
3
100,000
571
Once in fifteen day
9
9
C
8
100,000
571
Once in fifteen day
10
10
D
3
Not defined
Not defined
Once in fifteen day
11
11
D
5
Not defined
Not defined
Once in fifteen day
12
12
D
3
Not defined
Not defined
Once in fifteen day
Note: Perform non-viable count after completion of the activities in case of equipment. Alert and action limits are defined in case of class A.

5.7 Swab test of the surface

5.7.1 For sampling sites refer location chart of the site.
5.7.2 Prepare saline peptone solution and dispense 10 ml quantity in a test tube and put one cotton swab in it and sterilize in the autoclave at 15 lbs pressure and 121°C for 15 minutes.
5.7.3 Sanitize the outer surface of the saline tube with a filtered sporicidal agent and transfer the tube into cRABS through pass box.
5.7.4 Remove the excess solution from swab by pressing on side of the tube. Swab 5 X 5 cm2 area using parallel overlapping stroke with slow rotation of swab. Repeat the sampling by the stroke at the right angle to original stroke. After taking the swab put the swab stick back into saline peptone solution. The sample according to the sampling plan. After taking swab write the location of swab and date of swab with a marker.
5.7.5 Bring the tubes to quality control laboratory and vortex for 1 minute and perform the Bioburden according to membrane filtration method according to SOP.
5.7.6 Incubate the Petri dishes at 22.5 ± 2.5° C for 3 days followed by 32.5 ± 2.5° C for 2 days in an upright position.
5.7.7 After incubation count and observe the number of colonies on colony counter or under a light source with the help of a marker. Record the results.
5.7.8 Frequency of monitoring
After completion of activities
5.7.9 Acceptance criteria
Class           Action Limits
A                1 CFU / Plate
B                5 CFU / Plate

5.8 Personnel fingers dabs and cRABS gloves dab

5.8.1 Prepare the Soybean casein digest agar plates as per SOP “Procedure for Storage and Preparation of Microbiological Culture Media” SOP ”. Use the prepared plates after 48 hours of pre-incubation.
5.8.2 Mop the plates from outside using filtered sporicidal agent before entering into the aseptic area and label the plates indicating the name of the person, date of test, LHFD, RHFD.
5.8.3 Open the lid and take the finger impression of left-hand finger dabs (LHFD) and right-hand finger dabs (RHFD) by placing the finger and thumb on the agar surface.
5.8.4 Incubate the soybean casein digest agar in an inverted position at 22.5 ± 2.5° C for 3 days followed by 32.5 ± 2.5° C for 2 days.
5.8.5 After incubation count the number of colonies on colony counter or under a light source with the help of marker and record the results in the specified format.
5.8.6 In case of sterility cRABS take dab after completion of the activities. Gloves numbering from left to right
5.8.7 Mop the plates from outside using a filtered sporicidal agent and keep the plates in pass box of sterility cRABS, the plates shall be indicating the gloves No., date of the test.
5.8.8 First take the gloves dab in pass box, then pass the rest of the plates in the chamber by opening the inner door and take the dab of other gloves
5.8.9 Incubate the soybean casein digest agar in an inverted position at 22.5 ± 2.5° C for 3 days followed by 32.5 ± 2.5° C for 2 days.
5.8.10 After incubation count the number of colonies on colony counter or under a light source with the help of marker and record the results in the specified format.
5.8.11 Frequency of monitoring
After completion of activities
5.8.12 Acceptance criteria
Limits of Personnel fingers dabs.
Action limit: Class A: 1 CFU / 5 fingers
                   Class B: 5 CFU / 5 fingers

5.9 Contact plates of Personnel gowns

5.9.1 Prepare the Soybean casein digest agar RODAC (Replicate Organism Detection and Counting) plates as per SOP “Procedure for Storage and Preparation of Microbiological Culture Media” SOP ”. Pour the media in RODAC plates completely, make sure convex surface formed after solidification. The plates that are not having convex surface shall not be taken for monitoring. Use the prepared plates after 48 hours of preincubation
5.9.2 Decontaminate the external surface of the Petri plates with the help of lint-free sterile mop soaked in a filtered sporicidal agent
5.9.3 Mark the Petri plates at the base with the following details with a marker,
For negative control plate
For all the other plates
Media lot No
Name of the location
Date of use
Date of Sampling
Used by
Sampled by
5.9.4 Perform the microbial monitoring of gown by touching the convex surface of the media plate for the locations.
5.9.5 Incubate all the Plates and negative control plate in an inverted position at 22.5 ± 2.5 °C for 3 days and 32.5 ± 2.5°C for 2 days.
5.9.6 After incubation observe and count the number of colonies on the colony counter or under the light source with the help of a marker.
5.9.7 Note down the observation.
Limits: Class    Action limit
             B        5 CFU / Plate

6.0 ABBREVIATIONS

6.1 SOP - Standard Operating Procedure
6.2 °C - Degree Centigrade
6.3 CFU - Colony Forming unit
6.4 µ - Micron
6.5 LHFD - Left-hand finger dab
6.6 RHFD - Right-hand finger dab
6.7 RODAC - Replicate Organism Detection and Counting





Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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