GROWTH AND CULTIVATION OF BIOOBJECTS - V. M. Samygin - 2016

CHAPTER 13. STABILIZATION OF PROPERTIES AND PRESERVATION OF MICROORGANISMS

Proper culture preservation is a critical issue, as the loss or alteration of strain properties due to improper storage can disrupt many scientific research programs. The primary objective of preservation is to maintain Cell viability and culture purity while preventing changes and Mutations—essentially keeping the microorganisms as close as possible to the originally isolated strain. While numerous Methods for bacterial preservation exist, not all strains respond identically to every technique. The choice of method is frequently dictated by available equipment, storage space, and qualified personnel.

13.1. Short-Term Preservation Methods

The traditional method for maintaining bacterial cultures involves periodic subculturing onto fresh media. The interval between transfers depends on the specific microorganism, the medium used, and environmental conditions. Certain Bacteria must be subcultured every other day, whereas others require transfers only after several weeks or months. Successful application of this method relies on three key conditions:

1) a suitable maintenance medium;

2) an optimal storage Temperature;

3) the appropriate subculturing frequency.

Minimal media are generally preferred because they slow down cellular metabolic rates, thereby extending the intervals between transfers. However, some bacteria require complex media for growth, or the presence of sophisticated compounds in the medium to preserve their specific physiological properties. In such cases, more frequent transfers may be necessary due to accelerated growth or the accumulation of metabolic end products.

The simplest approach involves storing microbial cultures at room temperature in a rack or a specialized cabinet. This method requires constant monitoring because cultures tend to dry out rapidly. To mitigate evaporation, screw-cap tubes with rubber gaskets, further sealed with Parafilm, are commonly used. To reduce metabolic rates, microorganisms are kept in a refrigerator at 5-8 °С. Implementing these measures allows most bacteria to be preserved for 3-5 months without subculturing. Subculturing frequency should be kept to a minimum to prevent the Selection of variants. Following each transfer, the culture must be checked for purity, supplemented by periodic abbreviated checks of phenotypic properties. Single colonies should not be isolated from subcultured strains, as this increases the likelihood of mutant selection. The primary drawbacks of periodic subculturing include the risk of contamination, labeling errors or misidentification, the selection of variants or mutants, potential culture loss, and the requirement for dedicated storage space.

Many bacterial species can be successfully preserved for months or even years using a simple and inexpensive method: under a layer of sterile mineral oil (such as liquid paraffin with a specific gravity of 0.865-0.890). Nevertheless, inadequate sterilization of the mineral oil can lead to culture contamination. The oil should be sterilized in a hot-air oven at 170 °С for 1-2 hours; autoclaving is not recommended for this purpose. Cultures are grown on Agar slants, in agar stubs, or in liquid media of appropriate composition. Once robust growth is achieved, at least 2 cm of sterile mineral oil is poured into the tube under sterile conditions (completely covering the agar slants) to prevent desiccation, lower metabolic activity, and slow down culture growth. Oil-covered cultures are stored vertically in a refrigerator. This technique shares the same disadvantages as conventional subculturing.

Storing bacteria at temperatures between 0 °С and -20 °С is generally discouraged due to cellular Damage caused by ice crystal formation. Nevertheless, certain bacteria can withstand freezing and remain viable for periods ranging from 6 months to 3 years.

Most cultures perish when dried under laboratory conditions. However, some cultures—particularly spore-forming bacteria—can survive for years if dried in an appropriate matrix (such as soil, strips or discs of sterile filter paper, or dried droplets or discs of gelatin).

13.2. Long-Term Preservation Methods

Lyophilization, or freeze-drying, is among the most cost-effective and efficient techniques for the long-term preservation of bacteria and other microorganisms. It allows many physiologically diverse bacterial species and Bacteriophages to remain viable for 30 years or more. The process involves removing Water from frozen Suspensions via sublimation under low pressure, meaning the water evaporates directly without passing through a liquid phase. Dried Cells can be stored for extended periods provided they are protected from oxygen, moisture, and light. At any given moment, the cells can be rehydrated back to their initial state.

Lyophilization is carried out using various Procedures and instruments. The simplest setup is a desiccator cooled so that The Cell suspension remains frozen while connected to a vacuum pump. Centrifugal lyophilization and preliminary freezing are more commonly employed.

The success of lyophilization depends heavily on the quality, viability, and growth conditions of the cells used. A sufficiently large population of cells is grown to ensure a suspension density of at least 10⁸ cells/ml. Cells are harvested during the period of maximum stability and viability—specifically in the late exponential or early stationary growth phase.

To prepare cells for lyophilization, they are suspended in a medium containing cryoprotectants. Commonly used cryoprotectants include 20% skim milk, a 24% sucrose solution diluted with an equal volume of growth medium, 10% dextran, horse serum, and Inositol. Cells grown on an agar surface are harvested under sterile conditions. For cells grown in liquid culture, the biomass is separated aseptically by centrifugation and resuspended to yield a concentration of at least 10⁶ cells/ml. This protocol is applied when using 20% skim milk or a 24% sucrose solution as the cryoprotectant. Lyophilized cultures are stored at temperatures below 5 °С. Storage in freezers at -3 °С or -69 °С yields even better preservation results. Lyophilized cultures must never be stored at room temperature.

