Methods For Maintenance and Preservation of Pure Cultures

Discover essential techniques for the Maintenance and Preservation of Pure Cultures, focusing on bacterial and fungal preservation methods. This comprehensive guide covers best practices to ensure the longevity and purity of microbial cultures, making it an invaluable resource for researchers and laboratory professionals. The following points highlight the top 7 methods used for maintenance and preservation of pure cultures:

1. Refrigeration
2. Agar Slant Culture
3. Paraffin Method
4. Cryopreservation
5. Lyophilization
6. Preservation by Drying in Vacuum
7. Saline Suspension
8. Preservation at Very Low Temperature

Learn how these methods can help maintain the integrity of your samples for long-term use.

Method 1 – Refrigeration

Refrigeration is a widely used method for the short-term preservation of pure microbial cultures. By storing cultures at temperatures between 0°C and 4°C, the metabolic activities of microorganisms are significantly slowed down, which helps in extending their viability for a limited period. This method is particularly effective for both bacterial and fungal cultures, though the duration for which each can be stored varies.

• Storage Conditions:
  • Pure cultures are stored at low temperatures, typically in a refrigerator or cold room, where the temperature is maintained between 0°C and 4°C.
  • This environment is conducive to preserving the cultures by reducing their metabolic rate, thereby slowing down cellular processes.
• Duration of Preservation:
  • Bacteria: When stored under refrigeration, bacterial cultures can remain viable for approximately 2 to 3 weeks.
  • Fungi: Fungal cultures, due to their differing metabolic needs, can be preserved for a longer period, typically 3 to 4 months.
  • It is important to note that refrigeration is intended for short-term storage only, as the cultures continue to metabolize, albeit at a reduced rate.
• Metabolic Activity:
  • Despite the reduced temperature, the metabolic activities of the microorganisms are not completely halted.
  • Microbes continue to grow slowly, utilizing available nutrients in the medium and releasing waste products as a result of their metabolic processes.
  • Over time, the accumulation of waste products and depletion of nutrients leads to the eventual decline and death of the cultures.
• Practical Considerations:
  • Refrigeration is a practical method for laboratories that require temporary storage of cultures for ongoing experiments or between sub-culturing.
  • The ease of access and relatively simple requirements make refrigeration a convenient option for short-term culture maintenance.
• Limitations:
  • The primary limitation of refrigeration is the finite duration for which cultures can be effectively stored.
  • As metabolic activities continue, albeit slowly, cultures must eventually be transferred to fresh media or preserved using more long-term methods to avoid loss of viability.

Method 2 – Agar Slant Culture

Agar slant cultures are a widely utilized method for the maintenance and preservation of microorganisms, particularly in microbiology laboratories. This technique is favored for its simplicity, cost-effectiveness, and ability to maintain the viability of bacterial and fungal cultures over extended periods.

• Preparation and Inoculation:
  • Agar slants are prepared in vitro by pouring molten agar medium into test tubes or other suitable containers.
  • The tubes are then tilted to create a slanted surface as the agar solidifies, providing a larger surface area for microbial growth.
  • After preparation, the slants are inoculated with the desired microorganism using a sterilized inoculating loop.
  • The inoculated slants are incubated for 24 hours or until sufficient microbial growth is observed.
• Preservation Process:
  • Once the microbial culture has grown adequately, the slants are preserved by covering the culture with sterile mineral oil.
  • The mineral oil layer, typically about 1 cm deep, acts as a protective barrier, preventing desiccation and contamination of the culture.
  • This technique helps maintain the integrity of the microbial culture for several months or even years when stored properly.
• Storage and Maintenance:
  • The inoculated and preserved agar slants are stored in a refrigerator to slow down the metabolic activities of the microorganisms, further extending their viability.
  • Despite this preservation method, periodic transfer of cultures to fresh media is essential to prevent the loss of viability and maintain the culture’s genetic stability.
  • The time intervals between transfers vary based on the origin and condition of the microorganism, but typically, transfers are made every six months.
• Transfer Procedure:
  • When transferring the culture, a loopful of growth is carefully removed from the slant, ensuring that excess mineral oil is drained off by touching the loop to the glass surface of the container.
  • The culture is then inoculated into fresh agar medium to initiate new growth, while the original stock culture is preserved.
  • This cyclical process of incubation, preservation, and periodic transfer ensures the long-term maintenance of the culture, making agar slants an effective and economical preservation method.
• Advantages of Agar Slant Culture:
  • Economical: Agar slant cultures are a cost-effective method for maintaining microbial cultures, requiring minimal resources and equipment.
  • Simplicity: The process is straightforward, making it accessible to a wide range of laboratories.
  • Longevity: When properly maintained, cultures preserved on agar slants can remain viable for several years, particularly at room temperature.
• Applications in Microbiology:
  • Agar slant cultures are commonly used in research, clinical, and industrial microbiology settings.
  • They provide a reliable means of preserving pure cultures of bacteria and fungi for long-term use in various experiments and applications.

