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15 Preservation of Thermophilic Microorganisms
15 Preservation of Thermophilic Microorganisms Stefan Spring DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg1b, D-38124 Braunschweig,Germany ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Introduction Maintenance by subculturing and preservation Methods for the long-term preservation of thermophiles Vaccum drying Deep freezing
^^^^^ INTRODUCTION Thermophilic and hyperthermophilic microorganisms are highly diverse in respect of their metabolism and phylogeny. Besides high temperatures, most thermophiles have also adapted their lifestyle also to other environmental conditions which can be termed extreme from an anthropocentric point of view, e.g. acidic pH values, highly reduced conditions or hypersalinity. This versatility, among other reasons, may have caused an increasing interest in this group of prokaryotes. The mounting research activity in this field in many laboratories resulted in a need for reliable maintenance methods for thermophilic strains. Especially for comparative genetic or biochemical studies it is often desirable to work with stocks of preserved cultures, or it could be necessary to maintain a large number of mutant strains over longer periods of time. On the other hand, standard methods of preservation often fail in the case of thermophilic microorganisms, especially if they are also strictly anaerobic or acidophilic. Several methods which can be used for the long-term preservation of these fastidious microorganisms are discussed in this chapter. Most of these techniques were originally developed for the reliable preservation of extremophiles at culture collections, but with some expertise, it should be possible to adapt these procedures to the technical facilities available in most research or industrial laboratories. Resources for material and equipment useful for the described methods are listed at the end of this chapter. METHODS IN MICROBIOLOGY, VOLUME 35 0580-9517 DOI:10.1016/S0580-9517(05)35015-X
Copyright ß 2006 Elsevier Ltd. All rights reserved.
Preservation of Thermophilic Microorganisms
CONTENTS
^^^^^ MAINTENANCE BY SUBCULTURING
AND PRESERVATION In several laboratories studying thermophilic prokaryotes, important strains are only maintained by frequent subculturing. Subculturing means serial transfer of strains from media with depleted nutrient sources to fresh media. After inoculation into fresh media, cultures are incubated to obtain growth and then eventually stored. To prevent frequent subculturing, the metabolic rate of the organism during storage should be kept at a minimum. Many thermophilic strains stop growing already below 40 C, so that they can be stored easily without refrigeration at room temperature. Refrigeration to 4–8 C can be used to extend the interval of subculturing of some strains, but on the other hand may have a negative effect on the long-term stability of other thermophilic strains. Viability of distinct cultures over time may vary considerably and depends largely on the reached growth phase, storage conditions and quality of the used medium. Hence, time of storage between transfers is normally kept at a minimum to ensure survival of important strains. Subculturing is inexpensive in terms of equipment, applicable to all cultivable strains and avoids problems associated with the resuscitation of preserved stock cultures. However, it can be time-consuming if organisms are handled that require frequent transfers or a whole collection of strains has to be maintained. Besides the time necessary for preparation of media and manipulation of strains, subculturing implicates several risks and disadvantages. Contamination of pure cultures is a permanent threat and the mislabelling or transposition of vials can lead to an interchange of strains. The risk of mishaps increases with the frequency of the manipulation of a strain and can be only minimized by the consequent use of sound microbial techniques. Frequent subculturing can also have a negative effect on the genetic integrity of a strain. Genetic changes or the loss of plasmids may result in the selection of mutant strains and thereby, to a strain drift. A progressive genetic drift in a distinct culture may remain unrecognized if inconspicuous traits of the organism are affected. Only by using reliable long-term preservation techniques, most of these risks can be efficiently avoided. The investment in laboratory equipment and training of technical staff, which is necessary for the establishment of most preservation techniques, appears to be only a small effort in comparison to the damage caused by the potential loss of important reference strains.
^^^^^ METHODS FOR THE LONG-TERM
PRESERVATION OF THERMOPHILES Several well-established methods are available for the long-term preservation of thermophiles. The basic principle common to all of
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these techniques is the reduction of the amount of freely available water to a value where metabolism is suspended. A decrease in water activity can be achieved either by dehydration or freezing. Numerous procedures have been developed for the gentle cryopreservation or drying of cultures in order to minimize cell damage during preservation. Nevertheless, survival rates of some cultures can be rather low. In general, survival rates depend largely on the sensitivity of a strain against the harmful effects caused by the used preservation method and can vary considerably among closely related strains. Consequently, not all of the techniques described in this chapter are applicable to the whole range of thermophilic microorganisms. The main assets and drawbacks of the most common techniques are discussed below.
