Preserving and maintenance of Microbial Cultures in Food Microbiology Laboratory

 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.

•                      Freeze-drying

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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