Culture Preservation Strategies

Culture Preservation Strategies

Once a microorganism has been isolated and grown in pure culture, it is necessary to maintain the viability and purity of the microbial culture by keeping the pure culture free from contamination. The goal of preserving microbial cultures is to keep the organisms alive, uncontaminated, and without mutation. Microbial culture preservation is the process of storing microorganisms so they can be used for research and to maintain biodiversity.   It ensures reproducible results and continuity in research, allows microorganisms to be easily stored and retrieved and help to preserve biodiversity and genetic resources.

Culture preservation strategies include: regular subculturing, paraffin method, storage in soil, storage in silica gel, refrigerator/cold room storage, freeze-drying (lyophilization), preservation under liquid nitrogen, etc. 

Generally, in laboratories, the pure cultures are periodically sub cultured, that is, transferred periodically into a fresh medium to allow continuous growth and viability.  The transfer is always done under aseptic conditions to avoid contamination. This method is time-consuming, it is very difficult to maintain if there is a large number of pure cultures and there is a risk of genetic changes as well as contamination. So, this technique is replaced by methods such as refrigeration, paraffin method, cryopreservation, lyophilization, etc.

Examples for Short-term Storage methods include Periodic Transfer to Fresh Media, Refrigeration, Preservation in Glycerol at -20 °C, Stab Cultures, etc

Examples for Long -term Storage methods include Cryopreservation, Lyophilization (Freeze-Drying), Paraffin Method, etc

Regular subculture or Periodic Transfer to Fresh Media:

This involves periodically transferring a small portion of a growing culture to fresh media to maintain viability and prevent genetic drift. Transferring a small portion of a culture to a fresh, sterile medium at regular intervals replenishes nutrients and removes metabolic waste products, keeping the culture viable.  

The Frequency of the procedure depends on the organism's growth rate and storage conditions. Fast growing bacteria might need subculturing weekly, while slower-growing fungi might only need it monthly.  Cultures stored at lower temperatures (e.g., in a refrigerator) generally require less frequent subculturing.  Many common heterotrophs remain viable for several weeks or months on a medium like nutrient agar.

This is a Simple method and requires minimal equipment.  Disadvantages include High risk of contamination, chance of genetic changes, labor-intensive, risk of culture loss due to errors, etc., and this method is not suitable for long-term storage.  

Stab cultures at room temperature are used for non-fastidious organisms such as Staphylococci and Enterobacteriaceae. For this, tubes of carbohydrate-free agar with deep butt are prepared and the organism is stabbed into the agar.  After overnight incubation, the tube cap is sealed using molten paraffin wax and may be stores at room temperature for about one year. 

Stab culture in cystine trypticase agar (CTA) method is recommended for the preservation of Neisseria and Streptococci. For Neisseria, transfer at every two weeks and for Streptococci, transfer at every month is required.

Paraffin method:

This is a simple method where a layer of sterile mineral oil is placed over the culture on an agar slant, slowing down metabolism by limiting oxygen access. 

Covering the culture with a layer of sterile paraffin oil reduce oxygen availability and slow down metabolism and prevent desiccation.   For this, the culture is grown in a solid medium and sterile paraffin oil is added to cover the culture surface. This may be stored at room temperature or in a refrigerator.

This technique is Simple, relatively inexpensive and suitable for some fungi and bacteria.

Procedure:

•  Grow the microorganism on a solid agar medium.
•  Prepare sterile paraffin oil by heating it to 121°C for 2 hours (or by filter sterilization).
•  Once growth is visible, carefully pour sterile paraffin oil over the culture, forming a layer of about 1-2 cm.
• Seal the container to prevent contamination and evaporation.

Suitable Organisms: Primarily used for some fungi and yeasts. Less effective for bacteria.

Disadvantages - Not suitable for all organisms, can be difficult to revive the culture, risk of contamination.

Storage in soil:

Some microorganisms can be preserved by embedding them in sterilized soil, which provides a protected environment for long-term storage. 

Procedure:

•  Prepare a thick suspension of the microorganism in sterile water or buffer.
•  Mix the suspension with the sterile soil.
•  Allow the soil to air dry completely.
• Store the dried soil in a sterile container at room temperature or in a cool, dry place.

Primarily used for spore-forming bacteria and fungi that can withstand desiccation.

Disadvantages - High risk of contamination, Difficult to retrieve pure cultures, Not suitable for all microorganisms.

Storage in silica gel:

Cultures are dried on silica gel, a desiccant, to absorb moisture and preserve viability. 

Procedure:

Use commercially available silica gel desiccant. Dry it thoroughly in an oven and sterilize by dry heat sterilization.

• Prepare a concentrated suspension of the microorganism.
• Mix the suspension with sterile silica gel.
• Allow the silica gel to dry completely.
• Store the dried silica gel in a tightly sealed container at room temperature or in a refrigerator.

Suitable for some bacteria and fungi.

Advantages over Soil: Provides a more controlled drying environment, reducing the risk of contamination.

Limitations: Not suitable for all organisms, can be challenging to revive the culture, risk of contamination.