Freeze-drying or lyophilization is a process which extracts water from a sample by sublimation. Sublimation is the transition of a substance from the solid to the vapour state, without passing through an intermediate liquid phase. Lyophilization involves freezing of the cell pellet so that the water becomes ice, application of vacuum in order to sublimate the ice directly into water vapour and drawing off the water vapour. Cultures preserved by freeze-drying are relatively stable over time and can be stored without further attention. However, due to the low survival rates of susceptible strains, a selection of more resistant subpopulations with different genetic characteristics may take place during freeze-drying. To avoid a genetic drift by the continuous selection of subpopulations, subsequent batches should be always prepared from the same seed stock. Ampoules with lyophilized samples can be transported easily without the risk of damage caused by lyses of sensitive cultures. However, the resuscitation of vacuumdried samples can be labour-intensive, especially if anaerobic strains were preserved. In addition, most strains show a prolonged lag-phase after resuscitation and need two or three transfers until normal growth takes place. This preservation method requires an investment in special equipment, which is mainly useful for the purpose of freeze-drying. Some training of technical staff will also be necessary to ensure a safe and smooth flow of lyophilization procedures. Thus this method is only cost-effective for laboratories which have to prepare larger batches of preserved samples from thermophiles or intend to store a large collection of strains without further attention. Thermophiles that are suitable for preservation by lyophilization represent mainly aerobic or aerotolerant, heterotrophic microorganisms, which are relatively robust and show a good growth yield. In contrast, strains that are extremely sensitive to oxygen or reach only very low cell densities in most cases, hardly survive the harsh
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Freeze-drying
conditions during freeze-drying and should be better preserved by cryopreservation.
Liquid-drying Unlike lyophilization, liquid-drying (L-drying; Annear, 1956) involves vacuum-drying of samples from the liquid state without freezing. Liquiddrying has, in general, the same benefits and drawbacks as freeze-drying, but the procedure is less elaborate and may have advantages in the preservation of microorganisms that are particularly sensitive to the initial freezing step involved in lyophilization.
Deep Freezing The only technique which seems to be applicable to all thermophilic prokaryotes studied so far is the deep freezing of concentrated cell suspensions in fresh media supplemented with a suitable cryoprotectant. The survival rates of cultures preserved by this method are usually quite high and the long-term stability almost unlimited. The resuscitation of cultures preserved by deep freezing is uncomplicated and growth normally takes place without an extended lag phase. However, it is important that the temperature of storage should be kept below 139 C, because only then all physical or chemical reactions are suspended (Morris, 1981) and a satisfactory stability of the preserved culture can be achieved. In contrast, the storage of cryopreserved stocks in normal freezers at temperatures above 70 C is not recommended, because free water is still available enabling residual physical or chemical activity, which could damage DNA or other essential cell compounds causing a rapid loss of viability in some strains. Normally, storage in liquid nitrogen (196 C) or the nitrogen vapour phase (140 C) is used to achieve the required low temperatures. With exception of the liquid nitrogen storage tank, the equipment necessary for this method is easy to obtain and inexpensive. A major constraint of this preservation technique may be, however, the continuous need for liquid nitrogen to maintain the required storage temperature. A complete evaporation of liquid nitrogen will cause an inevitable temperature rise in the refrigerator which eventually may kill all sensitive stocks. Hence, care has to be taken to ensure that liquid nitrogen is replenished in regular intervals. Nevertheless, in some geographical regions the adequate supply with liquid nitrogen is problematic. In this case, storage of less sensitive cultures in deep freezers at 80 C may be an alternative. However, it has to be noted that although metabolism is suspended, cells can be damaged by the recrystallization of ice, which still takes place at this temperature. In the following sections, standard protocols for the preservation of thermophilic prokaryotes are exemplified on representatives of various physiological groups. 352
^^^^^ VACUUM-DRYING
Equipment Technical equipment necessary for vacuum-drying methods include a vacuum pump, a freeze-dryer equipped with a centrifuge head and manifold or an evacuation jar connected to a moisture or cold trap, constrictors and diverse glass ware. Vacuum-dried samples of cultures can be prepared either in double vial or single vial ampoules. The Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) delivers dried cell pellets exclusively as double vial preparations, sealed under vacuum (Figure 15.1). Double-vial ampoules are more elaborate to prepare, but have the advantage that a contamination of the atmosphere by aerosols that can be produced by sudden release of the vacuum in single-vial preparations is efficiently prevented. In addition, the cell pellet is protected from contamination, because inflowing air filters through the sterile cotton plug of the inner vial.
