How to store/preserve Microorganisms such as bacteria, fungi, protozoans and virus.

These are some simple and broadly applied methods are necessary to maintain organisms (bacteria, protozoa, fungi, and viruses) for short and long-term recovery.

Short-Term Preservation Methods

• Direct Transfer to Subculture

The simplest method for maintaining the short-term viability of microorganisms, most often used for bacteria, is periodic subculture to fresh medium.

Although simple, if microorganisms are saved for more than 1 week, this method is potentially labour intensive, requires extensive laboratory space, and may compromise a microorganism’s phenotypic profile.

Each transfer to a new subculture increases the likelihood of mutation with undesirable changes in a microorganism’s characteristics. Furthermore, plasmids may be lost with subculturing.

The interval between transfers varies among organisms.

Additionally, the rate of mutation is quite variable.

Some organisms appear stable indefinitely with repeated transfer, and others may change phenotypic traits after as few as two or three passages.

The actual rate of mutation, however, has not been studied until recently using sequencing technology.

Issues that must be addressed with direct transfer include the medium to be used, the storage conditions, and the frequency of transfer.

• Immersion in Oil

An alternative to capping tubes is to add a layer of mineral oil to the top of the specimen.

Many bacteria and fungi can be stored for periods of up to 2 to 3 years by this method, and transfers are not needed as frequently.

Microorganisms are still metabolically active in this environment, and mutations can still occur. Mineral oil should be medicinal-grade oil with a specific gravity of 0.865 to 0.890.

Contamination of the specimen can occur if the mineral oil is not adequately sterilized.

For sterilization, it should be heated to 170°C for 1 to 2 h in an oven.

Autoclaving is not considered acceptable.

Sterile mineral oil is also commercially available.

• Freezing at −20°C

Refrigeration or freezing in ordinary freezers at −20°C may be used to preserve microorganisms for periods longer than those that can be accomplished by repeated transfers.

Viability may be maintained for as long as 1 to 2 years for specific microorganisms, but overall, damage caused by ice crystal formation and electrolyte fluctuations results in poor long-term survival.

The medium used for storage appears to be important, since preservation times vary from a few months to 2 years depending upon which medium is used.

Modern self-defrosting freezers with freeze-thaw cycles must be avoided because cyclic temperature fluctuation will destroy the microorganism.

• Drying

Although most microorganisms do not survive drying, moulds and some spore-forming bacteria may be dried and stored for prolonged periods.

Soil has been described as a storage medium if it is autoclaved and air dried, but it is not a standardized, defined, and consistent product for use over long periods.

Instead, commercial silica gel can be used in small cotton-plugged tubes after being heated in an oven to 175°C for 1.5 to 2 h, with moderately successful recovery of fungi.

Alternatively, a suspension of 108 microorganisms can be inoculated onto sterile filter paper strips or disks.

The paper is dried in air or under a vacuum and is placed in sterile vials.

These vials can be stored in the refrigerator for up to 4 years, and then single strips or disks can be removed as needed.

This method is commonly used for quality control organisms.

• Storage in Distilled Water

Most organisms do poorly in distilled water, but some survive for prolonged periods.

Many fungi and Pseudomonas spp.

survive for several years in distilled water at room temperature.

According to McGinnis et al. with the exception of fungi that do not easily sporulate, 93% of yeasts, molds, and aerobic actinomycetes can be easily and inexpensively preserved this way.

Long-Term Preservation Methods

• Ultralow-Temperature Freezing

Microorganisms can be maintained at temperatures of −70°C or lower for prolonged periods.

Systems for achieving these temperatures include ultralow-temperature electric freezers and liquid nitrogen storage units.

With either system, unwanted heating can occur due to the loss of electrical power or liquid nitrogen.

Close observation of the system and an adequate alarm mechanism are essential, since any increase in temperature will reduce viability.

In the event that the temperature does rise, restoring power and returning to the target storage temperature as quickly as possible are essential.

The presence of a cryo-preservative such as glycerol may reduce the risk to microorganisms upon short exposure to higher temperatures.

If thawing does occur, there are no guidelines for rapid restoration of the storage condition.

Refreezing of the sealed vials may be considered.

For long-term storage, temperatures below −130°C are recommended for fastidious cells, such as fungal hyphae and protozoa.

Cellular activity and chemical reactions cease at these low temperatures, but at −70°C they may still continue to a limited extent.

Hence, for long-term cryopreservation of certain organisms, storage in liquid nitrogen (−196°C) or liquid nitrogen vapor (−150°C) is recommended.

Storage Vials

Storage vials must be able to withstand very low temperatures and maintain a seal for their contents.

Plastic (polypropylene) or glass (borosilicate) tubes may be used.

Plastic vials with screw tops and silicone washers are much easier to use than glass vials that must be sealed with a flame and then scored and broken open.

Several commercial suppliers’ stock acceptable vials, e.g.,

• Fisher Scientific Products (Pitts- burgh, PA),
• VWR Scientific (Radnor, PA),
• Wheaton Science Products (Millville, NJ),
• And Becton Dickinson and Co. (Franklin Lakes, NJ).
• Vials come in a variety of sizes.

Half-dram vials are available from several suppliers and can be conveniently packaged in a 12-by-12 grid so that 144 vials are stored in one box or layer.

Cryoprotective Agents

To protect microorganisms from damage during the freezing process, during storage, and during thawing, cryoprotective agents are often added to the culture suspension.

Whereas most bacteria, fungi, and viruses survive better with such additives, studies have shown that cryoprotective agents significantly damage others.