Method 3 – Saline Suspension

The Saline Suspension method is a preservation technique that leverages the inhibitory effects of sodium chloride to maintain bacterial cultures over time. This method is particularly useful for short to medium-term storage and is characterized by its simplicity and effectiveness in preventing microbial overgrowth.

• Mechanism of Preservation:
  • The Saline Suspension method involves suspending bacteria in a saline solution, specifically a 1% sodium chloride (NaCl) solution.
  • Sodium chloride, when used at a high concentration, acts as an inhibitor of bacterial growth. However, in this method, a sublethal concentration of 1% is used, which slows down bacterial metabolism without completely halting it.
• Preparation and Storage:
  • To prepare a saline suspension, bacterial cultures are first harvested and then suspended in the 1% NaCl solution.
  • The suspension is placed in screw-cap tubes to minimize evaporation, which is crucial for maintaining the concentration and effectiveness of the saline solution.
  • These tubes are then stored at room temperature, allowing for convenient access whenever the cultures are needed for further experimentation or sub-culturing.
• Advantages of Saline Suspension:
  • Ease of Use: The method is straightforward, requiring only basic laboratory equipment and readily available materials.
  • Storage Stability: The use of a screw-cap tube ensures that the saline suspension remains stable over time, preventing evaporation and maintaining the viability of the bacterial cultures.
  • Short to Medium-Term Preservation: This technique is suitable for preserving bacterial cultures for a period ranging from weeks to a few months, making it an effective choice for laboratories that need a reliable short-term storage solution.
• Practical Application:
  • When needed, a small portion of the bacterial suspension can be transferred to an agar slant or other growth medium to revive the culture.
  • The ease of transferring cultures from the saline suspension to a growth medium makes this method practical for routine laboratory use.
• Considerations:
  • While the Saline Suspension method is effective for preserving bacterial cultures for a limited time, it may not be suitable for long-term preservation.
  • The sublethal concentration of sodium chloride slows down bacterial growth, but over extended periods, the cultures may lose viability or undergo genetic changes.

Method 4 – Paraffin Method

The Paraffin Method is a straightforward and economical technique for preserving pure cultures of bacteria and fungi over an extended period. This method is widely employed in microbiology laboratories due to its simplicity and effectiveness in maintaining the viability of microbial cultures.

• Preservation Technique:
  • The Paraffin Method involves the application of sterile liquid paraffin over the surface of a culture slant.
  • The culture, typically grown on a slanted agar medium, is first allowed to grow under normal incubation conditions.
  • After sufficient growth is observed, the culture is covered with a layer of liquid paraffin, which is poured carefully to create a protective barrier.
• Function of Paraffin Layer:
  • The primary function of the paraffin layer is to create anaerobic conditions by limiting the availability of oxygen to the culture.
  • Besides this, the paraffin also plays a crucial role in preventing the dehydration of the agar medium, ensuring that the culture remains hydrated and viable.
  • These conditions help the microorganisms enter a dormant state, reducing their metabolic activities to a minimum.
• Storage and Longevity:
  • Once the paraffin layer is applied, the culture is stored upright at room temperature.
  • This method is particularly advantageous for long-term storage as it allows the culture to remain viable for several years without the need for frequent transfers or additional maintenance.
  • The paraffin layer effectively seals the culture, protecting it from environmental factors that could lead to contamination or degradation.
• Application and Benefits:
  • The Paraffin Method is highly suitable for laboratories that require long-term preservation of microbial cultures without incurring significant costs.
  • It is one of the most economical methods available, requiring only minimal resources and equipment.
  • The simplicity of the procedure also makes it accessible to a wide range of users, from research institutions to educational settings.

Method 5 – Cryopreservation

Cryopreservation is a highly effective method for the long-term storage of pure microbial cultures, utilizing extremely low temperatures to maintain cell viability over extended periods. This technique involves freezing cultures in liquid nitrogen at -196°C, providing a robust solution for preserving microorganisms for years.