Centrifugal Freeze-drying Centrifugal freeze-drying is the preferred method for the preservation of fastidious thermophilic strains that are susceptible to the initial freezing in skim milk intrinsic to standard freeze-drying protocols. This method relies on the freezing of the cell suspension by evaporative cooling caused by the loss of water during evacuation of the freeze-drying chamber. To avoid frothing of the suspension due to removal of dissolved gases before freezing is complete, the suspension is centrifuged during the initial stages of drying. In this way, the initial freezing of the cell suspension, which is a critical step of the normal shelf freeze-drying process, can be omitted. At the DSMZ, a special preservation mixture has been developed 353
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Figure 15.1. Double-vial preparation of a vacuum-dried sample, sealed under vacuum.
which is now routinely used for the protection of thermophilic strains during the centrifugal freeze-drying process. The use of this protective solution allows the lyophilization of most aerobic thermophiles, with the exception of strains that grow only to a very low cell density. The supplementation of this solution with amorphous ferrous sulfide also allows the lyophilization of some anaerobic thermophiles without using an oxygen-free gas atmosphere during the processing of samples. Preparation of vials 1. Plug clean glass vials (44 11 mm, flat bottom) loosely with nonabsorbent cotton wool plugs (dental rolls size no. 2, 40 mm in length) and sterilize at 121 C for 20 min. 2. Prepare outer glass tubes (135 215 mm, soft glass) by placing a few pieces of self-indicating silica gel (e.g. silicagel rubin) into the tubes which are covered with a small amount of cotton wool (see Figure 15.1). Preparation of the suspending medium 1. Preservation mixture: Dissolve 2% (w/v) gelatine, 2% (w/v) yeast extract and 24% (w/v) sucrose in distilled water, bubble with N2 gas for at least 30 min to remove oxygen and fill in a suitable container, which can be tightly closed, e.g. a serum bottle with butyl rubber stopper and aluminium crimp. Autoclave the protective solution at 121 C for 20 min. The suspending medium is prepared by mixing an equal ratio of freshly prepared sterile cultivation medium and preservation mixture. 2. Amorphous ferrous sulfide for anaerobic strains: The ferrous sulfide is prepared according to Brock and O’Dea (1977). Briefly, equimolar amounts of ferrous ammonium sulfate and sodium sulfide react in solution to form amorphous FeS, which precipitates. After settling of the FeS precipitate, the supernatant is discarded. This procedure is repeated several times with an anoxic solution of 0.9% (w/v) NaCl until the supernatant no longer contains detectable amounts of ferrous or sulfide ions. Finally, the purified aqueous suspension of FeS is dispensed under nitrogen gas atmosphere in suitable vessels and autoclaved. The final concentration of amorphous ferrous sulfide in the suspending medium should be approximately 1 mg ml1. Preparation of the cell suspension Depending on the oxygen relationship and acid tolerance of the strain to be preserved, different procedures are necessary for the preparation of the cell suspension. Ideally cells are harvested when they are most active and have reached the mid exponential growth phase. The processing of aerobes is exemplified on representatives of the genera Aeropyrum and Sulfolobus, whereas the lyophilization of anaerobes is illustrated by a protocol suitable for hyperthermophilic Pyrococcus species. 354
Aerobic heterotrophic thermophiles
Strictly anaerobic thermophiles Special care has to be taken to avoid exposure to oxygen during harvesting of Pyrococcus spp. and other obligate anaerobic thermophiles and hyperthermophiles. Either anaerobically grown cultures have to be transferred under an oxygen-free gas atmosphere to anoxic and gas-tight centrifuge bottles or in a more straightforward approach are cultured in heavy-walled, round-bottomed glass bottles, which can be used for growing of cells and centrifugation. Suitable glass bottles (50–70 ml volume) can be ordered from most glass blowers as custom-made product and should have necks that can be closed with a butyl rubber septum and screw cap as with Hungate-type anaerobe tubes. After centrifugation the centrifuge bottle is opened, a gassing cannula inserted and the supernatant is removed aseptically under a flow of oxygen-free gas. Normally, the gas-mixture used corresponds to the gas atmosphere is used to cultivate the respective strain. However, a mixture of N2 and CO2 instead of a H2 containing gas mixture should be used to avoid risks caused by the generation of flammable gas mixtures in the laboratory. Cell pellets of one or more bottles are collected using suspending medium that has been supplemented with 1 mg ml1 amorphous FeS and transferred to a tube continuously flushed with oxygen-free N2 gas. Filling of vials is carried out under oxic conditions using for instance an Eppendorf Multipipette with 2.5 ml Combitip. This procedure should be done quickly because the ferrous sulfide provides protection of cells against oxygen only for a limited time.
