To describe the procedure to revive and maintain the ATTCÒ Bacterial and Fungal Cultures by seed stock technique that are used as positive controls in Microbiological Quality Control Tests and also in verifying the response of each new batch of medium.
    2. Scope:-

             It is applicable to all ATCCÒ certified Bacterial and Fungal Cultures.  

3. HSE Statement:-

§     Gloves
§     Face Mask

4. Responsibilities:-

                               i)      Manager Quality Control is responsible to ensure that procedure & formats are followed entirely as approved.
                             ii)      Microbiologist is responsible to perform the test.
     5. Materials Required

5.1 Incubator (35±1°C)
5.2 Refrigerator (2-8°C)
5.3 Inoculating loops
5.4 Bunsen Burner
5.5 Sterile and Pre-incubated Tryptic Soya Agar Plates
5.6 Sterile and Pre-incubated Tryptic Soya Agar Slants
5.7 Sterile and Pre-incubated Sabouraud Agar or Potato Dextrose Agar Plates
5.8 Sterile and Pre-incubated Sabouraud Agar or Potato Dextrose Agar Slants
5.9 ATCCÒ certified Microbiologics Kwik-Stik Reference Bacterial and Fungal Cultures
5.10 70% filtered IPA
5.11 Biological Safety Cabinet   
6. Definitions:-

     6.1 Culture:-
      A growth of microorganisms, viruses, or tissue cells in a specially prepared nutrient medium  under supervised conditions.
     6.2 Fungal Culture:-
A fungal culture is a type of procedure that is used to determine if fungi are present in an area of the body.  Fungi are microorganisms that thrive in moist, dark places, such as in shoes, a damp locker room, or in the folds of the skin. Some fungi are not harmful, while other types can cause infections.
  6.3 Pure culture:- 
         A culture of a single cell species, without presence of any contaminants
  6.4 Primary culture:-
A cell or tissue culture started from material taken directly from an organism, as opposed to that from an explant from an organism.
  6.5 Slant culture:-
      A culture made on the surface of solidified medium in a tube which has been tilted to provide a greater surface area for growth.
     6.6 Plate culture :-
                         A culture grown on a medium, usually agar or gelatin, on a Petri dish
  6.7  ATCC:-
                 American Type Culture Collection Center
7.Flow Chart:-

8.0 Procedure:-
8.1 Revival of ATCC Cultures
8.1.1 Wear gloves and mask before handling the culture. All the activity should be performed under
         Biological Safety Cabinet.
8.1.2 Allow Kwik-Stik pouch to warm at room temperature.
8.1.3 Wipe the pouch with 70% filtered IPA.
8.1.4 Remove the Kwik-Stik from the pouch.
8.1.5 Tear off the pull-tab label and paste on the
8.1.6 Squeeze the cap of the Kwik-Stik to break the ampoule and release the hydrating fluid.
8.1.7 Tap the bottom of the Kwik-Stik to move the fluid down to the pellet.
8.1.8 Once hydrated, pinch the bottom of the Kwik-Stik to crush the pellet and mix it well with the fluid.
8.1.9 Use the saturated swab from the Kwik-Stik to streak onto the working and stock sterile agar plates
 (for Bacterial Cultures) or sterile Sabouraud Agar or Potato Dextrose Agar Plates as per Incubate the inoculated bacterial cultures at 30-35oC for 3-5 days and fungal cultures at 20-25oC for 5-7 days.
8.1.11After incubation, seal the plates with paper tape and store in refrigerator.
8.1.10    Always use the “Working” plates for quality control procedures. Use the untouched “Stock” plates for subculture transfers.
8.1.11    Subculture the stock cultures  within 1 month, from the date of preparation, on freshly prepared sterile and pre-incubated agar plates or slants. This will be the 2nd transfer of culture.
8.1.12    For each subsequent subculture use the “Stock” plate to transfer growth. After five transfers, start with a new reference culture. The reason of minimizing the number of passages or transfers to five is to avoid lab strains that may have different phenotypic characteristics from the reference strain.
8.1.13 After every transfer, check for purity by performing a simple and gram staining as per SOP
9. References:-
8.2              Standard Methods Online Edition, 9020B
8.3              Microbiologics Kwik-Stik Product Insert
8.4              NELAC Quality Systems Manual Appendix D.3.7
10. Record:-
           Revival and Maintenance of ATCCÒ  Cultures
11. Distribution:-

This SOP has to be distributed in below mentioned Departments:-

Sr. NO
Distributed to
Quality Control Department

Quality Management Department


12. Revision History:-