Table of Contents

Advantages of Preservation

Most bacterial strains can be stored for many years as freeze-dried cultures (lyophilization) or at temperatures below -130°C (cryopreservation).4 There are many advantages of preservation that far outweigh the required investment in the equipment and reagents needed to maintain living cultures. These advantages include:

  • Overall safety of bacterial stocks against loss due to equipment failure or contamination by other microbial organisms.
  • Elimination of time, energy, and material costs associated with the maintenance of bacterial strains not currently in use.
  • Insurance against phenotypic drift associated with prolonged passage, due to genetic instability and/ or selective pressures.
  • Creating a standard working stock that can be used for a series of experiments.

Cryopreservation

Overview

Generally, freezing a suspension of living cells may result in a decrease in viability.16, 17 As the bacterial cell suspension is cooled below the freezing point, ice crystals begin to form and the concentration of solutes in the suspension increases. The formation of intracellular ice crystals can result in the damage of cellular structures. This effect can be minimized if water within the cells is allowed to escape by osmosis during the cooling process. A slow cooling rate, generally -1°C to -10°C per minute, facilitates this progression. However, as the cells lose water, they shrink in size and will quickly lose viability if they surpass a minimum threshold volume. The addition of cryoprotectant agents, such as glycerol or dimethylsulfoxide (DMSO), will mitigate these effects.18, 19 For the preservation of bacterial cultures, ATCC recommends using glycerol as a cryoprotectant.

The standard procedure for cryopreservation is to freeze cells slowly until they reach a temperature below -70°C in medium that contains a cryoprotectant. Vials are then transferred to a liquid-nitrogen freezer to maintain cultures at temperatures below -130°C.

The recovery of cryopreserved cells requires the rapid thawing of the bacterial suspension in a 37°C water bath. The entire contents of the vial are then transferred to an appropriate growth medium. There are numerous factors that can affect the viability of recovered bacterial strains. These critical parameters can include the composition of the freeze material, the growth phase of the bacterial strain, or the concentration of bacterial cells within the solution. To obtain optimal cell viability upon recovery, modify the cryopreservation procedure for each bacterial strain, being sure to harvest cultures during the late logarithmic phase of growth.

Contact ATCC for more information on the cryopreservation of bacterial strains. ATCC bacterial strains that are frozen are commonly cryopreserved with a medium consisting of a final concentration of 10% sterile glycerol.

Freeze Medium

Glycerol and DMSO are the most common cryoprotectant agents. ATCC predominantly recommends using a 20% glycerol stock at a final concentration of 10%. However, if the bacterial strain is sensitive to glycerol, a 50% DMSO stock can be used at a final concentration of 5%. In contrast, glycerol can be sterilized via autoclavation whereas DMSO must be sterilized by filtration. Care should be used when handling any DMSO solution as it will rapidly penetrate intact skin and may carry toxic contaminants along with it.

Use only reagent-grade DMSO or glycerol. Store both in aliquots protected from light. ATCC offers DMSO (ATCC® 4-X™) that has been thoroughly tested for use, as well as TSB with 10% glycerol (ATCC® 20-2200™) for the cryopreservation of non-fastidious bacteria. Overall, the optimum formulations for individual bacterial strains must be determined empirically.

Equipment

  1. Cryopreservation vials

    There are two materials to choose from for cryopreservation vials: glass or plastic. Glass vials are more difficult to work with; they need to be sterilized before use, they need to be sealed with a hot flame, and they can be difficult to open. However, they are preferred for long-term storage (many years) of valuable cultures and are considered fail-safe once properly sealed.

    If cryopreservation in glass ampoules is not possible, plastic vials can be used. Plastic vials come in two varieties: those with an internal thread and silicone gasket, and those with an external thread. Vials with an internal-thread were the first commercially available, but have some disadvantages over the external-thread version. For example, while the silicone gasket provides an excellent seal, it needs to be tightened just right; the vial will leak if the seal is too tight or too loose. For the storage of cryopreserved stocks, ATCC uses plastic vials.

