Maintenance and preservation of stock cultures:10 Powerful & Essential Techniques

Learn the maintenance and preservation of stock cultures in microbiology with easy explanations, daily life examples, advantages, and short- and long-term preservation techniques.

Introduction

Imagine your mother prepares homemade mango pickle every summer. She stores it in a clean, airtight glass jar so it remains fresh and tasty for many months. If the jar is left open or moisture enters, the pickle gets spoiled.

Similarly, in a microbiology laboratory, scientists need to preserve microorganisms such as bacteria, fungi, and yeast so that they remain alive, pure, and unchanged for future use. If these microorganisms are not stored properly, they may die, become contaminated, or lose their original characteristics.

The preserved original culture is called a stock culture. It acts as a master culture from which new working cultures are prepared whenever required.

The challenge of keeping pure cultures viable for extended periods of time is linked to the research of pure cultures. This is required to eradicate genetic instability, guard against impurities, and preserve its characteristics. Often referred to as the stock culture collection, the majority of bacteriological laboratories typically keep a sizable collection of pure cultures. These facilities house microbial organisms that microbiologists have isolated and characterized. In most cases, all the traits are retained in the stock cultures. To assist microbiologists in getting pure cultures for a variety of studies, stock culture collection centers have been set up all around the world. A few of the centers also aid in the classification of recently isolated organisms.

Learn more about authenticated microbial cultures from the American Type Culture Collection (ATCC).

Maintenance methods for stock cultures

The process of keeping microbial cultures alive and pure for routine laboratory use.

A pure culture must be maintained after it is acquired. Using flawed or contaminated cultures is one of the most common ways that incorrect data and conclusions are acquired in microbiology. Stock cultures are cultures of interesting organisms kept in the lab for research and reference. It is necessary to keep the stock cultures free of contamination, viable, and true to type.

The most common way of maintaining stock cultures is either on agar slants or in agar stabs.

Agar slants

After the slants are inoculated and incubated until a respectable amount of growth is achieved, they are often refrigerated. Care is taken to ensure that the culture tubes are tightly sealed (with melted wax) to prevent drying out. Agar slant cultures kept in this manner may maintain viability for weeks or months with hardy organisms such as spore formers, Gram-positive cocci, yeasts, and fungi. Transferring cultures to new slants on a regular basis is standard procedure (sub-culturing).

Agar stabs

Agar stabs are more effective than agar slants at maintaining the viability of some organisms. Instead of standing at an angle as with slants, the tube for an agar stab, known as an agar deep, is left to stand vertically while the agar solidifies. A straight needle is used for inoculation, and it is inserted all the way to the bottom of the agar after being touched to the culture. The culture grows in a thin zone along the stab’s line during incubation.

The benefit of the agar stab method is that the organism’s waste products, such as acids, disperse and dilute into the agar from the growing zone. One of the primary causes of the decline of marly stock cultures is waste product accumulation. Even while a stab produces less overall growth than a slant, there is still enough growth to start new cultures when necessary.

Maintaining stock cultures on agar slants is generally not advised because so much of their surface is exposed to the air, making contamination very easy.

Preservation methods for stock Cultures

To provide better circumstances for the preservation of bacteria, a significant amount of research has been conducted. Not every species reacts to a given situation or procedure in the same way. A technique that works for one species might not work for another. Additionally, it is crucial that the preservation and maintenance techniques preserve every trait of the species as it existed at the time of preservation.

There are basically two major methods for the preservation and maintenance of cultures: –

1. Methods in which organisms are in a state of continuous metabolism and,
2. Methods in which organisms are in a state of suspended metabolism.

1. Organism in continuous metabolic state

When the previously used medium dries out or runs out, the organisms must be repeatedly subcultured onto new nutritive medium. This includes using the following techniques to preserve organisms:

i. Periodic transfer to fresh media

Microorganisms are preserved on agar slants in every microbiology lab. The slants are refrigerated after being incubated for at least 24 hours. Every now and then, these cultures are moved to new media. The organism and development conditions determine how frequently transfers occur.

ii. Overlaying culture with mineral oil

After being inoculated, the agar slants are incubated until healthy growth appears. After that, sterile mineral oil is applied to them up to a depth of one centimeter beyond the slanted surface’s tip. The special benefit of this technique is that it allows one to inoculate a new medium, extract some of the growth beneath the oil using a transfer needle, and maintain the original culture. This approach is quite appealing due to its simplicity. Using this technique, several species have been satisfactorily kept for 15–20 years.

iii. Storage in sterile soil

This technique is frequently used to preserve fungus and bacteria that produce spores. Bacterial cultures maintained by this method have been reported to be viable for 70–80 years. Spore suspensions are added to sterile soil (sterilized for 2-3 hours at intervals of 1-2 days), and the combination is dried at room temperature and refrigerated.

iv. Saline suspension

High doses of sodium chloride are commonly employed as a bacterial growth inhibitor. To stop evaporation, bacteria are suspended in a 1% salt solution (sublethal concentration) in screw-cap tubes. The tubes are kept at ambient temperature. The transfers are made on agar slants and incubated as needed.

