Preserving and
maintaining microbial cultures is an essential part of food microbiology.
Microbial cultures are used for internal quality control. In this article, we
will discuss in detail the procedure for preserving and maintaining microbial
cultures in food microbiology laboratories.
1. Authenticity
of cultures
Before preserving a microbial culture from any
source, it is important to identify and verify it correctly. Microbial cultures
can be identified using standard microbiological techniques, such as culturing,
staining, and biochemical tests. It is crucial to know the specific characteristics
of the culture, including its morphology, growth requirements, and any
potential pathogenicity.
2.
Prepare the culture
Before preservation,
the microbial culture should be prepared by ensuring that it is healthy,
active, and in the desired growth phase. This may involve sub-culturing the
microorganism and growing it in a suitable medium. The culture should be grown
to its maximum density in the medium to ensure that there are sufficient cells
for preservation.
3. Use a sterile technique
When handling microbial
cultures, it is important to use sterile techniques to avoid contamination.
This includes wearing gloves, working in a laminar flow hood or other sterile environments, and using sterile tools. Contamination can lead to the loss of the
culture or the introduction of unwanted microorganisms.
4.
Preservation method
There are several
methods for preserving microbial cultures, including Subculturing, freezing,
and Freeze-drying. The choice of method depends on the laboratory.
- . Subculturing is a method of preserving
microbial cultures by transferring a small amount of the culture to a fresh
growth medium periodically. This process can help maintain the viability of the culture and ensure that the microorganisms remain healthy and active over time.
Subculturing is a widely used technique in microbiology laboratories for
preserving frequently used microbial cultures. This can be stored at 2-8°C. However,
subculturing has some limitations. Frequent subculturing can lead to changes in
the morphology and physiology of microorganisms, which can affect their genetic
stability and alter their characteristics. Additionally, repeated subculturing
can increase the risk of contamination, as microorganisms can be introduced
during the transfer process.
- Freezing: Freezing is a common method
for preserving microbial cultures. The microorganisms are frozen at low
temperatures, usually -20°C or -80°C, which slows down their metabolic activity
and prevents growth. Before freezing, the culture is mixed with a
cryoprotectant, The role of cryoprotectants is to prevent the formation of ice
crystals, which can cause physical damage to the cell membrane and disrupt
cellular functions. Cryoprotectants work by lowering the freezing point of
water and stabilizing cell membranes. They also help to maintain the osmotic
balance within the cell and prevent dehydration. The principle behind
cryoprotection is that it provides a protective shield around the cells,
allowing them to survive the freezing and thawing process. Cryoprotectants can
be natural or synthetic compounds, such as glycerol, dimethyl sulfoxide, and
sucrose. In practice glycerol (15% - 20%), use as a Cryoprotectant.
Also
known as lyophilization, is a process of removing water from biological
samples, including microbial cultures, by sublimation under a vacuum.
Freeze-drying is a popular method of preserving microbial cultures because it
can prolong the shelf-life of the culture and reduce the need for frequent
subculturing.
To
prepare a culture for freeze-drying, a cryoprotectant, such as glycerol, is
added to protect the cells during freezing. The culture is then frozen and
placed in a freeze-dryer, where the frozen water is removed through
sublimation. The resulting freeze-dried culture is a stable, dehydrated product
that can be stored for extended periods at low temperatures.Freeze-drying
offers several advantages over other methods of preserving microbial cultures.
Freeze-dried cultures can be easily transported, as they are lightweight and
require little storage space. The process also enables cultures to be revived
quickly, by adding suitable media.
However,
freeze-drying does have some limitations. It can be an expensive process, additionally,
the process can damage some fragile microorganisms, resulting in decreased
viability. This process is generally adopted by the culture collection centers to
supply reference culture to the laboratory.
5.
Storage of the culture
The stored microbial
culture should be labeled clearly and stored in a suitable environment. The
storage conditions should be optimized for the specific culture and
preservation method. The culture should be periodically monitored for viability
and contamination.
Approximate time for
cultures to remain viable in different storage conditions.
|
Preservation
condition
|
Temp (°C)
|
Time
|
|
Agar
plates/Slant
|
2-8
°C
|
4
- 6 weeks
|
|
Deep
freezer
|
-20
°C
|
1
- 3 years
|
|
Ultra
deep freezer
|
-
80 °C
|
5-6
years
|
|
Freeze
dried
|
2-8
°C
|
15
years+
|
6.
Periodic maintenance
To ensure that the
microbial culture remains viable and active, it is important to perform
periodic maintenance. This may involve sub-culturing the culture at regular
intervals, testing its viability, and storing it under suitable conditions. The
frequency of maintenance depends on the specific culture and preservation
method.
Acceptable
number of passages for bacterial cultures in the laboratory
It is widely agreed
that minimizing the number of passages is crucial to prevent the occurrence of
phenotypic variations, genetic drift, and contamination in bacterial cultures.
However, standards organizations have different opinions regarding the
acceptable number of passages.
The U.S. Pharmacopeia
(USP) provides specific guidelines on this matter. According to USP General
Chapter: <1117> Microbiological Best Laboratory Practices, the number of
transfers of working control cultures should be monitored to prevent excessive
subculturing that may increase the risk of phenotypic alteration or mutation.
The USP further states that working cultures used for testing should not exceed
five passages from the American Type Culture Collection (ATCC) reference
culture.
USP 36-NF 31 <51>
also emphasizes that viable microorganisms used in testing must not be more
than five passages removed from the original ATCC culture. Additionally, the
USP 36-NF 31 General Notices: Terms and Definitions defines microbial strains
cited and identified by their ATCC catalog number should be used directly or,
if subcultured, should not be more than five passages removed from the original
strain.
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