  2. Controlled-rate freezing chambers

    There are several means to achieve a cooling rate of -1°C per minute. The best method involves the use of a computer controlled, programmable electronic freezing unit (such as Thermo Scientific* CryoMed Freezers), which rigorously maintains this rate of cooling. This is the method used exclusively at ATCC. Such equipment is relatively expensive and absolutely necessary for only the most sensitive strains.

    A less costly approach is to place the cryopreservation vials into an insulated chamber and cool for 24 hours in a mechanical freezer at -70°C or colder. There are several commercially available freezing chambers which achieve a cooling rate very close to the ideal -1°C per minute (CoolCell® LX; ATCC® ACS-6000™). Alternatively, the vials can be placed into a polystyrene box, with 15 mm (3/4 inch) thick walls and 1L capacity that is packed with paper, cotton wool, or foam peanuts for insulation.

Liquid Nitrogen Freezing Storage

The ultra-low temperatures (below -130°C) required for long-term storage can be maintained by specialized electric freezers, or more commonly, by liquid nitrogen freezers. There are two basic types of liquid nitrogen storage systems: immersing vials in the liquid or holding vials in the vapor phase above the liquid. The liquid-phase system holds more nitrogen and thus requires less maintenance. However, there is always a chance that some liquid will enter improperly sealed vials, which may then explode when retrieved. For this reason, ATCC strongly recommends storage in vapor-phase.

Vapor-phase storage systems create a vertical temperature gradient within the liquid nitrogen container. The temperature at the bottom of the container will be -196°C, whereas the temperature at the top will vary depending upon the amount of liquid nitrogen at the bottom and the length of time the container is open. To ensure the safe storage of cells, maintain sufficient levels of liquid nitrogen in the container so that the temperature at the top is -130°C or colder. All storage systems should be equipped with temperature alarms.

Gloved hand pulling out vials from a Cryopreservation tank

Cryopreservation Procedure

The procedure below will work for most non-fastidious bacterial strains and should be modified as needed. Additional information on the preservation of fastidious bacterial strains can be found in the latter segment of this chapter. Freeze medium formulations for ATCC bacterial strains can be found on the ATCC website.

  1. In preparation for freezing, grow the bacterial strain under optimal conditions in an appropriate medium as to retain the salient features of the strain. Bacterial strains should be grown to late log phase. 
  2. When freezing bacteria, add 5 to 10% glycerol or DMSO in culture medium. Glycerol is usually prepared in aqueous solution at double the desired final concentration for freezing. It is then mixed with an equal amount of cell suspension. 
  3. Label the appropriate number of vials with the name of the bacterial strain and the date. Aliquot 1 to 1.8 mL of the bacterial suspension to each vial and seal. Seal plastic ampoules tightly with the screw cap. Seal glass ampoules with a gas-oxygen torch, pulling the neck of the ampoule as it is rotated in the flame. 
  4. Allow the cells to equilibrate in the freeze medium at room temperature for a minimum of 15 minutes but no longer than 40. After 40 minutes, cell viability may decline if DMSO is used as the cryoprotectant. 
  5. Place the vials into a pre-cooled (4°C), controlled-rate freeze chamber and place the chamber in a mechanical freezer at -70°C (or colder) for at least 24 hours. Alternately, use a pre-cooled (4°C) programmable freezer unit set to cool the vials at -1°C per minute until a temperature below -40°C is achieved and then set the temperature to abruptly drop to -130°C. 
  6. Quickly transfer the vials to liquid nitrogen or a -130°C freezer. 
  7. Record the location and details of the freeze. 
  8. After 24 hours at -130°C, remove one vial, restore the bacterial strain in culture medium, and determine the viability and sterility.

Recovery of Cryopreserved Cells

The frozen bacterial solution needs to be warmed as rapidly as possible and then immediately transferred to an appropriate growth medium. Some bacterial strains may take more time than normal to fully recover from cryopreservation.