• Cryopreservation Process:
  • Freezing in Liquid Nitrogen: The core of cryopreservation involves rapidly cooling microbial cultures to temperatures of -196°C using liquid nitrogen.
  • Temperature Control: This ultra-low temperature halts all metabolic and enzymatic activities within the cells, effectively putting them in a state of suspended animation.
• Role of Stabilizing Agents:
  • Glycerol Addition: To prevent cellular damage during the freezing process, stabilizing agents such as glycerol are added to the microbial suspension.
  • Ice Crystal Prevention: Glycerol functions as a cryoprotectant by lowering the freezing point of the solution and inhibiting the formation of ice crystals within the cells.
  • Cell Survival: By preventing ice crystal formation, glycerol helps maintain cell membrane integrity, thereby promoting higher survival rates upon thawing.
• Advantages of Cryopreservation:
  • Long-Term Storage: Cryopreservation allows for the preservation of microbial cultures for several years without significant loss of viability.
  • Preservation of Genetic Material: This method maintains the genetic and phenotypic characteristics of the cultures, ensuring that they remain representative of the original strain.
  • Minimal Maintenance: Once cultures are cryopreserved, they require minimal maintenance, making this method highly efficient for long-term storage.
• Application and Procedure:
  • Preparation: Before freezing, microbial cultures are mixed with a cryoprotectant solution, typically containing glycerol or other suitable agents.
  • Freezing: The culture is then rapidly frozen by immersing it in liquid nitrogen. This rapid cooling prevents the formation of ice crystals that could damage the cells.
  • Storage: The frozen cultures are stored in cryogenic containers at -196°C until needed.
• Thawing and Reviving Cultures:
  • Thawing Procedure: When required, the cryopreserved cultures are quickly thawed in a warm water bath or at room temperature.
  • Revival: Post-thawing, the cultures are inoculated into suitable growth media to recover and resume normal metabolic activities.
• Considerations:
  • Cryoprotectant Selection: The choice and concentration of cryoprotectants are crucial for successful cryopreservation. Incorrect formulation can lead to reduced cell viability.
  • Equipment Requirements: Cryopreservation requires specialized equipment for handling liquid nitrogen and maintaining ultra-low temperatures.

Method 6 – Lyophilisation (Freeze-Drying)

Lyophilisation, commonly known as freeze-drying, is a preservation method used to maintain microbial cultures and other biological materials over long periods. This technique is widely employed in culture collection centers and other research facilities due to its effectiveness in preserving the viability of microorganisms.

• Lyophilisation Process:
  • Rapid Freezing: The initial step in lyophilisation involves rapidly freezing the microbial culture at an extremely low temperature, typically around -70°C. This rapid freezing is crucial for preventing the formation of large ice crystals, which can damage cellular structures.
  • Dehydration by Vacuum: Following freezing, the culture is subjected to a high vacuum environment. Under this vacuum, sublimation occurs, where ice transitions directly from a solid to a gas, bypassing the liquid phase. This process effectively removes the water content from the microbial cells.
• Microbial Dormancy and Viability:
  • Cellular Dehydration: The vacuum conditions ensure that the microbial cells are dehydrated while still in their frozen state. This dehydration halts all metabolic activities, placing the cells in a dormant condition.
  • Long-Term Viability: As a result of this dormancy, the microorganisms can retain their viability for several years. The lack of moisture prevents microbial growth and metabolic reactions, thereby preserving the integrity of the cultures.
• Storage and Preservation:
  • Sealing and Storage: After dehydration, the vials containing the lyophilized cultures are sealed under vacuum using a small flame. This sealing process protects the cultures from moisture and contamination.
  • Storage Conditions: The sealed vials are stored in the dark at 4°C in refrigerators. This low-temperature storage further ensures the long-term preservation of the cultures.
• Application and Benefits:
  • Culture Collection Centers: Lyophilisation is frequently used by culture collection centers due to its effectiveness in maintaining a large number of cultures with minimal variation in characteristics.
  • Versatility: Besides microbial cultures, this method is also employed for preserving toxins, sera, enzymes, and other biological materials.
  • Reduced Contamination Risk: The freeze-drying process significantly reduces the risk of contamination and allows for the maintenance of culture characteristics over extended periods.
• Revival of Cultures:
  • Thawing and Rehydration: To revive a lyophilized culture, the vial is aseptically opened, and a suitable growth medium is added. The culture is then incubated to allow recovery and growth.
  • Further Transfers: Once revived, the microorganisms can be further transferred and used for research or applications.

Method 7 – Preservation by Drying in Vacuum

Preservation at very low temperatures is a crucial technique for maintaining the viability of microorganisms and other biological materials over extended periods. This method effectively slows down or halts metabolic activities, thus preserving the integrity of the cultures.