Drying procedure Drying is carried out in two stages. Primary drying is achieved by centrifugal freeze-drying, whereas standard shelf freeze-drying is used to 355
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Aeropyrum spp. and other oxygen-tolerant hyperthermophiles can be harvested as usual by centrifugation of liquid medium in sterile centrifuge tubes or bottles. The cell pellet is resuspended in approximately 5 ml of suspending medium containing the preservation mixture and aseptically distributed in aliquots of 0.15 ml to each single vial. Several thermoacidophilic species, e.g. Sulfolobus solfataricus, are sensitive to low pH values at the end of the growth phase and easily lose viability. Therefore, the growth medium is neutralized prior to harvesting of the cells by adding a small amount of solid, sterilized calcium carbonate. After 10–15 min, the undissolved carbonate forms a deposit which then can be removed from the culture supernatant. In addition, the pH value of the thermoacidophiles growth medium is adjusted to a moderate value (pH 4.0–4.5) prior to mixing with the preservation mixture in order to prevent damage of cells during lyophilization.
obtain double-vial preparations in the second step. 1. Primary drying 1.1. Place vials in the centrifuge head of a freeze-drying machine, switch on the centrifuge and thereafter apply vacuum. 1.2. Centrifuge at approximately 750 rpm for 2–3 h. 1.3. Switch off the centrifuge and continue primary drying until the vacuum has dropped to 1–10 mbar. 1.4. Switch off refrigerator and vacuum pump, allow air to slowly enter the vacuum chamber and remove vials from the centrifuge head. 2. Secondary drying 2.1. Cut off parts of the cotton-wool plugs that are projecting from the vials and place vials in outer glass tubes containing self-indicating silica gel. 2.2. To protect the cotton-wool plugs from heat during constriction cover the vials with glass wool (Tempstran 475–106) slightly compressed to a layer of 1–2 cm thickness (see Figure 15.1). The outer tubes are constricted just above the glass wool either by hand or by using a semiautomatic ampoule constrictor. 2.3. After cooling, attach the double-vial preparations to the manifold of a freeze-drying machine for secondary drying for at least 2 h or overnight (see Figure 15.2). 2.4. At a vacuum of at least 0.1 mbar, the tubes are flame-sealed at the middle of the constriction.
Liquid-drying The L-drying method has been successfully applied at the DSMZ to several thermophilic, obligate chemolithoautotrophic bacteria, which are difficult to preserve using one of the standard freeze-drying protocols. Examples of successfully preserved thermophiles include Aquifex pyrophilus, Thermocrinis albus, Hydrogenobacter thermophilus and Sulfurihydrogenibium azorense. In general, members of these genera and related hydrogen-oxidizing thermophiles are microaerophilic and sensitive to elevated levels of oxygen. Thus, during handling of these cultures care has to be taken to ensure that exposure to atmospheric levels of oxygen is limited to a minimum. The following outline of an L-drying preservation method suitable for fastidious thermophilic strains is based on the description given by Malik (1990). A setup of the equipment necessary for primary L-drying is shown in Figure 15.3. Alternatively, this method can be carried out without a freeze-drying machine following the simplified procedure described by Malik (1991).
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Preservation of Thermophilic Microorganisms Figure 15.2. Secondary vacuum-drying. Constricted outer tubes containing inner vials are attached to a manifold and mounted on a freeze-drying machine. Adapted from Malik (1990) with permission.
Preparation of the carrier material A thin disc of carrier material is prepared in order to protect the cell suspension from freezing during the evacuation period. 1. Fill glass vials (44 11 mm, flat bottom) with 0.1 ml of 20% (w/v) skim milk containing 1% (w/v) neutral activated charcoal and 5% (w/v) meso-inositol. 2. The vials are loosely plugged with non-absorbent cotton wool and sterilized at 115 C for 13 min.
357
Figure 15.3. L-drying procedure. Setup of the necessary equipment: (a) freeze-drying machine; (b) vacuum pump; (c) water bath kept at a constant temperature of 20 C; (d) metallic evacuation jar equipped with vacuum valve; (e) aluminium Erlenmeyer cap; (f) inner vial with sample and cotton plug. Adapted from Malik (1990) with permission.
