Drain Point – Microbial Monitoring Procedure

Learn about the essential steps for microbial monitoring of drain point in the pharmaceutical industry. Ensure compliance and safety with our detailed procedures. Gain insights into the importance of microbial monitoring at drain points in the pharmaceutical sector. Enhance product safety and mitigate contamination risks effectively.

Microbiological Monitoring of Drain Points

1.0   Purpose

    • To lay down the procedure for Monitoring of drain points by analysis of pathogens.

2.0   Scope

    • This Standard Operating Procedure is applicable for the Monitoring of drain points at the Microbiology Department.

3.0   Responsibility

    • The officer or Executive of the Microbiology department shall be responsible for the preparation of new or revision of existing SOPs.
    • The head of the department/designee of respective areas & QA shall be responsible for reviewing the SOPs.
    • The Plant Head and head quality shall be responsible for approval of SOP.
    • QA shall be responsible for the distribution and control of SOPs to various departments.

4.0    Abbreviation

    • ATCC                  : American type culture collectionDrain Point
    • CC                       : Change Control
    • cm                       : Centimeter
    • CFU                    : Colony Forming Unit
    • °C                        : Degree Celsius
    • LAF                    : Laminar Air Flow
    • ml                       : Millilitre
    • mm                     : Milimeter
    • NCTC                 : National Collection of Type Cultures
    • SCDA                 : Soyabean Casein Digest Agar
    • µm                      : Micrometer

5.0   Procedure for Microbiological Monitoring of Drain Point

    • Preparation of Swabs for Drain Point
    • Aseptically add 3-5ml of sterilized 0.9% w/v saline solution into the sterile tube containing the swab.
    • Carry the set of swabs to the respective area as per standard entry and exit procedure.
    • Sampling at Drain Point
    • Dip the sterilized swabs in sterilized 0.9%w/v saline, sample approximately 25 cm2 area giving vertical and horizontal strokes from the drain points location As per Annexure 2.
    • Write the following information on the sampling container with the help of an ink marker pen.
      • Drain ID number
      • Date of sampling
      • Sampled By
    • Testing of Swab Sample of Drain Point

    • Transfer the samples to the microbiology laboratory and follow the procedure for analysis of Escherichia coli, Salmonella, P.aeruginosa, and S.aureus (Pathogens).
    • Vortex the tube containing saline and swab for 20-30 seconds before analysis.
    • Prewet the 0.45 µ membrane filter which has a 47 mm diameter with 50 ml of sterile Buffer peptone water or normal 0.9% w/v saline water before analysis.
    • Transfer the vortexed saline onto the membrane filter.
    • Pour 3-5 ml sterile Buffer peptone water into the test tubes to remove the traces of the remaining sample and transfer to the membrane filter.
    • Rinse the membrane with 2 x 50 ml of sterile buffer peptone water onto the membrane filter.
    • After Rinsing remove the membrane filter with the help of sterile forceps and inoculate in 100 ml Soyabean casein digest medium tube and incubate at 30-35°C for 18-24 hours.
    • Testing Procedure for Escherichia coli in Drain Point Swab

    • Inoculate 1.0 ml of the enriched culture into the tube containing 100 ml of MacConkey broth.
    • Incubate the tube at 42-44°C for 24-48 hours.
    • Subculture on a pre-incubated plate of MacConkey agar and incubate the plates at 30-35°C for 18-72 hours.
    • After completion of the incubation period observe the tubes for acid and gas formation and plates for the presence of Brick Red colonies. In case no characteristic results are observed, the sample meets the requirement for the absence of coli
    • Indole Test

    • Transfer 0.1 ml of enrichment medium to a tube containing 5.0 ml of Peptone water and incubate the tubes at 43-45°C for 24 hours.
    • After completion of the incubation period add 0.5 ml Kovac’s reagent shake well and allow it to stand for one minute.
    • If the red color ring is observed in the upper layer of the medium it confirms the presence of Escherichia coli.
    • Tests for Salmonella