2. Organisms in suspended metabolism state

This requires drying or storage at a low temperature to the point where the metabolism slows down or stops, but culture can still be revived. It consists of:

i. Drying in a vacuum

After being vacuum-dried on calcium chloride, the organisms are refrigerated. Compared to air drying, the organisms live longer.

ii. Lyophilization (Freeze Drying)

The best method for preserving cultures is lyophilization. For more than 20 years, numerous bacterial species that were maintained via this method have continued to be viable and unaltered. The cells are frozen and quickly dried during this procedure (Figure 9.4). Small vials containing the bacterial suspension are submerged in a combination of dry ice and alcohol at a temperature of -78°C. The vials are immediately attached to a high vacuum line. As a result, the organisms are dried while remaining frozen. Ultimately, the vials with a little name are scaled off in a vacuum. After that, these cultures can be kept at 4°C for a number of years.

Sera, loxins, enzymes, and other biological materials are also preserved using this procedure. To revive microbial cultures, just break open the vial aseptically, add a suitable sterile medium, and make additional transfers following incubation. This approach significantly lowers the risk of contamination. The need for a compact storage area is another benefit.

For detailed guidelines on microbial preservation techniques, refer to the American Society for Microbiology (ASM).

iii. Use of liquid Nitrogen

The microbiologists now have an additional way to sustain the cultures thanks to the readily available liquid nitrogen, which has a temperature of -196°C. This process involves freezing the cultures in sealed vials with a protective agent (glycerol or dimethyl sulfoxide). After that, the frozen cultures are stored in a liquid nitrogen refrigerator. This method has proven effective for numerous specimens that lyophilization is unable to preserve.

Explore global standards for microbial culture preservation from the World Federation for Culture Collections (WFCC).

iv. Storage in Silica Gel

For one to two years, bacteria and yeast can be kept in silica gel powder at a low temperature. This approach involves mixing a thick suspension (paste) of cells with finely ground, heat-sterilized, and cooled silica powder, then storing the mixture at a low temperature. This method’s fundamental idea is rapid desiccation at low temperatures, which prolongs the cells’ viability.

Role Of culture collection centres

A culture collection can be either a generic collection that aims to preserve a large variety of organisms of interest or a specialized collection of a limited set of species. A culture collection’s goal is to keep cultures in a viable, contaminant-free form so they may be shipped right away to researchers, educators, students, or businesses at a low cost. Therefore, the individual worker does not need to keep cultures that he utilizes infrequently.

Because various organisms need to be preserved in different ways, maintaining a big and diversified culture collection is difficult. It requires a high level of expertise and understanding. Although the cost of a stock culture may seem high, it is important to remember that the buyer is paying for the instant availability of a culture free of contaminants. It is important to stress that the investigator should not accept without question the purity and dependability of a stock culture obtained from a culture collection center.

The culture should be thoroughly examined as soon as it is received, and it should be returned if it does not match the requirements. It is entirely normal for mistakes to happen, resulting in the dissemination of flawed cultures, as culture collections frequently preserve thousands of distinct cultures.

Additionally, culture collection centers provide the services of identifying and verifying every new isolate that is acquired by researchers.

In addition to being listed by species name in culture collections, organisms are frequently assigned numerical identifiers. Not every pure culture of the same species is the same. Typically, each isolate of the same species is referred to as a strain, and arbitrary numbers or letters are used to differentiate one strain from another. A single strain of the species is identified by the culture collection’s unique culture number. When referring to research done with a particular stock culture, the precise strain number must be specified because strains of the same species can differ significantly.