When reviving lyophilized cultures, the ampoules are scored with a triangular file about 2.5 cm from the top before opening, then disinfected by wiping with 70% alcohol. The ampoule is wrapped in sterile gauze, and the scored tip is snapped off. This Procedure is typically performed in a fume hood, or in a specialized enclosed cabinet in the case of pathogenic bacteria. The broken tip is briefly flamed before introducing the liquid medium. The medium and the resulting bacterial suspension are injected and retrieved using a sterile syringe and needle.

The lyophilized culture is suspended immediately after opening the ampoule by adding 0.3-0.4 ml of an appropriate sterile liquid medium. The suspension within the ampoule is thoroughly mixed and transferred to test tubes containing 5 ml of liquid medium. After thorough mixing, a 0.2 ml aliquot is plated onto solid or semisolid medium of identical composition. Culture purity must be verified both before and after lyophilization. To achieve this, the cell suspension is serially diluted and streaked onto solid media. The tubes and agar plates are incubated at the optimal temperature for the bacteria, and as soon as growth appears, a transfer is made to fresh medium to confirm purity. Growth following lyophilization often begins after an extended lag phase; therefore, one should not conclude that a culture is dead based solely on insufficient incubation time.

To evaluate the efficiency of freeze-drying, cell viability is assessed both before and after lyophilization. Furthermore, periodic checks should be conducted to confirm the viability of stored lyophilized cultures and to screen for any alterations in bacterial properties resulting from the freeze-drying process or prolonged storage.

Bacterial cultures can also be stored for extended periods in ultra-low-temperature freezers (e.g., at -70 °С). This method proves highly suitable for a wide range of bacteria.

Certain bacterial species that do not survive lyophilization can be successfully preserved long-term in the liquid phase (-196 °С) or vapor phase (-150 °С) of liquid nitrogen using well-insulated containers (ultrafreezing). Many bacteria maintain their phenotypic traits under these conditions for over 15 years. Two Types of cryoprotectants are utilized when storing bacteria in liquid nitrogen. The first type includes glycerol and dimethyl sulfoxide (DMSO), which readily penetrate The cell membrane, providing both intracellular and extracellular protection against freezing injury.

The second type of cryoprotectant comprises substances such as sucrose, lactose, glucose, mannitol, sorbitol, dextran, polyvinylpyrrolidone, and polyglycol, which exert a protective effect exclusively on the outer surface of the cell membrane. Type one protectants, namely glycerol and DMSO, have proven more effective and applicable to a broader spectrum of bacteria. Glycerol and DMSO are typically added to the growth medium at concentrations of 10% and 5% (v/v), respectively. Glycerol is sterilized by autoclaving for 15 minutes at 10⁴ Pa. DMSO is sterilized by filtration through Seitz filter candles with a pore size of 0.3 µm; DMSO is collected in 10-15 ml portions into sterile tubes and stored frozen at 5 °С (DMSO freezes at 18 °С). Due to the accumulation of oxidative breakdown products, open bottles of DMSO should not be kept for more than a month.

The physiological state of the culture plays a crucial role in bacterial survival following liquid nitrogen freezing. Active growing cells harvested from the mid-to-late exponential growth phase are predominantly used for freezing. The cells are cultivated in an appropriate medium. For liquid cultures, cells are harvested aseptically by centrifugation, and the pellet is resuspended in sterile fresh medium containing either 10% (v/v) glycerol (prepared by mixing 20% glycerol with an equal volume of sterile medium) or 5% (v/v) DMSO (prepared by adding the appropriate amount of 100% DMSO to the sterile medium). When cultures are grown on agar, the cells are washed off the surface with a sterile liquid medium containing a suitable cryoprotectant, and 0.4 ml aliquots of the suspension—containing at least 10⁸ cells/ml—are dispensed into sterile, labeled ampoules. For non-pathogenic bacteria, the ampoule is cooled at 4 °С for at least 30 minutes prior to sealing. The Glass is then heated just above a pre-scored constriction by rotating it in a flame for a few seconds to drive off moisture. This step prevents internal pressure buildup during sealing, which could otherwise cause bubbles to form. Ampoules are preferably sealed using a glass-working workstation equipped with an oxygen-gas torch. Sealed ampoules are subsequently tested for hermetic integrity by immersion in a 0.05% methylene blue solution at 5 °С for 30 minutes. Defective seals are easily identified by the penetration of the dye.

The best results in survival and recovery of bacterial cultures were observed with slow cell cooling, for example, at a rate of 1 °C/min. Rapid heating of frozen microorganisms or spores leads to their prompt recovery (revitalization). To revive frozen cultures, they are rapidly thawed in a water bath at 37 °C with gentle shaking until all the ice has melted. For glass ampoules, this typically takes 40-60 s, while for polypropylene ones, it takes 60-120 s. Immediately after thawing, the ampoule is removed from the water bath and disinfected with 70% ethanol. The ampoule is opened, and the culture is transferred to fresh medium under sterile conditions. It should be kept in mind that when extracting frozen glass ampoules from liquid nitrogen, the possibility of an explosion cannot be ruled out; therefore, gloves and a face shield should be worn during this procedure. To determine the effectiveness of the storage method, each strain is tested for viability.