This document discusses various methods for preserving pure bacterial cultures, including periodic transfer to fresh media, storage at low temperatures, overlaying with mineral oil, lyophilization, and cryopreservation. Lyophilization, or freeze drying, involves freezing the culture, removing moisture under vacuum to leave a dried form, and storing at cold temperatures. It allows for long-term storage of viable cultures. Cryopreservation uses cryoprotectants like glycerol and dimethyl sulfoxide to suspend cultures before freezing at very low temperatures like in liquid nitrogen. This stops biological activity and preserves cultures in a dormant state for 10-30 years. Both lyophilization and cryopreservation allow for long-term preservation of viable microbial cultures

1. MMMMM
MAINTANANCE AND
PRESERVATION OF
PURECULTURE
BY:
V. Dhanalakshmi
I M.SC, MB
AJ College, Sivakasi.

2. Introduction:
• Biopreservation is the process of preserving the integrity and functionality of cells.
• The primary aim of culture preservation is to maintain the organism alive
uncontaminated and without variation or mutation.
• The cultures maintained in viable conditions are called “Stock culture
Collection”.
• Preservation here refers to maintenance of the pure culture to keep them viable
for extended duration of the time without any genetic change.

3. Objectives of Preservation:
1.Academic use.
2. Future use.
3.Biotechnology field.
4.Research purpose.
Methods of Preservation:
1.Periodic transfer to fresh media.
2.Storage @ low temperatures.
3.Storage in sterile soil.
4.Preservation by overlaying cultures with mineral oil.
5.Lyophilization.

4. Periodic transfer to fresh media.
•Strains can be maintained by periodically preparing a fresh culture from the previous
stock culture.
•The transfer is always subject to aseptic condition to avoid contamination .
•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 more common heterotrophs remain viable for several weeks or months on
a medium like Nutrient Agar.
•The transfer method has the disadvantage of failing to prevent genetic changes.

5. Storage @ low temperatures:
•Pure cultures can be successfully stored at 0-4°C either in refrigerators or in cold-
rooms.
•This method is applied for short duration (2-3 weeks for bacteria and 3-4 months for
fungi) because the metabolic activities of the microorganisms are greatly slowed
down but not stopped.
•Thus their growth continues slowly, nutrients are utilized and waste products released in
medium.
•This results in, finally, the death of the microbes after sometime.

6. Preservation by overlaying cultures with mineral oil:
This is a simple and most economical method of maintaining pure cultures of bacteria and
fungi.
In this method, sterile liquid paraffin is poured over the slant (slope) of culture and stored
upright at room temperature.
The layer of paraffin ensures anaerobic conditions and prevents dehydration of the medium.
This condition helps microorganisms or pure culture to remain in a dormant state and,
therefore, the culture can be preserved for months to years (varies with species).
The advantage of this method is that we can remove some of the growth under the oil with a
transfer needle, inoculate a fresh medium, and still preserve the original culture.

8. Lyophilization :
• Lyophilization is also known as freeze drying.
• It was discovered by Richard aldmann in 1980.
• Bacteria are stored in low temperature.
PRETREATMENT:
Pretreatment include concentrating the product to make it stable by adding certain
type of component depending on the product.
FREEZING :
Solidify the product.
Ice crystal formed at -50°C -80°C

9. PRIMARY DRYING :
Sublimation : Solid state is directly converted into gaseous state.
Ice will be removed from the product which is freezed.
Pressure and heat is supplied.
98 % 99 % of moisture will be removed.
SECONDARY DRYING :
It was also known as desorption.
Temperature at -50°C and pressure was applied.
100% moisture will be removed.
Lyophilized culture was stored in dark at 4°C in refrigerator.

10. Advantage of Lyophilization:
• Only minimal storage space is required; hundreds of lyophilized cultures can be
stored in a small area.
• Small vials can be sent conveniently through the mail to other microbiology
laboratories when packaged in special sealed mailing containers.
• Lyophilized cultures can be revived by opening the vials, adding liquid medium, and
transferring the rehydrated culture to a suitable growth medium.
• Freeze-drying method is the most frequently used technique by culture collection
centers.
• Many species of bacteria preserved by this method have remained viable and
unchanged in their characteristics for more than 30 years.

11. Cryopreservation:
It preserves the dense suspension of microbial culture by
employing cryopreservative agents like glycerol and dimethyl sulfoxide.
Enzymatic or chemical activity of the biological material will be totally stopped and
this leads to preservation of material in dormant state.
Liquid nitrogen freezers and mechanical cryogenic freezers are the two kinds of
cryo freezers. Liquid nitrogen freezers preserve cell culture in the liquid or vapour
phase (of liquid nitrogen) at a freezing temperature of -196 °C.
Cryopreservative agents serve as stabilizing agents that maintain the cell culture by
preventing ice crystal formation. The cell viability under cryopreservation is between 10
to 30 years.

12. It preserves the microbial cell of interest that could not be preserved under
lyophilization. It is quite an expensive method.
Factors like the type of microbial cell, choice of the cryoprotectant, rates of
cooling and thawing decide cryopreservation effectiveness.
Cryopreservation is comparatively easy in the long term preservation of the
microbial cells than lyophilization.

13. Advantages of cryopreservation :
• It is beneficial in storing a large range of disease-free biological samples for long periods of time.
• The national and international transportation of samples has become easier with cryopreservation
• Since this technique freezes the biological product at a particular stage, the sample remains viable for an
indefinitely long period of time.
• It also saves a lot of research time since scientists do not have to wait for fresh viable cells.
Disadvantages of cryopreservation :
• One of the major disadvantages of this technique is that ice crystals can form inside the cells thereby
causing cell damage.
• The use of unsuitable cryoprotectants also affects cell viability.
• Water migration can cause extracellular ice formation and cellular dehydration. Such stresses can
damage the cells directly.
• There is no standard protocol for every biological sample, and thus expertise and time are required to
store precious samples.

14. Freezing at -70°C :
Long-term storage of aerobes and anaerobes can be accomplished by freezing at -70°C.
Frozen, non-fastidious organisms should be thawed, reisolated, and refrozen every five
years; fastidious organisms should be thawed, reisolated, and refrozen every three years.
Acid-fast bacilli (AFB) may also be frozen at -70°C in 7H9 broth with glycerol.
Viruses may be stored indefinitely at -70°C in a solution containing a cryoprotectant, such
as 10% dimethyl sulfoxide (DMSO) or fetal bovine serum.

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