Microbial analysis of Compressed Air and Nitrogen
It describes the (Standard Operating procedure) Sop for Microbial analysis of Compressed Air and Nitrogen Gas
Sop for Microbial analysis of Compressed Air and Nitrogen Gas
I. Purpose & Scope:
- To provide a general methodology to be followed for Microbial analysis of Compressed Air and Nitrogen Gas in Quality control, Cephalosporine production block and microbiology laboratory of pharmaceutical industry.
- Applicable to all personnel who are working in Quality control, Production and microbiology laboratory.
II. Responsibilities:
• All Quality Control, Microbiologist, production personnel and related personnel shall be responsible to follow and implement this SOP.
III : Introduction and Procedural Part :
Introduction :
- Testing and monitoring of compressed and nitrogen gas that comes into contact with pharmaceutical products is vital to assuring the quality and safety of those products.
- The Compressor itself can function as culprit by creating a contaminated environment for the product, thereby resulting in contamination in the product.
- So Compressed and Nitrogen line should be tested on routine basis in order to determine microbiological cleanliness of the air supply. Testing should be conducted on a Periodic basis.
Microbiological SAMPLING & analysis of Compressed Air:
- Clean the outlet of Compressed air with 70 % IPA and allow flushing the air for 1 to 2 minutes.
- The collection of Compressed air should carry out by a person after wearing hand gloves and Mask. Refer Annexure I for location of Compressed air.
- After flushing the air, pass the compressed air for about 10 minutes in 100 ml of sterilized buffered peptone water solution pH 7.
- After collection, clean the air line and transfer the sample bottle to microbiology laboratory for further testing.
- Transfer 100 ml above sample in a sterile filtration assembly. Filter the whole sample and transfer the 0.45 µ membrane filter paper on the Pre incubated plate of Soybean casein digest agar (SCDA) /Dey engley Neutralizing Agar (DENA) Medium.
- Firstly ,Incubate the plates in inverted position at 20°C to 25°C for 72 hours for determination of total fungal count (TFC) and Further incubate the at 30-35°C for 48 hours for determination of total bacterial count (TBC).
- After 5 days incubation period, Count the number of colony forming unit (CFU) observed on the membrane filter of the plate.
- Total Viable aerobic count (TVAC) is the summation of the TFC and TBC. The count is expressed as cfu/m3. Record the results in Annexure-II.
- Positive control: For positive control, Refer the media fertility test performed during Media preparation.
- Negative Control: Keep one plate of the same lot of media used for testing, as negative control and incubate at respective temperatures along with test.
- Frequency of Monitoring: Once in Month, During Activity.
Microbiological SAMPLING & analysis IN ASEPTIC production Area:
- For sampling of Nitrogenand Compressed Air in Aseptic area (Grade A), Use Sterile preincubated Soybean casein digest medium (SCDM) and Fluid thioglycollate medium (FTGM) tube/bottle.
- Transfer the sampling apparatus/ bottles to aseptic production area through Dynamic pass box by keeping it into SS container/kit.
- Enter the aseptic area by following respective entry procedure. Take Sampling SS kit/container from Dynamic Pass box.
- Sampling of Nitrogen and Compressed air should be performed carefully.
- At first Sanitize the Outer surface of Sampling point by spraying 70% filtered IPA, then flush the air for 1-2 minutes.
- Then purge the air by opening the valve in Soybean casein digest medium and Fluid thioglycollate medium for minimum 5 minutes. Avoid splashing of media outside due to pressure of air by adjusting the valve.
- After collection of sample, close the valve and immediately close the bottle/tube. Then put all sampling apparatus in Sampling SS kit and transfer it to Microbiology laboratory through Dynamic pass box.
- Incubate Soybean casein digest medium tube/bottle along with negative control at 20-25°C for 14 days.
- Incubate Fluid thioglycollate medium tube/bottle along with negative control at 30-35°C for 14 days.
- Carry out Positive control on same lot of Fluid Thioglycollate medium using 10-100 cfu/ml viable micro-organisms inoculum of Clostridium sporogenus (ATCC 11437).
- Carry out Positive control on same lot of Soybean casein digest medium using 10-100 cfu/ml viable microorganisms inoculum of Bacillus subtilis (ATCC 6633).
- After incubation, observe the sampled tube/bottle for any growth/turbidity. Record the result in Annexure III.
- Acceptance Criteria: No evidence of growth should be observed in sampling bottle.
- Frequency of Monitoring: Once in Month, During Activity.
Annexure I
List of user points of compressed and Nitrogen Gas Sampling
A) Compressed Air sampling points:
| Sr.No | Name of Sampling Point | Location | Grade | Acceptance criteria |
| 1 | C | NMT 25 cfu/m3 | ||
| 2 | A | Should be Sterile |
B) Nitrogen Gas sampling points:
| Sr. No | Name of Sampling Point | Location | Grade | Acceptance criteria |
| 1 | A | Should be Sterile |
Annexure II
Microbial Analysis Report of Compressed Air
| Date of Sampling | Date of completion | ||
| Section : | Sampled by : | ||
| Frequency : | Next Due on : | ||
| Analyzed By : | Media Lot No. | ||
| Incubation Temp. | 20-25°C For 72 hours | Incubator ID No | |
| Incubation Temp . | 30-35°C For 48 hours | Incubator ID No |
| Sr.No |
Name of Sampling Points
|
Grade | Total viable aerobic count
TVAC (cfu/m3) |
|
| Total Bacterial Count | Total Fungal Count | |||
Acceptance Criteria : For Grade C : NMT 25cfu/m3
Negative Control: Growth observed/ not observed
Conclusion: Total viable aerobic count of Compressed air found Satisfactory/Not Satisfactory.
Observed By: Checked By:
Date: Date:
Annexure III
Microbiological Analysis report of compressed and Nitrogen Gas in sterile Area (Grade A)
- I) Sample and Incubation Details :
| Name of Sampling Point | Date of Testing | ||
| Sampled by | Date of Completion | ||
| Lot number of SCDM | Lot number of FTGM | ||
| Incubator I.D used for SCDM (20-25°C) | Incubator I.D used for FTGM (30-35°C) |
- II) Observation :
| Incubation Day | Date | SCDM | FTGM | Negative Control | Observed By | Checked By |
| 1st Day | ||||||
| 2nd Day | ||||||
| 3rd Day | ||||||
| 4th Day | ||||||
| 5th Day | ||||||
| 6th Day | ||||||
| 7th Day | ||||||
| 8th Day | ||||||
| 9th Day | ||||||
| 10th Day | ||||||
| 11th Day | ||||||
| 12th Day | ||||||
| 13th Day | ||||||
| 14th Day | ||||||
| 15th Day * |
* Column to be used for recording observation if 14th day is holiday.
H = Indicates Holiday, – ve Indicates no growth(sterile), +ve Indicates growth
III) Observation of Positive control and Negative controls :
- Soybean Caseine digest medium :
| Name of Organism | Positive control | Observed On/By |
Negative control :————————————–
- Fluid thioglycolate Medium :
| Name of Organism | Positive control | Observed On/By |
Negative control :————————————–
Conclusion : The Analyzed sample are found Satisfactory/Not Satisfactory.
Analyzed By: Checked By:
Date: Date:
