SOP of Growth Promotion Test of Microbiological Media

1.0 OBJECTIVE : To lay down the procedure for checking the nutritive property of the culture media to support the growth of microorganisms.

2.0 SCOPE : This SOP is applicable for the nutrient media used for all microbiological analysis in the Microbiological Laboratory of Quality Control Department of (Company Name).

3.0 RESPONSIBILITY
3.1 Each individual in the laboratory is responsible for carrying out the procedure.
3.2 Overall responsibility for training, implementation & follow-up with the QC Manager or his/ her nominee.

4.0 ACCOUNTABILITY
4.1 Head of Quality.

5.0 PRECAUTIONS
5.1 All activities of the Growth Promotion test is to be carried out under Laminar Air Flow in the microbiology testing room
5.2 Incubation of the plates/bottles containing media for growth promotion test shall preferably be incubated in separate incubators when available incubators are in place. If available separate incubators are not in place, the facility has to be developed, however, until then the incubation has to be continued in the same incubators. Once, the facility is developed for the incubation of the growth promotoin test separately, it has to be followed strictly.
5.2 Laminar airflow unit should be cleaned immediately with 70% isopropyl alcohol after the operations are over.
5.3 Use fresh sterile pipette for each transfer.
5.4 The medium to be poured in petriplates should have a temperature of 40-450c.
5.5 If any spillage of cultures, immediately wash with 70% IPA solution.
5.6 If growth promotion test is failed to meet the acceptance criteria during testing a sample, the test result will not be accepted and the same test shall be repeated. In that case, an incidence to be raised and the root cause to be identified prior to repeating the test.

6.0 PROCEDURE
6.1 Preparation of culture suspension
6.1.1 Prepare culture suspension containing 10-100 cells per ml as follows.
6.1.2 Remove the desired standard strain from the refrigerator and allow it to equilibrate to the room temperature.
6.1.3 Warm the hydration fluid and dilution fluid (Phosphate buffer) to 34ºC to 38ºC.
6.1.4 Pick a sterile forcep, remove two pellets and place into the 2.0 ml vial of hydration fluid.
6.1.5 Immediately replace the rubber stopper, recape the vial and return the remaining lyophilized materials to refrigerated storage.
6.1.6 Immediately recap the vial with the hydrated material and place into water bath at 34ºC to 38ºC for 30 minutes to assure complete hydration.
6.1.7 Immediately after completion of hydration, vortex the hydrated material to achieve equal distribution of the challenge strain throughout the hydrated suspension.
6.1.8 Proceed to the next step immediately.
6.2 Preparation of working dilution
6.2.1 With a sterile pipette, remove 1.0 ml of the well mixed hydrated suspension and transfer to 9.0 ml of pH 7.2 phosphate buffer/ Normal saline and mix well.
6.2.2 This is the working culture suspension that is this suspension contain 100 to1000 cfu/ml.
6.2.3 The hydrated microorganisms suspension must be used within 30 minutes to ensure microorganisms viability.
6.3 Preparation of inoculums (Cell suspension) from stock culture:
6.3.1 Streak a loopful of culture from the stock culture on a agar plate in a manner to get isolated colony and incubate the plates.
6.3.2 After the incubation select a colony and check the cell property by gram staining.
6.3.3 If the organisms meets the property of the specific organisms, pick morphologically same one or two colonies and transfer them to a tube containing 5.0 ml of sterile saline solution and make a suspension.
6.3.4 Serially dilute the cell suspension with sterile saline solution to obtain a cell suspension containing 100 to 1000 cfu per ml.
6.3.5 This is the working dilution and store it in the refrigerator at a temperature 2°C to 8°C for further use.
6.4 Growth promotion test of Agar media by Pour plate method
6.4.1 With a sterile pipette, remove 1.0 ml of the working culture suspension containing 100-1000 cells and transfer to 9.0 ml of pH 7.2 phosphate buffer/ normal saline and mix well. This is the working solution for pour plate method (10 to 100 CFU/ml). With a sterile pipette, remove 1.0 ml of the working solution of culture suspension containing 10-100 cells for each organism under test as specified in Table-1 into individual plate in duplicate.
6.4.2 Cool to about 45°C and pour sterile Agar media that is to be challenged in each plate.
6.4.3 Allow the plate to stand for 20 minutes to solidify and incubate the plates as per the time and temperature specified in Table-1.
6.4.4 Pour sterile agar media on a sterile plate having no culture suspensions as negative control and incubate it in the same condition.
6.4.5 Observe the plates for growth and note down the results in the record (FRM No.: FQC/269-01).
6.4.6 The media are suitable if a clearly visible growth of the microorganisms occurs.
6.5 Growth promotion test of Agar media by Spread plate method
6.5.1 Add 0.1 ml of the culture suspension containing 10-100 cells for each organism under test as specified in Table-1 into individual plate in duplicate.
6.5.2 Spread the culture suspension by using sterile glass spreader.
6.5.3 Allow the plate to stand for 1 hour and incubate the plates as per the time and temperature specified in Table-1.
6.5.4 Label a blank media plate (Refer 6.4.4) as negative control and incubate it in the same condition.
6.5.5 Observe the plates for growth and note down the results in the record(FRM No.: FQC/269-01).
6.5.6 The media are suitable if a clearly visible growth of the micro-organisms occurs.
6.6 Growth promotion test of liquid media
6.6.1 Add 0.1 ml of the culture suspension containing 10-100 cells for each organism under test as specified in Table-1 into individual broth in duplicate.
6.6.2 Simultaneously run negative control to verify testing conditions and incubate it in the same condition.
6.6.3 Observe the bottle for growth and note down the results in the record(FRM No.: FQC/269-01).
6.6.4 The media are suitable if a clearly turbid growth of the micro-organisms occurs.

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