IsoLab follows these methods as part of our bacterial denitrifier method.
OPS Diagnostics has several products which are very useful for freeze drying microorganisms. Microbial Freeze Drying Buffer is a formulation designed for high yields of viable bacteria. General excipients (mannitol, sucrose, and trehalose), lyophilization vials (including split stoppers and seals), as well as other cryogenic accessories are available. OPS Diagnostics also offers a freeze drying service for microorganisms .
Freeze drying bacteria (lyophilization) is a very well established method for the archiving and long-term storage. Initial reports of freeze drying bacteria can be found in the middle of last century. The approaches used vary widely, but they all following the standard process associated with lyophilization, namely the freezing of the sample, application of a high vacuum, warming of the sample while under vacuum which causes water sublimation, driving off excess water through a drying phase, and finally sealing of the sample to prevent water uptake. This general process is used to preserve bacteria, fungi, yeasts, proteins, nucleic acids, and any other molecules which may be degraded due to the presence of water.
There are four significant considerations for freeze drying bacteria. Culturing and preparing the cells is the first consideration. Generally this is not different than methods for typically culturing bacteria. The second aspect involves suspending the bacteria in a suitable freeze drying medium. Traditionally skim milk or sucrose is used, but these have been surpassed by commercial formulations and those developed by the ATCC. The third consideration is the freeze drying process itself. This is extremely dependent upon which freeze dryer is used and the quantity of samples which need to be preserved. The final aspect deals with post-lyophilization storage. Each one of these aspects will be examined below.
For a detailed protocol on freeze drying bacteria, visit the page Bacteria Freeze Drying Protocol.
Selecting a Freeze Drying Medium
Preserving bacteria by lyophilization requires that the bacteria are suspended in a medium that helps to maintain their viability through freezing, water removal, and subsequent storage. The ideal solution will have a component that helps to form a solid "cake" which gives body to the bacterial suspension once freeze dried. Common ingredients for this include mannitol, skim milk and bovine serum albumin (BSA). A second component of a good medium is a lyoprotectant which will help to preserve the structure of biomolecules throughout the lyophilization process. The classic lyoprotectant used with bacteria is sucrose, which is a glucose linked to fructose via its C1 (anomeric) carbon. A similar molecule, trehalose (glucose linked C1 to C1 with another glucose) is very popular for freeze drying proteins, but can be less effective for preserving bacteria. Salts are one component which should not be used in freeze drying media as the salts will concentrate during water sublimation which can destroy cells via severe localized dehydration.
Several basic freeze drying solutions are commonly used to produce good results. Skim milk at a concentration of 20% is a very traditional medium, but viability after processing may decrease up to 90% or more (depending upon the strain). However, if the sample originally contained upwards of 109 cells then plenty of viable cells remain. Alternatively, a solution of 5-10% sucrose is a traditional freeze drying medium. Cake formation with sucrose is not as good as solutions containing BSA or mannitol, but the lyoprotecting property of the sugar yields good viability. Several solutions are suggested by the American Type Culture Collection (ATCC). They have published several recipes for freeze drying solutions, such as Reagent 18, which combines sucrose and BSA that together generate samples that freeze dry well and are effectively preserved. The Microbial Freeze Drying Buffer formulated by OPS Diagnostics is built off the Reagent 18 formulation, but substitutes plant protein for BSA.
Preparation of the Bacteria
Freeze drying is best performed on healthy, actively growing cells which are collected and suspended in freeze drying medium. Cells are usually cultured in liquid medium, and then collected by centrifugation. Alternatively, cells can be washed off a recently streaked agar plate. In either case, it is best to suspend and freeze dry at high cell densities, around 109/ml, as many strains may experience significant drop in viability immediately as a result of the freeze drying. Using higher cell concentrations ensures that the culture will retain at least some viable cells after prolonged storage. It is also possible to simply inoculate cultures into a freeze drying medium and lyophilize at very low cell densities, however the long-term survival of such preparations should be carefully scrutinized.
Freeze drying of bacteria should be done in glass vials or ampoules. Plastic should never be used as water can actually diffuse across many plastics over time. The type of glass vessel used may be dependent upon the configuration of the freeze dryer as well. A basic freeze drying apparatus may simply be a high efficiency vacuum pump connected to a cold moisture trap which in turn is attached directly to the sample. In such cases, long neck, heat sealable ampoules should be used. Dried bacteria are sealed under vacuum in the ampoules with a propane or acetylene flame. Flame sealing is labor intensive, but the most secure method of preserving the samples. If the freeze dryer has a drying chamber, such as with shelf dryers, then samples can be lyophilized in serum vials and sealed with rubber stoppers (called bungs). These stoppers often have a notched, or split, base that is inserted in the vial opening which allows water to escape during drying. Shelf freeze dryers are often equipped with a stoppering mechanism that pushes stoppers into the vials effectively sealing the vial while under vacuum.
Curators of culture collections may use glass tubes instead of vials or ampoules. Many elaborate (but useful) configurations are used to preserve culture collections, including sealed tubes placed in large tubes containing labels and desiccant, which in turn are flame sealed closed. These techniques are not discussed here, instead the reader should consult such manuals as the ATCC Freeze Drying guide.