    • Add 0.1 ml of enrichment culture to a tube containing 10.0 ml of Rappaport Vassiliadis Salmonella enrichment broth.
    • Incubate the tubes at 30-35°C for 18-48 hours and observe the tubes for color change or turbidity.
    • After completion of the incubation period from the tube of Rappaport Vassiliadis Salmonella enrichment broth, streak on the surface of pre-incubated plates of Xylose lysine deoxycholate agar medium.
    • Cover and invert the Petri plates and incubate at 30-35°C for 18-48 hours.
    • After completion of the incubation period if the medium shows red colonies with or without black centers it confirms the presence of salmonella
    • Tests for Pseudomonas aeruginosa

    • Streak one loop full of the enrichment culture on the surface of an incubated plate of Cetrimide agar.
    • Incubate the plates in an inverted position at 30-35°C for 18-72 hours.
    • Examine the presence of Greenish colonies which give florescence under UV light.
    • If no specific colonies are observed then the sample passes the test for the absence of Pseudomonas aeruginosa.
    • Oxidase test

    • Place 2 to 3 drops of freshly prepared 1% w/v solution of N, N N, N-tetra Methyl-P-Phenylene Di ammonium Dichloride on a piece of filter paper (Whatman No.1) or use ready to use Oxidase test disc and smear with the suspected colony.
    • If a purple color is produced within 5 – 10 seconds, the test confirms the presence of Pseudomonas aeruginosa.
    • Test for Staphylococcus aureus

    • Streak one loop full of the enriched culture on the surface of pre incubated plates of Mannitol-Salt agar.
    • Incubate the plates in an inverted position at 30-35°C for 24 to 48 hours.
    • If upon examination no colony shows characteristics colonies of yellow/white surrounded by a yellow zone, the test meets the requirement for the absence of staphylococcus aureus.
    • If any colony shows the characteristics as described above then perform.
    • Coagulase test.

    • Transfer representative suspected colonies from the agar surface of Mannitol salt agar media to individual tubes containing 0.5 ml of mammalian preferably rabbit or horse plasma, with or without additives.
    • Incubate the tubes in the water bath at 37°C. Examine the tubes after 3 hours and subsequently at suitable intervals up to 24 hours
    • If there is no coagulation, the sample meets the requirements of the absence of Staphylococcus aureus.
    • Negative Control

    • Put one un-inoculated plate/Tube of respective media as the negative control, and incubate the plate/Tube along with the other plates/Tube at the same temperature these plates should not exhibit any growth.
    • Analyze the sample as quickly as possible on arrival at the laboratory. If immediate analysis is not possible, store the sample under refrigeration at 2 to 8ºC. These stored samples should be analyzed within 4 hours.
    • Frequency: Quarterly ± 7 Days
    • Acceptance Criteria: Pathogen Should be Absent

Annexure 1:   List of Sampling Points of Drain Points

S. No.

Area Name of Location

Sample ID No.

Annexure 2 :   Sampling Schedule  of Drain Points

Year:                                                                                                                                        P= Purpose, A= Actual

S.

No.

Name of Sampling Point Sampling ID Jan. Feb. Mar. Apr. May. Jun. Jul. Aug. Sep. Oct. Nov. Dec.

Annexure 3   :   Drain Point Monitoring Report

Date of Sampling: Time of Sampling:
Sampled By: Incubator ID:
Date of Incubation: Incubation Completed on:
Media Used Prepared Media Lot No. Media Used Prepared Media Lot No.
Soybean Casein Digest Medium Rappaport Vassiliadis Salmonella Enrichment Broth
MacConkey Broth Xylose Lysine Deoxycholate Agar
MacConkey Agar Tripple Sugar Iron Agar
Mannitol Salt Agar Cetrimide Agar
Observations:
S.No. Sampling Point/Location Pathogens
E. coli Salmonella P. aeruginosa S. aureus
       
       
       
       
       
       
       
       
       
       
       
       
       
Negative Control        
Positive Control        

Limit: Pathogens should be absent.

Conclusion: The sample complies /does not comply with the above test.

pharmabeginers

Janki Singh is experienced in Pharmaceuticals, author and founder of Pharma Beginners, an ultimate pharmaceutical blogging platform. Email: [email protected]

Leave a Reply