There are two types of cryoprotective agents:

• those that enter the cell and protect the intracellular environment
• And others that protect the external milieu of the organism.

Preparation of Microorganisms for Freezing

Microorganisms are inoculated into a medium that adequately supports maximal growth.

Cultures are allowed to mature to the late growth or stationary phase before being harvested.

Broth specimens are centrifuged to create a pellet of microorganisms.

The pellet is withdrawn and resuspended in 2 to 5 ml of broth with the appropriate concentration of cryoprotectant additive.

For agar specimens, broth containing the cryoprotectant is placed on the surface of the agar.

The surface is scraped with a pipette or sterile loop to suspend microorganisms, and then the broth mixture is pipetted directly into freezer vials.

Alternatively, the agar surface can be scraped with a sterile loop.

The microorganisms can then be transferred directly into the vial of cryoprotectant and emulsified into a final dense suspension.

The volume of the aliquots to be frozen is typically 0.2 to 0.5 ml.

Thawing

Damage to microorganisms occurs as they are warmed from the frozen state.

Critical temperatures appear to be between −40 and −5°C.

Studies suggest that rapid warming through these temperatures improves recovery rates.

For optimal results, stored culture vials should be warmed rapidly in a 35°C water bath until all ice has disappeared (3, 6).

Once a vial is thawed, it should be opened and the organism should be transferred to an appropriate growth medium immediately.

Great care must be exercised during the thawing phase, since rapid temperature changes and resulting air pressure changes inside vials can cause the vials to explode.

Protective clothing and eyewear must be worn during this process.

For most practical purposes in the clinical laboratory, however, thorough thawing of stored bacteria or yeast in a water bath is not practical or necessary.

The frozen vial can be thawed at room temperature and plated with good results for most routine organisms.

If the organism vial must be saved for reuse at a later date, one may scrape off a small portion of the frozen contents with a sterile loop or pipette tip and then inoculate the appropriate media.

The vial may be returned to frozen storage immediately without thawing and may be reused at a later date with limited damage to the organism.

• Freeze-Drying (Lyophilization)

Freeze-drying is considered to be the most effective way to provide long-term storage of most bacteria, yeasts, sporulating molds, and viruses.

Better preservation occurs with freeze-drying than with other methods because freeze- drying reduces the risk of intracellular ice crystallization, which compromises viability.

Removal of water from the specimen effectively prevents this damage.

Among bacteria, the relative viability with lyophilization is greatest with Gram-positive bacteria (spore formers in particular) and decreases with Gram-negative bacteria, but overall, the viability of bacteria can be maintained for as long as 30 years.

In addition, large numbers of vials of dried microorganisms can be stored with limited space, and organisms can be easily transported long distances at room temperature.

The process combines freezing and dehydration.

Organisms are initially frozen and then dried by lowering the atmospheric pressure with a vacuum apparatus.

Freeze- drying has been extensively reviewed in the past, and the required equipment includes a vacuum pump connected in line to a condenser and to the specimens.

Specimens can be connected individually to the condenser (manifold method) or can be placed in a chamber where they are dehydrated in one larger air space (chamber or batch method).

Alexander et al. and Heckly have both published detailed descriptions of equipment options.

Storage Vials

Glass vials are used for all freeze-dried specimens.

When freeze-drying is performed in a chamber, double glass vials are used. In the chamber method, an outer soft-glass vial is added for protection and preservation of the dehydrated specimen.

Silica gel granules are placed in the bottom of the outer vial before the inner vial is inserted and cushioned with cotton.

For the manifold method, a single glass vial is used.

For both methods, the vial containing the actual specimen is lightly plugged with absorbent cotton.

The storage vial in the manifold method or the outer vial in the chamber method must be sealed to maintain the vacuum and the dry atmospheric condition.

All vials are sterilized prior to use by heating in a hot-air oven.

Cryoprotective Agents

In general, the two most commonly used agents are skim milk and sucrose.

Skim milk is used most often for chamber lyophilization, and sucrose is used most often for manifold lyophilization.

Skim milk is prepared by making a 20% (vol/vol) solution of skim milk in distilled water.

The solution is divided into 5-ml aliquots and autoclaved at 116°C, with care taken to prevent overheating and caramelization of the solution.

The preparation is then used in smaller volumes as described above for freezing.

Sucrose is prepared in an initial mixture of 24% (vol/vol) sucrose in water and added in equal volumes to the microorganism suspension in growth medium to make a final concentration of 12% (vol/vol).

Storage

Individual vials need to be appropriately labelled and sorted.

Storage at room temperature does not maintain viability and is not recommended.

Storage at 4°C in an ordinary refrigerator is acceptable, but survival rates may be improved at temperatures of −30 to −60°C

Reconstitution

Care must be taken when opening vials for reconstitution because of the vacuum inside the vial.

Safety glasses should always be worn, and vials should be covered with gauze to prevent injury if the vial explodes when air rushes in.

Reconstitution should also be conducted in a closed hood to avoid dispersal of microorganisms.

The surface of the vial should be wiped with 70% alcohol, and then the top of the glass vial can be scored and broken off or punctured with a hot needle.

A small amount (0.1 to 0.4 ml) of growth medium is injected into the vial with a needle and syringe or a Pasteur pipette, the contents are stirred until the specimen is dissolved, and then the entire contents are transferred with the same syringe or a pipette to appropriate broth or agar media.

A purity check must be done on each specimen because of the possibility of either cross contamination or mutation during the preservation process.

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