Method: Long Term Storage of Bacterial Strains
Nov. 14
1990
C. Helms
Purpose:
Bacterial strains may be stored indefinitely at low temperatures (-
20 degrees C and
-80 degrees C) in 15 to 40% glycerol. It is lab policy to prepare a
frozen stock of
newly acquired or created strains for the archives as soon as
possible.
Time required:
Procedure:
Day 1
- Inoculate a 15 ml culture tube containing 5 ml of LBM or
LBM+antibiotic
selective medium
with a freshly grown
isolated colony. Incubate at 37 degrees C
until culture is in
late log or stationary phase (usually 5 hours to overnight).
Day 2
- For each strain to be stored at -80 degrees C for the archives
prepare a
sterile
labeled cryovial. Pipet 225 ul sterile 80% glycerol into
the
cryovial. Add 1.0 ml of the bacterial culture (frozen stock will be
15%
glycerol). Mix well (vortex)
and place tube at -80 degrees C.
- For each strain to be stored at -20 degrees C as a liquid
glycerol " working"
stock
pipet equal volumes 80% glycerol and bacterial culture into a
labeled
polypropylene tube. Mix the contents well (if not well mixed ice
crystals
will form
decreasing the viability of the cells). Place the tube in
a -20 degrees C
freezer. If possible
check the viability of the cells after 1
week.
To recover a strain from the -80 degrees C glycerol stock
use a
sterile toothpick
to scrape some of the ice
then streak out the cells on the
appropriate medium
e.g.
LBM + ampicillin. Do not thaw the frozen stocks
because each
freeze-thaw cycle will result in a 50% loss in cell viability.
To use the -20 degrees C working stocks
pipet 50 to 100 ul as
inoculum for a 5 ml
overnight culture.
References: none
