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Preservation of the isolatesDr. Yuting Deng
Pearl River Fisheries Research Institute (PRFRI), Chinese Academy of Fishery Sciences (CAFS), China
OUTLINE
Choice of preservation5.3.1
Short-term preservation5.3.2
Long-term preservation5.2.3
Choice of preservation5.3.1
Purpose of preservation
For the study of various biological processes, the preservation of bateria plays a key
role in ensuring reproducible results and continuity in research.
Maintaining a library of microbial stocks also enables microorganisms to be easily
stored and retrieved, as compared to continuously sub-culturing microbial cultures
on plates or in tubes.
Characteristics of bacteria is with easy variation, therefore, the isolates should be
preserved in inactive or relative state within a certain period of time.
Advantages of preservation
Overall safety of bacterial stocks against loss due to equipment failure or contamination by
other microbial organisms.
Elimination of time, energy, and material costs associated with the maintenance of bacterial
strains not currently in use.
Insurance against phenotypic drift associated with prolonged passage, due to genetic
instability and/or selective pressures.
Creating a standard working stock that can be used for a series of experiments.
Preservation
Long time Preservation
Short time Preservation
Passage culture method
Liquid paraffin covered method
CryoperservationFreeze drying
method
Depending on the length of storage and type of bacteria, different preservation methods are used.
Methods of preservation
Long term preservation is to keep microorganisms in a dormant state, free from
contamination and genetic changes, until ready to be brought back to life later.
This is conventionally accomplished by using low temperatures, and freezing or
drying techniques to reduce cellular metabolic activity.
Short term preservation, continuous growth of microorganisms can be maintained for short
periods of time on agar medium. Bacteria can be stored using this method and it is
recommended for cultures which are used regularly. It is easy to recover the
microorganisms later.
Short-term preservation5.3.2
1. Passage culture method (Repeated subculturing)
The isolates were inoculated on slant culture,
puncture culture, or blister meat culture (for
preservation of anaerobic bacteria), etc.
After culture, it is kept in the refreigerator at 4-6℃
for storage about one month.
Repeated subculture the isolats to a fresh medium
every other month.
Easy to operate, without specialized equipment, check at any time
× Easy contaminated, occupied lots of space, phenotypic drift associated with prolonged passage
2. Liquid paraffin covered method
This is a modified method of subculturing, can prolong the
preservation time.
It is the culture and puncture in the slant culture covered by
sterile liquid paraffin, which can prevent the medium moisture
evaporation caused by bacterial death, on the other hand can
prevent the entry of oxygen to reduce bacterial metabolism.
Preserved for about 3 months in the refreigerator at 4-6℃ or
at room temperature.
Easy to operate, without specialized equipment
× Keep stand upright, occupied lots of space, inconvenience to carry
Long-term preservation5.3.2
1. Cryopreservation
Low temperature protects proteins and DNA from denaturation and damage and slows the
movement of cellular water.
Cryopreservation refers to the preservation of biological materials at cryogenic temperatures,
generally at -80℃ or -196℃ for storage of five years. .
This is appropriate for most non-fastidious bacterial strains.
(1) Cryoprotectant agents
Cryoprotectant agents are essential in cryopreservation.
Glycerol and dimethyl sulfoxide (DMSO) are the most common agents.
Stock concentration: 20 %
Final concentraion: 10 %
Sterilized by autoclaving
Stored in aliquots protected from light
Stock concentration: 50 %
Final concentraion: 5 %
Sterilized by filtration
Stored in aliquots protected from light
(2) Cryopreservation Procedure
a) In preparation for freezing, grow the bacterial strain under optimal conditions in an
appropriate medium as to retain the salient features of the strain. Bacterial strains
should be grown to late log phase.
b) When freezing bacteria, add 5 to 10% glycerol or DMSO in culture medium.
c) Label the appropriate number of vials with the name of the bacterial strain and the
date. Aliquot 1 to 1.8 mL of the bacterial suspension to each vial and seal.
d) Allow the cells to equilibrate in the freeze medium at room temperature for a
minimum of 15 minutes but no longer than 40. After 40 minutes, cell viability may
decline if DMSO is used as the cryoprotectant.
e) Place the vials into a pre-cooled (4°C) freeze chamber and place the chamber in a
mechanical freezer at -70°C (or colder) for at least 2 hours.
f) Quickly transfer the vials to liquid nitrogen or a -130°C freezer.
g) Record the location and details of the freeze.
Recovery of the isolates
Storage of the isolates
(3) Commercial cryovials
Designed for the storage of biological material at temperatures as low as –196 ºC (liquid nitrogen ).
The sterile vials contained 25 chemically treated breeds to which the mircroorganisms can adhere.
The hypertonic cryopreservation solution guarantees high recovery rates, even for fastidious species.
Easy to operate, easy removal of cryovials from
freezer without riks of defrosting.
Transfer the conlonies to the cryovial
Reverse the cryovial to mix the content well
Pipette out the redundant cryoprotectant agents
Lable and place into a chamber
Storage of the isolates
Pick up a breed Transfer to a liquid or solid medium Replace the tube to the chamber
Recovery of the isolates
2. Freeze-drying or lyophilization
Lyophilization is the process where water and other solvents are removed from a
frozen product via sublimation which occurs when a frozen liquid goes directly to a
gaseous state without entering a liquid phase.
This is often the preferred long-term preservation method in most microbial
resource centres because of the low cost of maintenance and ease of transportation
of lyophilized cultures.
The freeze-drying process results in a stable, readily rehydrated product.
Storage for decades at refrigerated temperatures (4˚C)
× Complicated operation, need specialized equipment (freeze dryer)
REFERENCES
Preservation of bacteria: ATCC. 2015. ATCC® bacterial culture guide: tips and techniques for culturing bacteria and bacteriophages. Virginia, USA,
American Type Culture Collection.
Discussion and Q&A
THANKS FORYOUR ATTENTION!
