Preservation of the isolates

Preservation of the isolatesDr. Yuting Deng

Pearl River Fisheries Research Institute (PRFRI), Chinese Academy of Fishery Sciences (CAFS), China

dengyt@prfri.ac.cn2021.07.29

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!