Whether ampoules or vials are used, these should be filled to no more than 1/3 volume. Smaller volumes are permissible and speed the freeze drying process, thus dispensing 250 µl of cell suspension into a 3 ml vial creates a high surface area as compared to the volume, which will allow for faster freeze drying processing. Once ampoules are filled, sterile cotton or glass wool is inserted into the neck of the ampoule to prevent contamination of the sample. With vials, bungs are inserted and the samples are ready for processing.
Freeze Drying Process
A basic freeze drying process can be divided into three stages: freezing, primary drying, and secondary drying. There can be many complicated variations on this basic process, but most bacteria will freeze dry well using a simple process.
The act of freezing bacteria prior to applying a vacuum to sublime water can be as easy as dipping a prepared ampoule into a dry ice/ethanol bath. Rapid freezing works well for preserving cell viability, however it makes the removal of water more difficult. When the frozen culture is placed under a vacuum, water jumps from the ice and into the headspace over the sample. Obviously the surface of the culture loses water first followed by water in the center of the sample. For water to sublime from the interior of the sample, small pores or channels must form so it can escape. Rapid freezing (with dry ice baths or even liquid nitrogen) tends to create a solid block where channel formation is minimized. Consequently rapidly frozen samples require greater drying times.
Samples can be frozen more slowly by placing a rack of vials/ampoules in a ultralow freezer and allowing the culture to cool more slowly. Shelf dryers often have programmable temperature control that can be used to freeze cultures slowly as well. A slower rate of cooling results in larger ice crystal formation in the sample, which essentially creates the channels for water escape.
Though different strains of bacteria may behave differently, dropping the temperature of prepared cells from ambient to -40°C over 30-60 minutes will typically be effective. However, before committing to freeze drying large numbers of samples, test the freezing step first.
Once the bacterial samples are frozen, the vacuum can be applied. Only high efficiency vacuums, i.e., pumps that can reduce the pressure to under 200 mtorr, will freeze dry samples effectively. The key to primary drying is to raise the temperature of the sample so it is higher than the temperature of the cold trap. In basic systems, the cold trap is often a flask which is immersed in a dry ice ethanol bath. Ampoules connected to basic systems are initially cold (-70°C which is the temperature of the dry ice batch) but warm as they absorb ambient heat. The heat creates sufficient molecular motion to allow water molecules to sublime, i.e., go from solid ice to gas, as long as a vacuum is present. With high efficiency vacuums, the trick is to remove water faster than the sample absorbs heat. The sublimation of the water thus keeps the bacterial solution frozen. If the sample increases in temperature too rapidly, the solution will melt which negates the value of freeze drying.
Shelf freeze dryers have a refrigerated condenser which serves as a cold trap. The condenser is also used to control the temperature of the shelf. For primary drying, the shelf temperature is raised so that the water in the sample sublimes, but melting doesn't occur. In this arrangement, a condenser may hold a temperature at -50°C while the shelf temperature is raised to -10°C. It is important that water moves from the warmer location (sample) to the colder location (condenser) without the sample melting.
The use of matrix forming agents, such as BSA or mannitol, is very useful for helping to form a frozen sample that maintains its shape as water is removed. Without the additives, the sample would collapse. Additionally, the use of small volumes also benefits this primary drying stage in that water is more rapidly removed from small samples with large surfaces areas, such as with 0.5 ml in a 3 ml vial.
Primary drying can take anywhere from 3-4 hours for a small sample to overnight for a fully loaded shelf freeze dryer. The length of time required should be determined empirically.
Drying of bacterial cultures is performed in two stages. Primary drying, described above, removes readily available frozen water. Secondary drying forces out residual water by increasing the temperature of the sample. In shelf dryers, the samples can be increased to 20°C for several hours prior to stoppering. It is important not to over dry the bacteria as this can be detrimental. The use of higher temperatures is also not recommended for the same reason. Freeze drying with a basic system does not allow a separation between primary and secondary drying. As the frozen water is driven off from the sample, its temperature will rise to match that of ambient. This period of ambient drying will serve as a secondary drying phase. Following secondary drying, both vials and ampoules must be sealed. For shelf dryers with a stoppering mechanism, press the stoppers into the vials while under full vacuum. With ampoules, an acetylene or propane torch is used to heat the long neck of the ampoule to seal it. The vacuum will help to pull the glass closed. Label all vials and ampoules with ink that won't rub or wash off. Be advised that labels can fall off leaving samples questionable.
Freeze dried proteins can be stored at relatively warm temperatures as long as no moisture gets to the sample. This is not true for bacteria. Holding bacteria at temperatures above 4°C for prolonged periods of time will dramatically decrease the viability of the cells. Bacteria which would otherwise be stable for years if kept in a refrigerator can die within a week at room temperature. Consequently, accelerated shelf life studies where the sample is held at 37°C to mimic long term storage conditions will not work with lyophilized bacteria.
For long term storage, keep vials and ampoules at 4°C. Periodically remove a vial/ampoule and assess the number of viable cells remaining. Rates of decay should be measured which will help to determine when the sample needs to be resuscitated and subsequently freeze dried.
For the most part, freeze dried bacteria should be viable for several years.