  1. Prepare a culture vessel that contains at least 10 mL of the appropriate growth medium equilibrated for both temperature and pH. 
  2. Remove the vial from the liquid nitrogen freezer and thaw by gentile agitation in a 37°C water bath (or a bath set at the normal growth temperature for that bacterial strain). Thaw the strain rapidly until all ice crystals have melted (approximately 2 minutes). 
  3. Remove the vial from the water bath and decontaminate it by dipping in or spraying with 70% ethanol. Follow strict aseptic conditions in a laminar flow hood for all further manipulations. 
  4. Unscrew the top of the vial and transfer the entire contents to the prepared growth medium. 
  5. Examine the cultures after an appropriate length of time.

Lyophilization

Overview

Freeze-drying is a process where water and other solvents are removed from a frozen product via sublimation.20 Sublimation occurs when a frozen liquid goes directly to a gaseous state without entering a liquid phase. The freeze-drying process results in a stable, readily rehydrated product. This process consists of three steps: pre-freezing the product to form a frozen structure, primary drying to remove most water, and secondary drying to remove bound water.

During the initial freezing process, ice crystals begin to form and the concentration of solutes in the suspension increases. The method used during freezing can greatly affect the ability to freeze-dry the material. It is recommend using slow rates of cooling as this will result in the formation of vertical ice crystal structures, thus allowing for more efficient water sublimation from the frozen product.

Freeze-dried products are hygroscopic and must be protected from moisture during storage. Additionally, these products are sensitive to other factors including oxygen and temperature, which can significantly decrease the shelf life. It is important to store freeze-dried material in a manner that protects the product from exposure to moisture and oxygen, and to store the material at refrigerated temperatures (4°C).

Contact ATCC for more information on the lyophilization of bacterial strains. Most ATCC bacterial strains for distribution are prepared as freeze-dried cultures.

Equipment

  1. Lyophilization Vials

    For the storage of freeze-dried organisms, ATCC uses both double-vialed glass ampoules and stoppered serum vials. Double-vialed glass ampoules are used for the lyophilization of batch cultures via a component freeze-dryer. Samples prepared within these vials are initially freeze-dried in cotton-plugged inner vials, which are then sealed in outer vials under vacuum. Outer vials are supplied with a small amount of a silica gel desiccant, which sustains a state of dryness. Samples preserved in this fashion can be stored indefinitely.

    In contrast, stoppered serum vials are used for lyophilization using the Preceptrol® method. This particular technique was developed as a way to produce a large number of vials in a cost-effective manner. Bacterial strains preserved in this manner are popular strains often used in quality control, teaching, and testing. In this method, material is freeze-dried in a glass serum vial then sealed with a rubber stopper and metal cap. Because these samples are usually distributed in serum vials capped with butyl rubber stoppers rather than the double-vialed ampoules, they are cheaper to produce and allow ATCC to reduce the usual fees. Samples prepared with the Preceptrol® method have a shelf-life of approximately 5 years.

  2. Lyophilization Apparatuses

    There are several methods available for the lyophilization of bacteria. Two such methods employed by ATCC include the Preceptrol® method and the use of a Component Freeze-dryer. In both freezedrying procedures, samples are mixed with a suitable preservative, dispensed into the appropriate ampoule, and allowed to slowly freeze into a solid mass. Once frozen, samples are lyophilized within a freeze-drying system (VirTis® Genesis Pilot Lyophilizer, Millrock Technology® Max85 Freeze Dryer).

    During the primary drying phase, water is removed from the frozen product via sublimation. This is accomplished through the use of a vacuum pump, which allows water molecules to migrate from the frozen product and condense on a moisture trap called a condenser. For this to be possible the temperature of the condenser must be colder than the product temperature; the difference in these temperatures will affect the rate of sublimation. When primary drying is complete, all residual moisture is removed by directly heating the product. During this secondary drying phase, water must be desorbed to a residual moisture content of 1% or less. This process requires a low pressure, low condenser temperature system. Once dried, the ampoules are properly sealed and stored at refrigerated temperatures (4˚C).

