SOP for surface swab test procedure for microbiology laboratory
- Kazi
- Last modified: July 18, 2024
1. Purpose of SOP
This standard operating procedure (SOP) describes the swab test procedure for determining microbiological contamination on equipment surfaces such as tanks.
2. Scope of SOP
The SOP is used for swabbing/sampling from the pharmaceutical production tank surfaces. This laboratory procedure is used to determine counts and recovery.
3. Abbreviations
3.1. cfu/CFU – colony forming units
3.2. NMT – not more than
3.3. TSA – Tryptone Soy Agar
3.4. TSB – Tryptone Soy Broth
3.5. PPE – Personal Protective Equipment
4. Materials required
4.1. Safety
4.1.1 The swabs and other materials used must be, at all times, considered potentially hazardous.
4.1.1. Disposable gloves should always be worn when handling live cultures.
4.1.2. All work with open live cultures should be performed in a Bio-safety cabinet.
4.1.3. Recommended PPE to be worn.
4.2. Equipment
4.2.1. Incubator, 30 to 35 0C
4.2.2. Bio-safety Cabinet
4.2.3. Colony Counter
4.2.4. Vortex mixer
4.2.5. Water bath set at NMT 45 0C
4.2.6. 10cm x 10cm nylon template
4.3. Materials
4.3.1. Nylon Regular Flocked Swabs in Tube 80mm Breakpoint, sterile.
4.4. Media and reagents
4.4.1. Tryptone Soy Agar, molten at NMT 45 0C
4.4.2. Tryptone Soy Broth, 9mL dispensed into 10mL sterile tubes
4.4.3. MacConkey Agar plates, 90mm x 15mm
4.4.4. Pseudomonas-CFC selective agar plates, 90mm x 15mm
4.4.5. 5mL vials of Normal Saline (0.85%), sterile – to be used as diluent
4.4.6. Normal Saline (0.85%), sterile, 2mL dispensed into 10mL sterile tubes
4.4.7. 70% Isopropyl Alcohol
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5. Sampling method
5.1. Swab test procedure of a 10cm x 10cm flat surface (100cm2)
5.1.1. The swab is aseptically pre-moistened in a biosafety cabinet no more than 2 hours before use by a Technician.
5.1.2. Aseptically remove the swab from the plastic applicator tube, being careful not to touch the shaft or the swab tip. Ensure the plastic tube is kept clean and sterile and handled as little as possible.
5.1.3. The swab is moistened with the sterile diluent and pressed against the side of the diluent bottle to remove the excess diluent, leaving it moist but not saturated.
5.1.4. Return the swab aseptically to the plastic applicator tube.
5.1.5. Place the required amount of pre-moistened swabs into a sealed plastic pouch for transport to and from the QC Microbiology Laboratory.
5.1.6. The swabs are transported immediately to the sampling area for use by the production technician.
5.1.7. The production technician must aseptically remove the swab from the plastic applicator tube, careful not to touch the shaft or the swab tip. Ensure the plastic tube is kept clean and sterile and handled as little as possible.
5.1.8. A 10cm x 10cm nylon template that has been wiped down with 70% Isopropyl Alcohol before use is placed over the area to be sampled to ensure the correct surface area is swabbed.
5.1.9. The moistened swab is placed on the test site and sampled across the selected surface.
5.1.10. The swab is held at a slight angle to the surface, and moderate pressure is applied.
5.1.11 The angle and pressure should allow as much contact between the swab head and surface as possible without damaging the swab or shaft.
5.1.12. The surface should be sampled using parallel strokes from right to left (horizontally) from top to bottom of the sampling area, rolling the swab approximately 180 degrees clockwise to ensure the previously swabbed area is slightly overlapped. Ensure that the entire surface of the swab head comes into contact with the surface.
5.1.13. Repeat the same procedure vertically, i.e., at 90 degrees to the initial swabbing.
5.1.14. Repeat the same procedure diagonally, i.e., at 45 degrees to the initial swabbing.
5.1.15. The swab will be returned aseptically to the plastic applicator tube for transport to the QC Microbiology laboratory for further processing.
