It is essential to have a high fidelity strain collection which will be stable over the life of the lab. Anaerobic stocks are frequently prone to contamination due to the constraints of working in an anaerobic chamber and their frequent slower growth than common contaminants like Enterococcus sp. The goal of this protocol is to create 2 copies of each strain to be stored: 1 in a crimp top vial for long-term archival storage, and a 2nd in a conventional screw top vial for routine use. Crimp top vials may be preferable for anaerobes; however, they are difficult to sample in a sterile fashion and the seal can only be punctured so many times before the integrity of the stock is lost.
- Crimp Top Vials (Sigma 854964)
- Crimp Seals (Sigma 854144)
- Crimping Device (Ex. Sigma Z114243-1EA)
- 1.5 mL Cryovial (External threads, ex Simport T310-2A)
- Appropriate growth media
- Sterile 50% glycerol (mix 50:50 with water and autoclave)
- 1-2 days ahead of time, transfer all needed supplies into anaerobic chamber
- Transfer a well-separated single colony from an agar plate to 5 mL media in an appropriate tube.
- Incubate 24-48h until a turbid culture has formed
- Transfer 1mL to each of a crimp top vial and a screw top vial
- Add 0.5 mL 50% glycerol to each tube and mix by pipetting
- Seal both vials
- Affix computer-printed labels with the following information: Species, Strain, Box Position, and Date of Preparation
- Remove vials and transfer to appropriate position of freezer box in -80C
- Remove remaining liquid culture to bench and proceed with 16S rRNA isolate sequencing protocol to confirm strain identity.