swab testing of various surfaces for bioburden determination

1.0 OBJECTIVE
1.1 The objective of this SOP is to define the procedure for collection and testing of swabs of various surfaces for Bioburden determination in Microbiology lab.
2.0 SCOPE
2.1 This SOP is applicable for person taking the samples of swabs routine monitoring of aseptic area and analyst performing swab test in Microbiology
3.0 RESPONSIBILITY
3.1 Microbiologist-Quality control- To follow the SOP for swab test.
3.2 Head- Quality Control – Training on SOP.
3.3 Head- Quality control- Implementation & follow up of SOP
4.0 ACCOUNTABILITY
4.1 Head – Quality Control

5.0 ENVIRONMENT, HEALTH AND SAFETY
5.1 As per procedure

6.0 PROCEDURE
6.1 Materials, Equipment :
6.1.1 Materials:
6.1.1.1 Sterile swabs tubes containing 0.9% normal saline
6.1.1.2 Sterile gloves
6.1.1.3 Filtered 70% IPA
6.1.1.4 Marker pen
6.1.1.5 Soyabean casein digest agar plates
6.1.1.6 Sterile 0.45 micron membrane filter
6.1.1.7 Sterile forceps
6.1.2 Equipments:
6.1.2.1 Autoclave
6.1.2.2 Laminar air Flow
6.1.2.3 30-350C Incubator
6.1.2.4 20-250C Incubator
6.1.2.5 Manifold filtration unit
6.2 Preparation of swabs:
6.2.1 Prepare 0.9% normal saline solution and sterilize by autoclaving at 1210C and 15 Psi pressure for 20 minutes.
6.2.2 Pour 5 ml of sterilized normal saline into each of the swab tube.
6.2.3 Wrap the swabs in aluminum foil.
6.2.4 These swab tubes can be stored in refrigerator at 2-80C for 15 days.
6.3 Transfer of Swabs to Aseptic Area:
6.3.1 Take the required quantity of prepared swabs from refrigerator or freshly prepared deliver to pass-box outside the aseptic area.
5.1.1 Enter into the aseptic area following the respective area entry exit and gowning procedure.
5.1.2 Open the pass box door from inside the aseptic area.
5.1.3 Remove the outer aluminum packing of the swabs in pass box and deliver the swabs to aseptic area.
6.4 Procedure for sample collection:
6.4.1 Disinfect the swab tubes and hands with 70% IPA or other suitable hand sanitizer provided.
6.4.2 Unscrew the swab tube cap and remove the swab stick from the tube and rotate the swab around the top portion of the tube to express the excess solution from the swab.
6.4.3 Swab the designated approx 2 inch by 2 inch (25-30 cm2) area by pressing the swab tip to the surface to be sampled.
6.4.4 Move the swab in back and forth motion until the entire designated area has been sampled.
6.4.5 Roll the swab cap in your fingertips exposing a new surface of the swab tip.
6.4.6 Repeat swabbing of the designated area in a top to bottom motion.
6.4.7 Immediately place the swab back in its tube containing sterile normal saline and screw.
6.4.8 Write the Location/Equipment name, date of sampling, and sampled by on the outside surface of swab tube with permanent marker.
6.4.9 Wipe the sampled surface area with filtered 70% IPA solution.
6.4.10 Keep one swab as unused and mark it as process negative control.
6.4.11 Transfer the swabs to microbiology lab for analysis.
6.4.12 Record the following information in format No.CLS/QCD/042/F01:
6.4.12.1 Date of Sampling
6.4.12.2 Date of test carried
6.4.12.3 Swab prepared on
6.4.12.4 Sampled by
6.4.12.5 Product In filling
6.4.12.6 Batch No.
6.4.12.7 Remarks
6.5 General Procedure for sample testing:
6.5.1 Follow proper gowning procedure of microbiology lab.
6.5.2 Place the swab samples under LAF after sanitizing with filtered 70% IPA or other suitable sanitizing agent provided.
6.5.3 Ensure that the LAF is turned on at least 15 minutes before start of any work.
6.5.4 Sanitize the LAF work bench with filtered 70 % IPA.
6.5.5 Connect the manifold to the filtration flask and then filtration flask to the vacuum line.
6.5.6 Vortex the swab tube for 30 seconds on vortex mixer to get the cells come in the solution from the swab.
6.5.7 Sanitize the outer surface of sterilized filtration assembly with filtered 70% IPA.
6.5.8 Aseptically remove the butter paper from the filtration assembly and place to the manifold unit inside the laminar air flow hood.
6.5.9 Open the cap of swab tube and transfer the content to the filtration assembly.
6.5.10 Open the knob of the manifold to start the filtration.
6.5.11 Rinse the membrane with 100 ml of 0.1% peptone water or Diluting Fluid A.
6.5.12 After filter the swab sample and rinse the membrane with 100 ml of 0.1% peptone water or Diluting Fluid A.
6.5.13 After completion of filtration open the filtration assembly and aseptically remove the filter and place onto the surface of pre incubated Soyabean Casein Digest Agar in Petri plate.
6.5.14 Incubate the plate at 20-250C for NLT 3 days following the incubation at 30-350C for NLT 2 days.
6.5.15 After incubation record the results as total fungal count (TFC) and total bacterial count (TBC) in format No.
6.5.16 If any deviation found in the obtained results from the limits stated below, then intimate it to the production department to take necessary action.
6.6 Frequency:
6.6.1 Swab sampling of fill needles shall be carried out at the end of each batch filling when there is product change over. But in case of batch changeover when the same product of different batch is going to be filled, the sample shall be taken after the completion of complete filling activity when there is no more filling activity.
6.6.2 Swab sampling as a part of routine environment monitoring shall be carried out on the day of operational activities.
6.6.3 Swab sampling of other equipment and for other studies shall be carried out as per the plan and schedule given in the respective study document or protocol.
6.7 Precautions:
6.7.1 During swab sampling swab should not touch any other surface or hand, if happens then don’t use such contaminated swab and sample with a new swab.
6.7.2 Sample should be tested as early as possible after sampling, if not possible it should be stored in refrigerator at 2-80C for not more than 4 hours.
6.7.3 When going to sampling in any area the respective area gowning and entry procedure should be strictly followed.

6.8 Acceptance Criteria

S. No.	Location	Alert Limit	Action Limit
1.	Grade A surface like Fill needle, rotating turn table, product hopper, inside surface of rubber plug container and filling LAF surface.	1 cfu/25cm²	1 cfu/25cm²
2.	Grade B surface like room floor, wall, door etc.	2 cfu/25cm²	3 cfu/25cm²

7.0 ABBREVIATIONS

SOP : Standard Operating Procedure
QAD : Quality Assurance Department
QCD : Quality Control Department
IPA : Isoproply alcohol
NA : Not Applicable
No. : Number

ANNEXURE No.	Title of Annexure
ANNEXURE I	Swab Test Report

swab testing of various surfaces for bioburden determination

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