The preservation of microorganisms by different methodologies has been employed for decades.

The primary methods of culture preservation are continuous growth, drying, and freezing. Freeze-
drying and freezing below 135˚C are excellent methods for permanent preservation of bacteria,
actinomycetes, yeasts, and fungi. For long-term storage, cultures are usually preserved by
lyophilization or by ultra-freezing. In these methods, two basic approaches are employed to slow
down the rate of deleterious reactions in microbial culture. The first is to lower the temperature
which decreases the rate of all chemical reactions. This can be done using refrigerators and liquid
nitrogen freezers. The second option is to remove water from the culture, a process which can be
tricky and involves sublimation of water using a lyophilizer.

Periodical Transfer to Fresh Media

Microbial strains can be maintained by periodically preparing a fresh culture from the previous
stock culture. The culture medium, the storage temperature, and the time interval at which the
transfers are made vary with the species and must be ascertained beforehand. The temperature and
the type of medium chosen should support a slow rather than a rapid rate of growth, so that the
time interval between transfers can be as long as possible. Many of the microbes remain viable for
several weeks or months on a medium like nutrient agar or potato dextrose agar (PDA). This
method is generally used for maintenance of cyanobacteria. The periodic transfer method has the
disadvantage of failing to prevent changes in the characteristics of a strain due to the development
of variants and mutants.

Refrigeration

Microbes can be preserved for a short period of time at 4 ˚C. The cultures require frequently in
active growth on agar slants or plates can be stored in a refrigerator and precaution has to be taken
to avoid contamination. Cultures should be prepared using standard techniques and then sealed
before storing. For slants, it is recommended to use screw-capped tubes. For cultures on Petri
dishes, the plates need to be sealed with Parafilm. Sealing the plates not only helps to prevent
molds from sneaking into the plates, but it also slows the drying of agar. For short term over a
week or two, cultures can be stored as stabs in small, flat-bottomed screw-capped vials. In this
technique, vials are filled with a small amount of agar medium (e.g., 1 ml) and sterilized. Microbes
(e.g., bacteria) are then introduced into the solidified agar with a sterile needle. The culture is
incubated overnight and then stored at 4 ˚C. Cultures stored in stabs are more resistant to drying
and contamination, but they will lose viability more quickly than frozen stocks. The length of time
a stab can remain viable is dependent upon the strain.
Mineral Oil or Liquid Paraffin Storage

Covering the fresh growth in media slants with sterile mineral oil or liquid paraffin can preserve
many bacteria and fungi. The method’s basic idea is covering the well-grown culture on agar
nutrient medium with sterile mineral oil. The most common used oil is paraffin or vaseline with
layer thickness of 1–2 cm. The aim is to limit the oxygen access that reduces the microorganism’s
metabolism and growth, as well as to restrict the cell drying during preservation. The cell viability
in this method is high as compared to frequent transfer and storage at low temperature.

Freezing

Freezing is a good way to store bacteria and most of fungi. Generally, the colder the storage
temperature, longer the culture will remain viable. Freezers can be split into three categories:
laboratory, ultralow, and cryogenic. However, ice crystal formation is the major problem, when
bacteria are stored at low temperature. Ice can damage cells by dehydration caused by localized
increases in salt concentration. As water is converted to ice, solutes accumulate in the residual free
water and this high concentration of solutes can denature biomolecules. To lessen the negative
effects of freezing, glycerol is often used as a cryoprotectant. With bacteria, adding glycerol to
final concentration of 15 % will help to keep cells viable under all freezing conditions.

Freezing Bacteria Using Glycerol

Bacteria can be frozen using 15 % glycerol. The process is simple and requires screw cap
microfuge tubes and sterile glycerol. The glycerol is diluted to 30 % and an equal amount of
glycerol and culture broth are mixed, dispensed into tubes, and then frozen.

Freeze-Drying (Lyophilization)

Freeze-drying (lyophilization) is a well-established method for long-term storage. It is a method

of removing water, which not only serves as the medium for enzymatic reactions but also
spontaneous negative reactions such as free radical formation. Many bacteria and spore forming
fungi can be preserved very effectively by freeze-drying. By freezing the cells in a medium that
contains a lyoprotectant (usually sucrose) and then pulling the water out using a vacuum
(sublimation), cells can be effectively preserved. This method is laborious and requires specialized
equipment, but it has the advantage of generating stock cultures that are unaffected by power
outages and empty liquid nitrogen tanks.
Furthermore, if cultures are routinely shipped to other labs, freeze-dried cultures do not require
special handling. The downside on freeze-drying is that not all cultures react the same way thus
some experimentation is required to optimize the process for each strain. There are four significant
considerations for freeze-drying microorganisms. Culturing and preparing the cells is the first
consideration. Generally this is not different than methods for typically culturing bacteria. The
second aspect involves suspending the bacteria in a suitable freeze-drying medium, commonly,
skim milk or sucrose is used. The third consideration is the freezedrying process, which is
extremely dependent upon the type of freeze-dryer used and the quantity of samples to be
preserved. The final aspect deals with post-lyophilization storage. This process can be used to
preserve bacteria, fungi, yeasts, proteins, nucleic acids, and any other molecules which may be
degraded due to the presence of water.

Cryopreservation

Cryopreservation refers to the storage of a living organism at ultralow temperature (196 ˚C) such
that it can be revived and restored to the same living state as before it was stored. It is a very
reliable method and is generally considered superior to other preservation methods. Bacteria
preserved in liquid nitrogen normally show high survival rates and good strain stability during
long-term storage. In liquid nitrogen storage of microorganisms, polypropylene cryotubes, glass
vials, glass capillaries, and polypropylene straws are generally used. Several factors can affect cell
viability and stability during cryopreservation. During cryopreservation, dehydration of cells
results and osmotic imbalance is created due to the changes in the concentration of salts and other
metabolites. During the cooling process, rupture of the cellular membranes can also occur by the
formation of large ice crystals. Successful preservation can be achieved by the use of
cryoprotective agents (e.g., dimethylsulfoxide and glycerol), maintaining a controlled rate of
cooling (about 1 ˚C/min to about 30 ˚C), and an appropriate rewarming protocol (rapid thawing in
a 37 ˚C water bath which takes about 1 min for a glass ampoule and somewhat longer for a plastic
vial). In practice, a relatively slow cooling rate can be easily obtained by keeping ampoules/vials
in mechanical deep freezers for 1–2 h or in the neck of the liquid nitrogen storage unit for some
minutes and then lowering containers into it. It is, however, not good practice to plunge cultures
directly into liquid nitrogen, as the liquid nitrogen may seep into any imperfectly closed or sealed
capillaries, ampoules, or vials containing the bacterial suspensions. On removal from storage,
nitrogen (inside an ampoule) will virtually instantly change to the gaseous phase causing an
explosion. For safety reasons it is thus recommended that cultures should be stored in the gas phase
of liquid nitrogen. While preparing cells for cryopreservation, several factors such as optimal
growth conditions, physiological state of the cells (preferably from the late logarithmic to early
stationary phase of growth), high cells density should be considered as these can affect cell
viability after cryopreservation. After mixing, cell suspensions should be kept for equilibration
with the cryoprotective agent. For harvesting, liquid cultures are centrifuged. However, vigorous
pipetting and high-speed centrifugation should be avoided and cells should be handled gently.
Viability assays should be performed on all cultures before and after cryopreservation to assure
long-term viability. To assure purity, identity of the preserved cultures should be verified and after
freezing cultures should be recharacterized to assure their stability.