3. Freeze the vials at 20 C for several hours and thereafter transfer them to the drying chamber of a freeze-drying machine. Apply vacuum and freeze-dry overnight until vacuum has dropped to 0.1 mbar or less. Preparation of the cell suspension Cells are concentrated by harvesting and suspension in a protective medium. A mixture of activated charcoal and meso-inositol has proven most effective in preventing harmful effects on the cells during liquid-drying. 1. Suspend 1% (w/v) activated charcoal and dissolve 3% (w/v) mesoinositol in distilled water (pH adjusted to 7.0), bubble with N2 gas for at least 30 min to remove oxygen and fill in a suitable container, e.g. a serum bottle with butyl rubber stopper and aluminium crimp, which is tightly closed. Autoclave the protective solution at 115 C for 13 min. 2. Harvest cells as described in the section on centrifugal freeze-drying by aseptic centrifugation and suspend cell pellet in the protective solution, while maintaining anoxic conditions. The obtained cell suspension should have a concentration of about 108–109 cells ml1. Drying procedure L-drying is carried out in two stages. In the following L-drying procedure, the primary drying is achieved in two steps. 1. To the thin carrier disc of each vial, one drop (25–30 ml) of the cell suspension is transferred aseptically with care so as not to touch the side of the vial. 358
Storage and Recovery It was shown that the viability of vacuum-dried samples depends on the storage temperature (Banno and Sakane, 1981). As a rule of thumb, it was found that survival values were comparable when ampoules were stored at 5–9 C for a number of years or at 30–37 C for the same number of weeks (Malik, 1999). Thus ampoules should be kept in a dark cold place, or even better, a refrigerator. Resuscitation Please observe general safety precautions and wear protective goggles when opening ampoules. To open double-vial ampoules, the pointed part of the outer tube is heated in a Bunsen flame. Place two or three drops of water onto the hot tip to crack the glass. Strike off the glass tip with an appropriate tool (e.g. forceps). The inner vial is taken out and about 0.2–0.5 ml of fresh medium is added to the dried sample in order to dissolve and resuspend it. After about 2–5 min the content of the inner vial is usually rehydrated and can be transferred to a tube with the appropriate cultivation medium. In several cases it was observed that the ingredients of the protective medium can inhibit growth of fastidious strains in the first culture tube. Hence, a serial dilution of at least three or four tubes should be prepared. If the preserved culture can be grown easily on solid media, a few drops of the rehydrated sample should also be tranferred to an agar plate or slant to obtain single colonies in order to check the purity of the strain. In the case of obligate anaerobic strains, it is important to retain anoxic conditions during all steps after opening of the ampoule. This can be achieved in several ways depending on the used anaerobic technique and available equipment in the laboratory. If the Hungate technique is used, it is recommended to keep the inner 359
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2. Place ready vials quickly in aluminium Erlenmeyer caps and then transfer into a metallic jar maintained at 20 C in a water bath. 3. After 20–30 min of equilibration, apply a vacuum of 30–80 mbar and dry for 4–6 h. 4. For further drying, adjust vacuum to approx. 20–25 mbar and dry overnight while maintaining the temperature at 20 C. 5. Replace vacuum in the metallic jar with N2 gas and transfer the vials to soft glass tubes (135 15 mm) containing silica gel and cotton plugs. Add glass wool, constrict outer tubes, attach to the manifold of a freeze-drying machine and vacuum-dry for further 2–3 h at 0.01–1 mbar as described in the section on centrifugal freeze-drying. 6. At a vacuum of at least 0.1 mbar, the outer tubes are flame-sealed at the middle of the constriction.
vial under a flow of oxygen-free gas by inserting a gassing cannula until the cell pellet is completely resuspended. If the ampoule should be opened in an anaerobic gas chamber, it is necessary to score the ampoule with a sharp file at the middle of its shoulder about one cm from the tip. Transfer the ampoule with the file mark in the anaerobic chamber and strike the ampoule with a file or large forceps to remove the tip. If necessary, wrap the ampoule in tissue paper and enlarge the open end by striking with a file or pencil, then remove the glass wool insulation and the inner vial. Gently raise the cotton plug and sterilize the upper part of the inner vial by wiping it with tissue paper soaked in 70% ethanol. After opening of the ampoule, add approximately 0.5 ml of anoxic medium to resuspend the cell pellet and transfer the suspension to a vial with the recommended cultivation medium (5–10 ml).
Viability testing It is advisable to determine the survival rate of a preserved culture in order to determine the success of the vacuum-drying procedure. Sometimes, if no living cells can be recovered from lyophilized samples, it has turned out that cultures were incubated too long prior to preservation and thus were already inactive. For determination of the viability before the preservation process, an aliquot of the suspension used to fill the inner vials is inoculated in the appropriate culture medium and serially diluted to extinction in order to determine the approximate number of living cells. The same procedure is then repeated with a vacuum-dried sample. Although the stability of most dried cultures is satisfactory, there are also examples of fastidious strains that lose viability within several years of storage. Therefore, the survival rate of important vacuum-dried stock cultures should be checked in intervals of at least 5 years.
^^^^^ DEEP FREEZING Freezing and storage of cultures in liquid nitrogen has the advantage that the survival rates of the more susceptible microorganisms are usually higher as with methods based on vacuum-drying and that the procedures can be carried out easily under an oxygen-free atmosphere. Therefore, cryopreservation is the most effective method for the maintenance of fastidious thermophiles which grow only to a very low cell density or strains that are extremely sensitive to oxygen, e.g. most representatives of the hyperthermophilic methanogens (Methanocaldococcus spp. etc.). Moreover, this procedure is in general applicable to all known prokaryotes and hence has been established as standard preservation method for most seed stocks of prokaryotes held at the DSMZ culture collection. 360
Equipment The only major investment which is necessary to establish this method is a cryogenic storage tank for liquid nitrogen or alternatively a mechanical deep freezer, which can cool below 70 C. However, deep-freezers are only second quality and should only be used if a regular supply with liquid nitrogen cannot be guaranteed. The liquid nitrogen container has to be equipped with storage canes and canisters for the storage of glass capillaries in the liquid phase of nitrogen or racks with dividers for the storage of plastic cryotubes in the vapour phase. Further useful equipment includes standard laboratory accessories and diverse glass ware which can be obtained easily at low costs.