Difference between Maintenance and preservation of stock cultures

Feature	Maintenance of Cultures	Preservation of Cultures
Definition	The process of keeping microbial cultures alive and pure for routine laboratory use.	The process of storing microbial cultures for a long period while maintaining their original characteristics.
Purpose	To ensure cultures are readily available for regular experiments and teaching.	To conserve cultures for future research, industrial, or clinical use.
Storage Duration	Short-term (days to a few months).	Long-term (years to decades).
Methods Used	Periodic subculturing, refrigeration, agar slants.	Cryopreservation, lyophilization (freeze-drying), deep freezing, mineral oil storage.
Frequency of Handling	Cultures are handled and transferred frequently.	Cultures are rarely handled until required.
Risk of Contamination	Higher due to repeated transfers.	Lower because cultures remain undisturbed.
Risk of Genetic Changes	Higher because of repeated subculturing.	Very low, as metabolic activity is minimized.
Cost	Generally low.	Moderate to high, depending on the preservation method.
Applications	Routine laboratory work, practical classes, and short-term research.	Culture collections, long-term research, industrial production, and reference laboratories.

Conclusion

Stock cultures are the foundation of microbiological research, education, clinical diagnosis, and industrial applications. Proper maintenance ensures that microbial cultures remain viable, pure, and readily available for routine laboratory work, while preservation techniques help retain their original characteristics for many years with minimal genetic or physiological changes.

The choice of maintenance or preservation method depends on the type of microorganism, the intended duration of storage, available laboratory facilities, and the purpose of use. Techniques such as agar slants, agar stabs, periodic subculturing, mineral oil storage, lyophilization, cryopreservation in liquid nitrogen, and silica gel storage each offer specific advantages for different microbial species. By adopting appropriate maintenance and preservation methods, microbiologists can prevent contamination, reduce genetic variation, ensure reproducible experimental results, and safeguard valuable microbial resources for future scientific, medical, and industrial applications.

Frequently Asked University Questions (Previous 5 Years)

Long Answer Questions (10–15 Marks)

1. Explain the maintenance and preservation of stock cultures with suitable examples.
2. Describe the different methods of maintenance of stock cultures in detail.
3. Explain the long-term preservation methods of microbial cultures.
4. Discuss the principles, advantages, and limitations of lyophilization (freeze-drying).
5. Describe the cryopreservation of microorganisms using liquid nitrogen.

Medium Answer Questions (5–8 Marks)

1. Explain the agar slant method for maintaining stock cultures.
2. Describe the agar stab method and state its advantages.
3. Write a note on storage under mineral oil.
4. Explain the storage of microorganisms in silica gel.
5. Describe the saline suspension method of culture preservation.
6. Explain the storage of microorganisms in sterile soil.
7. Write a short note on periodic transfer to fresh media.
8. Explain the role of culture collection centers in microbiology.

Short Answer Questions (2–5 Marks)

1. Define stock culture.
2. What is maintenance of microbial cultures?
3. What is preservation of microbial cultures?
4. Define lyophilization.
5. What is cryopreservation?
6. What is the purpose of using mineral oil in culture preservation?
7. What are culture collection centers?
8. State two advantages of agar stab cultures.
9. Mention any four methods of stock culture preservation.
10. Why is repeated subculturing discouraged?

Very Short Questions (1–2 Marks)

1. Define stock culture.
2. What is an agar slant?
3. What is an agar stab?
4. Expand ATCC.
5. Expand MTCC.
6. Name any two preservation methods.
7. At what temperature is liquid nitrogen used? (−196°C)
8. Which method is also called freeze-drying?
9. Which preservation method provides the longest storage period?
10. Name one Indian culture collection center.

FAQs

References

1. Prescott’s Microbiology. Joanne M. Willey, Kathleen M. Sandman, & Dorothy H. Wood (2020). McGraw-Hill Education.
2. Brock Biology of Microorganisms. Michael T. Madigan, Kelly S. Bender, Daniel H. Buckley, et al. (2021). Pearson Education.
3. Microbiology: An Introduction. Gerard J. Tortora, Berdell R. Funke, & Christine L. Case (2019). Pearson.
4. Ananthanarayan and Paniker’s Textbook of Microbiology. R. Ananthanarayan & C. K. Jayaram Paniker. Universities Press.
5. Manual of Clinical Microbiology. American Society for Microbiology (2019).
6. Bergey’s Manual of Systematic Bacteriology. William B. Whitman (Editor). Springer.
7. World Federation for Culture Collections. Guidelines for the Preservation of Microbial Cultures.
8. Microbial Type Culture Collection and Gene Bank. Culture Preservation Guidelines.
9. American Type Culture Collection. Best Practices for Culture Preservation and Handling.
10. National Collection of Industrial Microorganisms. Microbial Culture Maintenance and Preservation Protocols.