5.2. Swab test procedure of an irregular surface/ crevice
5.2.1. Proceed as in sections 5.1.1, 5.1.2, and 5.1.4 to 5.1.6.
5.2.2. As much of the relevant surface as possible is to be swabbed.
5.2.3. The swab will be returned aseptically to the plastic applicator tube for transport to the QC Microbiology Laboratory for further processing.
6. Swab test procedure in the laboratory
6.1. Receipt and storage
6.1.1 Swab samples must be signed in and assigned a unique Quality Control Department sample number.
6.1.2 If swabs cannot be processed immediately, they must be kept at 2 to 80C for NMT 24 hours after sampling.
6.2. Swab recovery
6.2.1. Place the swab into a sterile tube containing 2mL sterile saline in a Bio-safety cabinet.
6.2.2. Aseptically break off the portion of the swab handle touched by the gloved hand.
6.2.3. Vortex the tube at high speed for 2 minutes in 10-second bursts.
6.2.4. Transfer 1mL of the liquid content of the swab tube to a sterile 90mm Petri dish, add approximately 20mL molten TSA media to the plate, and swirl to mix and distribute the sample.
6.2.5. Allow the TSA to cool down and solidify, then invert.
6.2.6. Incubate the inverted plates at 30 to 350C for NMT 5 days.
6.2.7. Count and record the number of CFU present, using a Colony Counter, if necessary.
6.2.8. Add 0.5mL of the remaining liquid content of the vortexed tube to 9mL of TSB in a sterile tube and incubate (enrichment step) at 30 to 350C for 5 days.
6.2.9. After incubation, streak 0.1mL of the broth onto Pseudomonas-CFC selective agar plates and MacConkey Agar plates to check for the presence/absence of Coliforms/pseudomonads.
6.2.10. Incubate the Pseudomonas-CFC selective agar plates at 20 to 250C for 48 hours.
6.2.11. All grown colonies are suspect Pseudomonas spp. and are counted as such.
6.2.12. Confirm the identification of the colonies using Gram staining and the Vitek II system.
6.2.13. Incubate the MacConkey Agar plates at 37±10C for 48 hours.
6.2.14. All grown colonies are suspect Coliforms and are counted as such.
6.2.15. Confirm the identification of the colonies using Gram staining and the Vitek II system.
6.3. Swab test negative control
6.3.1. Simultaneously, inoculate a separate swab directly with the sterile diluent used to moisten the test swab, with the same volume (0.1mL) as was used on the test swab.
6.3.2. Proceed as in section 6.2.1 to 6.2.10.
6.3.3. Incubate the inverted plate at 30 to 350C for NMT 5 days.
6.3.4. Count and record the number of CFUs present, if any.
6.4. Absence/presence of coliforms and pseudomonads
6.4.1. If any bacterial colonies are recovered from the TSA pour plates in section 6.2, streak each colony onto Pseudomonas-CFC agar and MacConkey agar plates to confirm the presence or absence of Pseudomonads and Coliforms.
6.4.2. Incubate the Pseudomonas-CFC agar plates at 20 – 250C, and the MacConkey agar plates at 37±10C for 48 hours, respectively.
6.4.3. All colonies recovered on the streak plates in 6.4.2 will be identified using Gram staining and the Vitek II system.
7. Acceptance criteria
7.1.1. NMT 400 CFU per 100 cm2
7.1.2. Absence of Coliforms and Pseudomonads
8. Results
8.1. Swab counts
8.1.1 All results are entered into the data worksheet, which facilitates reporting and trending of collected Microbiological swabbing data.
8.1.2 Calculate the final microbial count by multiplying the number of colonies on the TSA pour plates (section 6.2) by a factor of 2 to give a final count in cfu/100 cm2
240 SOPs, 197 GMP Manuals, 64 Templates, 30 Training modules, 167 Forms. Additional documents included each month. All written and updated by GMP experts. Checkout sample previews. Access to exclusive content for an affordable fee.
Author: Kazi Hasan
Kazi is a seasoned pharmaceutical industry professional with over 20 years of experience specializing in production operations, quality management, and process validation.
Kazi has worked with several global pharmaceutical companies to streamline production processes, ensure product quality, and validate operations complying with international regulatory standards and best practices.
Kazi holds several pharmaceutical industry certifications including post-graduate degrees in Engineering Management and Business Administration.
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