Preparation of Cell Suspension
Fresh culture medium containing 10% (v/v) glycerol or 5% (v/v) dimethyl sulfoxide (DMSO) is used as a suspending medium. DMSO is often more satisfactory than glycerol, because it requires less time to penetrate the organism. The cryoprotectant is sterilized separately by autoclaving in anoxic test tubes under nitrogen gas atmosphere (DMSO: 10 min, 115 C). Add cryoprotectant aseptically just before use to the appropriate sterile, aerobic or anaerobic cultivation medium. Preparation of cultures The cultivation and harvesting of aerobic and anaerobic thermophiles can be done as described in the corresponding paragraphs of the section on centrifugal freeze-drying. Again, the survival rates of aerobic thermoacidophiles like Metallosphaera or Sulfolobus species can be increased by neutralizing the growth medium prior to harvesting with some solid calcium carbonate.
Distribution in Aliquots and Freezing Cell suspensions can be distributed for freezing and subsequent storage either in screw cap plastic ampoules (cryotubes) or glass capillaries. Cryotubes have the advantage that the filling with suspension and the recovery of cultures is easy and needs no special training or equipment. However, there is always the risk of an imperfect sealing that could cause seepage of liquid nitrogen into the screw cap vial with subsequent explosion upon thawing. Therefore, it is safer to store cryotubes in the vapour phase, instead of submersing them in liquid nitrogen. In addition, plastic cryotubes can not be used for strictly anaerobic thermophiles. The material of the ampoules is not gas tight so that oxygen can penetrate the vial and irreversibly damage the cells. In contrast, glass capillary tubes can be hermetically sealed and are then absolutely impermeable to liquid 361
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Preparation of suspending medium
nitrogen and gases. They can be stored in the vapour phase as well as in the liquid phase of nitrogen and need very little storage place due to their small size. It has been found that for the cryopreservation of various filamentous fungi cooling rates in the range of 0.5 to 200 C min1 over the critical period from þ5 to 50 C were optimal (Smith and Thomas, 1998). However, it seems that controlled cooling rates may not have a significant effect on the survival of the much smaller cells of prokaryotes and hence this technique is not applied in the protocols given below. Plastic ampoules Polypropylene screw cap ampoules (1.8 ml vol.) can be obtained sterilized from the manufacturer. Aliquots of the concentrated cell suspension (approx. 0.5–1.0 ml) are distributed aseptically in the cryovials, which are then secured by tightly screwing down the lids. Place the cryotubes in suitable racks and then transfer in the vapour phase of liquid nitrogen for freezing. Glass capillary tubes The procedure for the freezing of microorganisms in glass capillary tubes is based on the description given by Hippe (1991) and illustrated in Figure 15.4A. Preparation of glass capillary tubes Glass capillary tubes (length 90 mm, outer diameter 1.4 mm, wall thickness 0.26 mm) are rinsed several times in distilled water and then dried. Mark capillaries at approx. 2.5 cm from one end with a permanent marker (water resistant ink), place capillaries in test tubes that are closed with aluminium caps and autoclave at 121 C for 20 min. Filling and sealing ofcapillaries 1. Transfer 0.5–1.0 ml of the concentrated cell suspension into a small sterile vial, which is placed in an ice bath. For the preservation of anaerobes, the vial is kept anoxic by gassing with an oxygen-free gas mixture that complies with the gas atmosphere used to cultivate the respective strain. However, CO2 gas should be replaced with a mixture of 80% N2 and 20% CO2, in order to avoid problems related to the high solubility of CO2 in water, which eventually could lead to the explosion of frozen capillaries upon thawing. 2. One glass capillary is taken from the sterile stock by fitting it to the tip of a micropipettor (e.g. micro-classic pipette controller; Brand GmbH, Wertheim) and enough of the cell suspension is aspirated to fill one-third of the length of the capillary. The volume taken up is approximately 25 ml. The suspension within the capillary is further aspirated until it is about 1 cm from the free end, which is sealed in a fine, hot gas flame. The second seal is made at 2.5 cm from the other end of the capillary (at the mark, which was made prior to sterilizing) 362
Preservation of Thermophilic Microorganisms
A.