Storage and Viability of Lyophilized Strains

To maximize the recovery of viable cells, bacterial cultures must be in optimum condition before the lyophilization process21. Generally, the age of a culture can affect its ability to survive freeze-drying. Bacterial cultures that are harvested from late log phase or early stationary phase have the greatest chance for survival during low-temperature stresses. In addition to proper preparation of bacterial cultures, the recovery of viable cells may also be affected by the use of appropriate growth conditions and media during reconstitution.22 Nutritive, non-selective growth media best aids in maximizing recovery.23

Because lyophilized products are hygroscopic, they must be stored under moisture-free conditions. Exogenous factors such as oxygen content and storage temperature can affect the shelf-life of freeze-dried strains. Oxygen can negatively react with the product, affecting culture viability, and is directly proportional to storage temperature. Therefore, long-term storage of lyophilized products should be kept at refrigerated temperatures (4°C) under conditions that protect the sample from exposure to moisture or oxygen.

Lyophilization Procedure

Preparing glass ampoules
  1. Component Freeze-dryer
    1. Wash inner vials (Glass Vials, Inc., 11.5 x 35 mm) and stopper with an oil-free cotton plug. Subsequently, label each vial and autoclave. 
    2. Prepare outer vials (Glass Vials, Inc., 14.25 x 85.0 mm) by placing a small amount of silica gel granules (Fisher Scientific, grade 42, 6-16 mesh) in the vials to cover about half the bottom. Add a small cotton wad to cushion the inner vial. Heat this overnight at 100°C. 
    3. Prepare the bacterial strain under optimal conditions in an appropriate medium as to retain the salient features of the strain. Bacterial strains should be grown to late log phase. 
    4. Suspend cells in Reagent 20 (ATCC medium formulation 9520) and mix thoroughly. Dispense 0.2 mL of this suspension into each inner vial. Replace the cotton plug and trim it so that it is flush with the edge of the vial. 
    5. Next, place the inner vials in a stainless steel pan and slowly freeze the sample. 
    6. Once frozen, lyophilize the samples for 18 hours within a freeze-dryer. Before use, the condenser should be pre-chilled and the system evacuated to below 30 µm of Hg. 
    7. When the lyophilization cycle is complete, remove the inner vials from the freeze-dryer and insert them into the prepared outer vials. Tamp a 1/4 inch plug of fiber paper above the cotton-plugged inner vial. This should be performed in a dry cabinet.
    8. Subsequently, heat the outer vial using a torch, rotating the vial until the glass just above the fiber paper begins to constrict. This should create a narrow capillary tube. Once the glass cools, attach the vial to a port of a manifold. Evacuate the system to less than 50 µm of Hg. 
    9. Seal the vials at the site of the capillary using a torch. Store vials at 4°C. After 24 hours, restore the bacterial strain in culture medium, and determine the viability and sterility.
  1. Preceptrol® Method
    1. Label 2 mL serum vials (Wheaton Scientific). Place these vials into a tray, cover with a 6.5 inch diameter filter and aluminum foil, and bake at 180°C for four hours. Meanwhile, autoclave the slotted butyl rubber stoppers (West). 
    2. Prepare the bacterial strain under optimal conditions in an appropriate medium as to retain the salient features of the strain. Bacterial strains should be grown to late log phase. 
    3. Suspend the cells in Reagent 18:
      • 0.75 g Trypticase Soy Broth
      • 10.0 g Sucrose 
      • 5.0 g Bovine Serum Albumin Fraction V 
      • 100 mL Distilled water 
      • Filter-sterilize through a 0.2 µm filter 
    1. Dispense 0.4 mL of the suspension into each vial and gently place a sterile stopper on each vial. Do not fully push stoppers in as vapor must be allowed to escape. 
    2. Next, slowly freeze the sample in a mechanical freezer. 
    3. Once the samples are frozen, cover the vials with sterile cotton, 0.5 to 1 inch thick. Then, lyophilize the samples for 18 hours within a freeze-drying system. Before use, the condenser should be pre-chilled and the system evacuated to below 100 µm of Hg. 
    4. When the lyophilization cycle is complete, remove the vials from the freeze-dryer and spray thoroughly with a disinfectant such as Amphyl® (Sterling Drug, Inc.). Place vials under ultraviolet light in a biological safety cabinet for at least 30 minutes.
    5. Seal the vials with aluminum caps (Wheaton) and store at 4°C. After 24 hours, restore the bacterial strain in culture medium, and determine the viability and sterility.