• Process Overview:
  • Drying with Calcium Chloride:
    • Preparation: The microorganisms are first dried over calcium chloride. Calcium chloride acts as a desiccant, absorbing moisture from the environment to prevent microbial degradation.
    • Vacuum Application: The drying process is conducted under a vacuum. The reduced pressure helps in removing moisture more efficiently, ensuring that the microorganisms are thoroughly desiccated.
  • Storage Conditions:
    • Refrigeration: Once dried, the microorganisms are stored in a refrigerator. This low temperature further minimizes metabolic activity and helps in maintaining the stability of the dried cultures.
• Mechanism of Preservation:
  • Moisture Removal: The primary function of calcium chloride in this process is to absorb excess moisture, which is crucial for preventing microbial growth and spoilage. By drying the microorganisms, the process reduces the potential for biochemical reactions that could lead to their degradation.
  • Reduced Metabolic Activity: Storing the dried microorganisms at low temperatures slows down or halts any residual metabolic processes. This preservation method keeps the microorganisms in a dormant state, thereby extending their viability.
• Benefits and Applications:
  • Long-Term Stability: This method is particularly useful for long-term storage of microorganisms, as it effectively prevents moisture-related damage and microbial growth. By maintaining cultures in a dry and cool environment, their viability can be preserved for extended periods.
  • Practical Application: This preservation technique is applicable in various research and industrial contexts where long-term storage of microbial cultures is required. It is commonly used in laboratories and culture collection centers to ensure the availability of stable cultures for future use.

Method 8 – Preservation at Very Low Temperature

Preservation at very low temperatures is a critical method for maintaining the viability of microorganisms and other biological materials over extended periods. This technique employs temperatures significantly below freezing to inhibit metabolic processes and preserve cultures.

• Method Overview:
  • Suspension Preparation: Microorganisms are first suspended in a nutrient broth containing 15% glycerol. Glycerol serves as a cryoprotectant, which helps to protect the cells from damage during the freezing process.
  • Freezing and Storage: The prepared suspension is then frozen and stored at temperatures ranging from -15°C to -30°C. This range of temperatures is effective in slowing down or halting microbial metabolic activities, thus preserving the cultures.
• Cryoprotective Agents:
  • Glycerol: Glycerol is a commonly used cryoprotectant that prevents the formation of ice crystals within the cells. These crystals can puncture cell membranes, leading to cell death. By adding glycerol, the damage caused by ice formation is minimized.
  • Dimethyl Sulfoxide (DMSO): Another cryoprotectant used is dimethyl sulfoxide. It functions similarly to glycerol by protecting cells from ice crystal damage and aiding in cell survival during the freezing process.
• Liquid Nitrogen Storage:
  • Extreme Low Temperature: For even more extended preservation, cultures can be stored in liquid nitrogen, which maintains a temperature of approximately -196°C. This extremely low temperature provides an optimal environment for long-term storage by virtually halting all cellular activity.
  • Sealed Ampoules: In this procedure, the cultures are frozen with a protective agent in sealed ampoules. These ampoules are then kept in a liquid nitrogen refrigerator. The sealing ensures that the cultures are protected from contamination and moisture.
• Benefits of Very Low Temperature Preservation:
  • Long-Term Viability: Preservation at very low temperatures, especially in liquid nitrogen, enables the cultures to remain viable for many years. This method is particularly useful for maintaining genetic stability and minimizing the risk of contamination.
  • Cellular Protection: The use of cryoprotectants, such as glycerol and DMSO, plays a crucial role in preventing cellular damage during freezing and thawing processes. This protection helps to maintain the integrity of the microorganisms.
• Application and Usage:
  • Research and Biotechnology: This method is widely used in research laboratories, biotechnology companies, and culture collection centers. It ensures the long-term availability of microbial strains for various applications, including genetic research and industrial processes.
  • Revival of Cultures: To revive preserved cultures, the ampoules are thawed and the microorganisms are reintroduced into a suitable growth medium. This process restores the microorganisms to an active state for further study or application.

References

• https://www.biologydiscussion.com/microorganisms/culture-microorganisms/maintenance-and-preservation-of-pure-cultures-4-methods/55037
• https://spada.uns.ac.id/pluginfile.php/266059/mod_resource/content/1/Maintenance%20and%20Preservation%20of%20Pure%20Cultures%20of%20Bacteria.pdf#:~:text=Pure%20cultures%20can%20be%20successfully,slowed%20down%20but%20not%20stopped.
• https://www.biologydiscussion.com/micro-biology/preserving-microbial-cultures-top-5-methods/17821#google_vignette
• https://www.biologydiscussion.com/organism/preservation-organism/top-6-methods-used-for-preservation-of-organisms/66008