Figure 15.4. Glass capillary tube method for the low-temperature preservation of microorganisms. From Hippe (1991) with permission. (A) Filling, sealing and storage of capillary tubes. (B) Removal of capillaries from freezing storage, opening, and recovery of cell suspension.
by heating and subsequent tearing of the softened glass. The making of the second seal is a critical step of this method and needs some training in order to achieve capillaries that are hermetically sealed. 3. As capillaries are prepared, they are placed in a vial with 75% ethanol for disinfection. The vial is placed in an ice/water bath in order to cool down capillaries immediately after the sealing procedure. 4. All capillaries are examined for correct seals under a stereomicroscope. As with ampoules, improperly sealed glass capillaries will take up nitrogen and will explode on removal from the cold. Nevertheless, a perfect seal is easier to achieve with capillaries. To avoid that moist 363
B.
Figure 15.4. Continued.
capillaries stick together when frozen, they are dried by placing them between absorbent paper and gently pressing and rolling with the flat hand. 5. The capillaries are stored in a capillary holder (aluminium or polypropylene tube), which is labelled with the strain designation on the outside and then placed in the vapour phase above liquid nitrogen for freezing. The time lapse between preparation of the suspension and freezing should be kept as short as possible. Rapid freezing by immersing capillaries directly in liquid nitrogen should be avoided, because not all capillaries may withstand the developing pressure and could break. 364
Storage and Recovery Cryotubes can be easily labelled with special cryo-markers prior to freezing and subsequent storage in racks in the vapour phase above liquid nitrogen. In contrast, the permanent labelling of capillaries is more difficult, but may be sometimes desirable if samples of different strains should be stored in the same capillary holder. In this case, capillaries can be colour coded by putting them into PVC straws (outer diameter 2 mm) of different colours, which are cut to length and squeezed together at one end. For storage, the capillary holder can be placed into an aluminium cane that is immersed into liquid nitrogen. Usually, several canes are collocated into one canister.
General safety precautions should be observed for exposure to liquid nitrogen or cryogenic equipment. Wear protective clothing and goggles while handling frozen samples! It has been found that slow warming may cause damage of cryopreserved samples due to the recrystallization of ice, therefore rapid thawing is recommended. To achieve the required fast thawing rates, frozen samples are immersed in warm water (30–37 C) immediately upon removal from the liquid nitrogen container. It has been observed in several cases that, strictly chemolithoautotrophic strains are inhibited by a remaining amount of the cryoprotectant in the first inoculated culture tube. Therefore, it is recommended to prepare a serial dilution of two or three tubes for the resuscitation of sensitive autotrophic strains. Plastic cryotubes Thaw the cryotube in a suitable container filled with warm water until the last visible ice has melted. Unscrew the cryotube, remove sample aseptically by using a Pasteur pipette and inoculate a suitable growth medium. Glass capillary tubes For the recovery of a strain, one capillary is removed from the liquid nitrogen tank and thawed rapidly in a container with warm water. The capillary is removed from the water bath, dried and opened at one end as shown in Figure 15.4B. In the case of aerobic thermophiles, the small volume of cell suspension is aspirated with a sterile Pasteur pipette that has been drawn out very finely to a length of 4 cm. While aspirating the suspension, the tip of the pipette is slowly moved further into the capillary. The suspension is then transferred to 5 ml of the freshly prepared appropriate medium. For the recovery of anaerobic strains, it is recommended to use a 1 ml disposable (tuberculin) syringe with a 25G hypodermic needle. Prior to use, the syringe is flushed with oxygen-free gas. The capillary is opened at both ends and the contents aspirated into 365
Preservation of Thermophilic Microorganisms
Resuscitation
the syringe while avoiding to uptake air bubbles along with the cell suspension. After aspirating the cell suspension from the capillary, the needle is inserted through the rubber closure of an unopened tube with 5–10 ml of anoxic medium. The tube is inverted and the suspension along with some medium is drawn into the syringe. Then, the tube is still in an inverted position, the contents of the syringe is injected into the tube. Viability testing The determination of survival rates after cryopreservation can be done according to the description given above in the section on vacuum drying. However, the testing of preserved cultures in regular intervals is not so important than with vacuum-dried samples, because the stability of frozen cultures stored in liquid nitrogen is practically unlimited. Further detailed information on preservation methods is available from the CABRI consortium, which has developed several guidelines for the maintenance of microorganisms (URL: http://www.cabri.org).
