Maintenance and Preservation of Microbial Cultures

A pure culture is formed from a mixed microbial population in a solid

nutritional medium. Pure culture is the isolation and cultivation of an isolated
colony from a mixed microbial population.
To research the morphology and physiology of the microbial colony isolated
from the mixed culture, a single colony or pure culturing is required. The
isolation colony must also be preserved and maintained in order to improve
the vitality of the microbial cell.
Pure Culture:
Pure culture is a technique widely utilised in the microbiological study of a
single cell or organism. The atmosphere contains a diverse microbiological
community. To isolate a single colony from a mixed population, pure culture
methods have been established. It entails aseptically transferring a single cell
to fresh nutritional agar medium.
Pure culture of microorganisms protects and revitalises the bacteria' various
features such as shape, size, and growth pattern. Isolation of the microbial
culture is critical before maintenance and preservation.
Pure culture simply refers to a single colony or cell development that is free of
contaminants and cell variations. The following are five types of pure culture:

1. Streak culture: By streaking the small inoculum from the diluted

microbial cell suspension, streak culture isolates distinct colonies.
2. Stab culture: The microbial culture is stabbed down the centre of a test
tube holding a solid nutritional medium using an inoculating needle or
an earbud.
3. Stock culture: Stock culture preserves the vitality of microorganisms by
subculturing a single microbial cell into a fresh media.
4. Starter culture: The creation of live organisms that commence
fermentation by modifying the chemical composition of the substrates
or raw materials is known as starter culture.
5. Enrichment culture: By providing ideal growing conditions, enrichment
culture separates certain microbial cells or organisms of interest.
The pure culture technique tries to achieve the following results:
  It ensures the survival of the culture.
 Furthermore, the pure culture approach preserves the microbial cell
properties.
 The preserved culture might be used for taxonomic research or
investigations.
 It also serves as an assay tool for vaccine development and industrial
manufacturing methods such as enzymes and organic compounds.

Preservation of Pure Culture

As shown in the diagram below, four standard procedures are used to preserve
distinct strains of microorganisms.

Refrigeration

Paraffin coating
Preservation
Techniques of
Pure Culture
Cryopreservation

Lyophilization

Refrigeration
The pure culture can be kept or conserved at temperatures ranging from 0 to 4
°C. The discrete colony of interest is streaked from the solid nutritional
medium to create slant cultures.
Refrigerators can keep bacterial cultures alive for up to 2-3 weeks and fungal
cultures alive for 3-4 months. The refrigeration temperature significantly slows
but does not entirely stop the metabolic activities of the organisms.
As a result, the microorganisms gradually consume the nutrient medium,
multiply, and eventually die due to nutrient scarcity and toxic waste
accumulation. The disadvantage of chilled microbial culture is that the cells are
only kept for a brief period of time.
Paraffin Coating
It is a technique for covering microbial cultures with mineral oil or paraffin.
Mineral oil should be put up to the slant's height to completely cover the
microbiological growth. The paraffin coating approach keeps the cell culture
alive for around 1-2 years. It is a simple and low-cost approach.
The slant cultures must be stored vertically in sterile liquid paraffin at room
temperature. The moisture content of the nutritional medium is maintained by
liquid paraffin. It also keeps anaerobic conditions.
Thus, a layer of paraffin keeps the bacterial and fungal cells inactive for several
years. It is a cost-effective method of preserving microbial cells for a longer
period of time.
Cryopreservation
It uses cryopreservative chemicals such as glycerol and dimethyl sulfoxide to
maintain the thick suspension of microbial culture. Small vials or ampoules are
used in cryopreservation to keep the microbial culture at an extremely low
temperature.
Cryo freezers are classified into two types: liquid nitrogen freezers and
mechanical cryogenic freezers. At a freezing temperature of -196 °C, liquid
nitrogen freezers preserve cell culture in the liquid or vapour phase (of liquid
nitrogen).
Mechanical cryogenic freezers, on the other hand, keep cell culture viability at
-150 °C. Cryopreservative chemicals act as stabilising agents, preventing ice
crystal formation and so preserving cell culture. Cell viability in
cryopreservation ranges from 10 to 30 years.
It preserves the microbial cell of interest that lyophilization could not preserve.
It is an extremely pricey procedure. The efficiency of cryopreservation is
determined by factors such as the kind of microbial cell, the cryoprotectant
used, and the rates of cooling and thawing. Cryopreservation is a less difficult
method of long-term microbial cell preservation than lyophilization.
Lyophilization
A dense cell suspension of microbial culture can also be preserved by freeze-
drying or lyophilization. In contrast to cryopreservation, it employs a
lyoprotectant (often sucrose) to stabilise the microbial cell culture. The
microbial cell suspension must be collected in tiny vials for freeze-drying.
Lyophilization requires freezing the cell culture at temperatures ranging from -
60 to -78 °C. The cell culture is next vacuumed and subjected to sublimation,
which dehydrates the moisture content of the microbiological sample.
It produces small distortions in microbial cell structures and keeps cells alive
for a longer period of time (more than 30 years). Lyophilized cultures require
less storage space. Small vials can now be easily transported to other labs or
collection centres.
Nonetheless, lyophilized cultures do not necessitate any particular care. It is a
low-cost method that necessitates the use of specialised equipment and
reagents. The disadvantage of the lyophilization method is that it must be
optimised for each strain because the method does not function in the same
way for all strains.

Maintenance of Pure Culture

Subculturing is the most effective method of preserving pure culture viability.
It entails the systemic transfer of a little amount of inoculum from the pure old
culture to the new nutritional agar slants. Stock culture is prepared on a
regular basis from the preceding stock culture.
Subculturing is just the passage of cells. Subculturing of suspended bacteria
and fungus refers to the cultivation of non-adherent or suspension cells.
Microorganisms that do not adhere to a surface proliferate in liquids.
Thus, non-adherent cell subculturing entails diluting a tiny amount of the old
stock culture in a new growth medium. It allows for cell line proliferation.
However, due to the emergence of variations and mutations, it fails to
maintain the properties of a strain.
Conclusion
As a result, we can conclude that biopreservation is critical in maintaining cell
integrity, functionality, and features. For different microbial strains, we might
utilise different preservation strategies. Thus, culture preservation efficiently
keeps cells inactive by preventing the formation of contaminants and
variations.