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1.0 OBJECTIVE : To lay down a general procedure for preservation and maintenance of microbial cultures by sub culturing. Sub culturing is an important method of storage and maintenance of the microbial cultures; employed as reference strains. Sub culturing of microbial cultures after stipulated period regains the viability of microbial action, which is then employed for analytical purpose.
2.0 SCOPE : This SOP is applicable for the procurement and maintenance of the microbial standard culture for Microbiological Laboratory of Quality Control Department of (Company Name).
3.0 RESPONSIBILITY
3.1 Microbiologist/ QC officer shall be responsible to ensure proper and timely execution of the procedure.
3.2 Overall responsibility for training, implementation & follow-up with the QC Manager or his nominee.
4.0 ACCOUNTABILITY
4.1 Head of Quality.
5.0 PRECAUTIONS
5.1 Maintain aseptic conditions at each step of sub culturing.
5.2 Transfer of lyophilized microbial culture should be done properly without spillage only in the broth culture media before transferring it to agar slant.
5.3 Subcultured microorganisms for routine use should be always stored in a refrigerator at 2 to 8° C.
5.4 Subcultured microorganisms for long term preservation should be kept in a freeze at -15 to -20° C.
5.5 Sub culturing of each microbial culture from stock solution should not be done more than 5 times. As for example if it is in passage three then only two passages can be done later.
6.0 PROCEDURE
6.1 Prerequisites
6.1.1 Ensure that the LAF workstation is cleaned and sanitized.
6.1.2 Prepare the media required, as per the SOP: Preparation of microbiological media (SOP No. QC101).
6.1.3 Microbiologist should follow the appropriate gowning procedure rinse hand gloves with 70% filtered IPA.
6.1.4 All Glasswares should be cleaned properly, dried and sterilized by dry heat sterilization before proceeding for sub culturing.
6.2 Procurement of standard cultures
6.2.1 The microbial culture should be procured from reliable source.
6.2.2 The microbial culture must have the tracibility certificate.
6.2.3 The date and source of procurement should be entered in the register.
6.2.4 The culture should be stored in the condition prescribed by the manufacturer.
6.2.5 The culture must not be used after the expiry date.
6.3 Preparation of culture suspension from the lyophilized culture
6.3.1 Prepare culture suspension containing 10-100 cells per ml from EZ-CFUTM Microorganisms as follows:
6.3.2 Remove the desired standard strain from the refrigerator and allow it to equilibrate to the room temperature.
6.3.3 Warm the hydration fluid and dilution fluid to 34ºC to 38ºC.
6.3.4 Pick a sterile forcep, remove two pellets and place into 2.0 ml vial of hydrating fluid.
6.3.5 Immediately replace the rubber stopper, recap the vial and return the remaining lyophilized materials to refrigerated storage.
6.3.6 Immediately recap the vial with the hydrated material and place into 34ºC to 38ºC incubator for 30 minutes to assure complete hydration.
6.3.7 Immediately following incubation, vortex the hydrated material to achieve equal distribution of the challenge strain throughout the hydrated suspension.
6.3.8 Proceed to the next step immediately.
6.4 Preparation of working dilution
6.4.1 With a sterile pippette, remove 1.0 ml of the well mixed hydrated suspension and transfer to 9.0 ml of pH 7.2 phosphate buffer and mix well.
6.4.2 This is the working culture suspension that contains 10 to 100 cfu/ml.
6.4.3 The hydrated suspension must be used within 30 minutes to ensure microorganisms viability.
6.5 Preparation of stock culture and working culture
6.5.1 Transfer 50 μl of the working dilution to 50 ml of sterile broth media.
6.5.2 Incubate the broth contaning the culture. The media used and the incubation conditions for different organisms are given in Annexure-I.
6.5.3 After incubation streak a loopful of culture on at least three agar plate in a manner to get isolated colony and incubate the plates.
6.5.4 After the incubation select a isolated colony (if any) and check the cell property by Gram staining.
6.5.5 If the organisms meet the property of the specific organisms, pick morphologically same one or two colonies and transfer them to a tube containing 5 ml of sterile saline solution and make a suspension. If the cuture is not the specific ones do the steps 5.6.1 to 5.6.4 again.
6.5.6 Immerse a sterile cotton swab into the suspension and streak agar plate uniformly to get confluent growth. Also take a loopful suspension and streak on a tryptone soy agar slant. Prepare 4 tubes and 4 plates for each organisms.
6.5.7 Incubate both the plate and tube.
6.5.8 After incubation check the growth. If significant growth is visible, tightened the cap of the tube and keep slant in the refrigerator at a temperature 2°C to 8°C with proper labeling (as per Annexure-IV).
6.5.9 Scrap the growth on each individual plate with a sterile cotton swab and suspend the culture in 4 different cryovial containing broth media with 10 % glycerol.
6.5.10 Keep the cryovials into the freeze at a temeparature range from -15 to -20 °C with proper labeling.
6.5.11 Preserve the slant and cryovials upto 3 months from date of preparation.
6.5.12 At this stage this culture is mentioned as first pass.
6.5.13 Use first slant/ cryovial for current month, second for next month and third for the third month for preparation of working slant and fourth for the preparation of the next stock slant/ cryovial. The stock prepared from this fourth slant is second pass of the cultue. So, at the end of the six months the prepared stock will be third pass and at the end of 9 months the stock will be fourth pass and so at the end of 12 months it will be fifth pass.
6.5.14 To prepare the stock culture from the previous stock culture, flow the steps from 5.6.3 to 5.5.10.
6.5.15 To prepare working culture streak a loopful culture from the stock culture to a new slant and incubate as per Annexure-I.
6.5.16 Prepare working culture at least once a week or as perweekly requirement. The sheif life of working culture shuld be maximum 7 days.
6.5.17 Label the individual slants and cryovials as per Annexure-IV
6.5.18 Incubate the slants at the temperature for the time specified in Annexure-I.
6.5.19 After use or after expiry, dispose the culture slants/cryovials as per SOP: Disposal of contaminated microbial culture media and biohazard materials. (SOP No.: QC102)