Refrigerator/cold room storage:

Keeping cultures at low temperatures in a refrigerator or cold room slows down metabolic activity, allowing for short-term preservation.

Refrigeration temperature slow down metabolic activity.  Here culture is stored in a refrigerator (4°C) or cold room (e.g., 10-15°C) and is suitable for short-term storage of many microorganisms.

Many bacteria, fungi, and yeasts can be stored for short periods (weeks to months).

Advantages: Simple, readily accessible.

Disadvantages: Not suitable for long-term storage, risk of contamination, some organisms are sensitive to cold.

Freeze-drying (lyophilization):

This method involves rapidly freezing a culture followed by dehydration under vacuum, which allows for long-term storage.  The principle is the removal of water from a frozen culture by sublimation, the dried culture is then sealed in a vial.  This preserves the culture in a dormant state and is suitable for many bacteria, fungi, and viruses.

Procedure:

• Prepare a concentrated cell suspension in a cryoprotective agent (e.g., glycerol, skim milk).
• Rapidly freeze the culture in liquid nitrogen or a dry ice-ethanol bath.
• Remove water by sublimation under vacuum in a lyophilizer.
• Seal the dried culture in a sterile vial under vacuum or with an inert gas.
• Store the lyophilized culture at room temperature or in a refrigerator/freezer for long-term preservation.

Cryoprotective Agents are used to prevent ice crystal formation, which can damage cells. Commonly used agents include glycerol, dimethyl sulfoxide (DMSO), and skim milk.

Advantages: Excellent for long-term storage, minimizes genetic changes, high viability upon rehydration.

Disadvantages: Requires specialized and expensive equipment (lyophilizer).  

Preservation under liquid nitrogen (Cryopreservation):

This technique is considered as the most effective method for long-term storage, cultures are suspended in a cryoprotectant and stored in liquid nitrogen at very low temperatures. This effectively stops all metabolic activity.  

Procedure:

• Cryoprotectant Addition: Mix the culture with a cryoprotective agent (e.g., glycerol, DMSO) to a final concentration of 5-10%.
• Freezing: Use a controlled-rate freezer or directly plunge vials into liquid nitrogen (-196°C). Controlled-rate freezing is often preferred to minimize ice crystal formation.
• Storage: Store the vials in liquid nitrogen.
• To revive the culture, thawing is done, Thaw rapidly in a water bath at 37°C. Remove the cryoprotectant as quickly as possible.

This is Suitable for a wide range of microorganisms, including bacteria, fungi, viruses, and cell lines.

Advantages: Best method for long-term storage, minimal genetic changes, high viability.

Disadvantages: Requires specialized equipment and careful handling of liquid nitrogen, can be expensive.

Choice of a culture preservation method depend on the type of organism, storage duration, cost and accessibility.  Different microorganisms have varying tolerance to different preservation methods. For short-term needs, refrigeration might be enough, while long-term preservation usually requires cryopreservation. Some methods like the paraffin method are inexpensive and readily available, while others like liquid nitrogen storage might require specialized equipment. 

Any culture preservation methods have the following concerns - Culture Purity, Sterility, Labelling, Record Keeping and Revival

Culture Purity -Start with a pure culture.

Sterility - Maintain sterile conditions throughout the preservation process.

Labelling - Clearly label all cultures with the organism's name, strain number, date of preservation, and any other relevant information.

Record Keeping -Maintain detailed records of the cultures, including source, characteristics, preservation method, and date.

Revival - Develop a standardized procedure for reviving the preserved cultures. Test the viability of the cultures periodically.

Microbial culture collections:

Microbial culture collections are repositories of microorganisms, cell lines, and other materials used for research. The key components of microbial culture collections include: 

These are specialized laboratories that maintain diverse microbial cultures, often with detailed information about each strain.  They play a crucial role in preserving biodiversity and providing cultures for research, education, and industry.   Their functions include Acquisition, identification, preservation, and distribution of microbial cultures. They often provide information about the cultures, such as their characteristics and uses.  Examples are American Type Culture Collection (ATCC), National Collection of Type Cultures (NCTC), Microbial Type Culture Collection and Gene Bank (MTCC), etc.

Key Functions and Importance of Microbial Culture Collections

• Acquisition: Collecting and identifying microbial strains from various sources.
• Authentication: Verifying the authenticity of the collected cultures
• Characterization: Identifying and characterizing microbial strains using various methods (morphological, physiological, genetic).
• Cataloguing: Recording information about the cultures
• Preservation: Employing various preservation techniques to maintain the viability and purity of cultures.
• Production: Creating cultures of microorganisms
• Distribution: Providing cultures to researchers, educators, and industry.  Provide essential resources for research in various fields (medicine, agriculture, biotechnology) and supplying cultures for various industrial processes (food production, pharmaceuticals, etc.).
• Research: Conducting research on microbial diversity, taxonomy, and other related areas.
• Education: Providing training and resources on microbial culture handling and preservation.

Examples of Culture Collections:

ATCC (American Type Culture Collection): Global bioresource centre.

NCTC (National Collection of Type Cultures): UK-based collection.

DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen): German collection.

MTCC (Microbial Type Culture Collection and Gene Bank): Indian collection.