Microbial analysis of Compressed Air and Nitrogen

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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)

 

  1. 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)  

    

  1. 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 :

 

  1. 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:

                    

 

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