Recovery of Lyophilized Cells

  1. To rehydrate freeze-dried strains, add 0.3 to 0.4 mL of broth. 
  2. Mix well and transfer to a tube containing 5 to 6 mL of broth medium. 
  3. A few drops of this suspension may also be added to an agar slant or place. Growth medium should be chosen to maximize the recovery of cells. The optimum medium is listed for each strain in the ATCC Catalogue of Bacteria and Bacteriophages. 
  4. Incubate strains under the appropriate temperature and atmospheric conditions.

Preservation of Fastidious Bacteria

Most bacterial strains can be freeze-dried, and almost all strains can be cryopreserved and maintained in liquid nitrogen vapor. In preparation for cryopreservation or lyophilization, bacterial strains should be grown under optimal conditions. This can include growth on agar, in shaken broth cultures, or in static broth cultures. Additional care must be taken when preparing fastidious bacterial species for preservation.

Anaerobes

To properly preserve anaerobic bacteria, anaerobic conditions must be maintained during growth, harvesting, dispensing, and freezing. The cryoprotectant and suspending medium must be pre-reduced, and anaerobic conditions should be maintained with oxygen-free gas flow using a sterile cannula.

Bacteriophages

Most bacteriophages can be freeze-dried successfully; exceptions are stored in liquid nitrogen.24, 25 Prior to preservation, bacteriophages can be propagated in a soft-agar layer or in broth; a titer of 108 pfu/mL is desirable. Bacteriophages can be cryopreserved in the absence of a cryoprotectant, performed under a controlled freezing rate. However, bacteriophages that require a cryoprotectant can be mixed with an equal volume of 20% glycerol. To freeze-dry, filtered bacteriophage suspensions should be mixed with 20% skim milk and lyophilized according to the component freeze-dryer method.

Mollicutes

Prior to cryopreservation or lyophilization, mollicutes should be grown in broth. Following growth, cell suspensions should be centrifuged and resuspended in equal amounts of broth and 20% glycerol. Alternatively, cells can be resuspended in equal amounts of broth and 24% sucrose for lyophilization in the component freeze-dryer method.26

Neisseria, Haemophilus, Campylobacter, and Helicobacter

These genera of bacteria are very sensitive to damage during preservation.27 To maximize cell viability and recovery, cells should be grown under optimal conditions and harvested at the proper point in the growth curve. To stabilize these strains during lyophilization, supplement the suspending medium with 0.5% sodium ascorbate prior to freezing.

Spirochetes

Spirochetes are very difficult to lyophilize. It is recommended that spirochetes be cryopreserved with 10% glycerol and stored in liquid nitrogen vapor.28

Special Hazards

Care must be taken during the cryopreservation or lyophilization of bacterial strains. Problems such as contamination, breakage of glass ampoules during handling and storage, dispersal of freeze-dried bacteria when opening glass ampoules, and the handling of liquid nitrogen must all be considered. To prevent contamination and the dispersal of bacteria, aseptic technique must be followed. This can include the decontamination of all equipment and vials as well as performing all preparations in a biological safety cabinet. Additionally, protective clothing should be worn during preparation to prevent contamination as well as to guard against harm due to contact with liquid nitrogen.