List of suppliers Air Liquide, Division Mate´riel Cryoge´nique 75 Quai d’Orsay 75321 Paris cedex 07, France http://www.airliquide.com Cryogenic storage vessels A. Albrecht GmbH & Co. KG Hauptstrasse 6-8 D-88326 Aulendorf, Germany PVC straws Chart Industries, Inc., MVE Bio-Medical Division 1800 Sandy Plains Industrial Parkway Marietta, GA 30066, USA http://www.chartbiomed.com Cryogenic storage vessels FGT Feingera¨tetechnik GbR Ernst-Tha¨lmann-Str. 27 D-99510 Apolda, Germany http://www.fgt.de Ampoule constrictor Hilgenberg GmbH Strauchgraben 2 366
D-34323 Malsfeld, Germany http://www.hilgenberg-gmbh.de Glass capillary tubes Lehmann & Voss & Co. Alsterufer 19 D-20354 Hamburg, Germany http://www.lehvoss.de Glass wool Martin Christ Gefriertrocknungsanlagen GmbH P.O. Box 1713 D-37507 Osterode am Harz, Germany http://www.martinchrist.de
Nalge Nunc International Corp. 75 Panorama Creek Drive Rochester, New York 14625-2385, USA http://www.nuncbrand.com Cryotubes Ochs Glasgera¨tebau GmbH Pappelweg 26 D-37120 Bovenden/Lenglern, Germany http://www.labor-ochs.de Glassware Paul Hartmann AG P.O. Box 1420 D-89504 Heidenheim, Germany http://www.hartmann-online.com Cotton plugs Gebr. Rettberg GmbH Rudolf-Wissell-Strasse 17 D-37079 Go¨ttingen, Germany http://www.rettberg.biz Glassware Sigma-Aldrich Chemie GmbH Eschenstrasse 5 D-82024 Taufkirchen bei Mu¨nchen, Germany http://www.sigmaaldrich.com 367
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Freeze-drying machines
Chemicals, Silicagel rubin Vacuubrand GmbH & Co. KG Alfred-Zippe-Str.4 D-97877 Wertheim, Germany http://www.vacuubrand.de Vacuum pumps
References Annear, D. I. (1956). The preservation of bacteria by drying in peptone plugs. J. Hyg. 54, 487–508. Banno, I. and Sakane, T. (1981). Prediction of prospective viability of L-dried cultures of bacteria after long-term preservation. Inst. Ferment. Osaka Res. Commun. 10, 33–38. Brock, T. D. and O’Dea, K. (1977). Amorphous ferrous sulfide as a reducing agent for culture of anaerobes. Appl. Environ. Microbiol. 33, 254–256. Hippe, H. (1991). Maintenance of methanogenic bacteria. In Maintenance of Microorganisms and Cultured Cells (B. E. Kirsop and A. Doyle, eds), 2nd edn., pp. 101–113. Academic Press, London. Malik, K. A. (1990). A simplified liquid-drying method for the preservation of microorganisms sensitive to freezing and freeze-drying. J. Microbiol. Meth. 12, 125–132. Malik, K. A. (1991). Maintenance of microorganisms by simple methods. In Maintenance of Microorganisms and Cultured Cells (B. E. Kirsop and A. Doyle, eds), 2nd edn., pp. 121–132. Academic Press, London. Malik, K. A. (1999). Preservation of some extremely thermophilic chemolithoautotrophic bacteria by deep-freezing and liquid-drying methods. J. Microbiol. Meth. 35, 177–182. Morris, G. J. (1981). Cryopreservation. Institute of Terrestrial Ecology, Cambridge. Smith, D. and Thomas, V. E. (1998). Cryogenic light microscopy and the development of cooling protocols for the cryopreservation of filamentous fungi. World J. Microbiol. Biotechnol. 14, 49–57.
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Introduction Maintenance by subculturing and preservation Methods for the long-term preservation of thermophiles Vaccum drying Deep freezing
^^^^^ INTRODUCTION Thermophilic and hyperthermophilic microorganisms are highly diverse in respect of their metabolism and phylogeny. Besides high temperatures, most thermophiles have also adapted their lifestyle also to other environmental conditions which can be termed extreme from an anthropocentric point of view, e.g. acidic pH values, highly reduced conditions or hypersalinity. This versatility, among other reasons, may have caused an increasing interest in this group of prokaryotes. The mounting research activity in this field in many laboratories resulted in a need for reliable maintenance methods for thermophilic strains. Especially for comparative genetic or biochemical studies it is often desirable to work with stocks of preserved cultures, or it could be necessary to maintain a large number of mutant strains over longer periods of time. On the other hand, standard methods of preservation often fail in the case of thermophilic microorganisms, especially if they are also strictly anaerobic or acidophilic. Several methods which can be used for the long-term preservation of these fastidious microorganisms are discussed in this chapter. Most of these techniques were originally developed for the reliable preservation of extremophiles at culture collections, but with some expertise, it should be possible to adapt these procedures to the technical facilities available in most research or industrial laboratories. Resources for material and equipment useful for the described methods are listed at the end of this chapter. METHODS IN MICROBIOLOGY, VOLUME 35 0580-9517 DOI:10.1016/S0580-9517(05)35015-X
Copyright ß 2006 Elsevier Ltd. All rights reserved.
Preservation of Thermophilic Microorganisms
CONTENTS
^^^^^ MAINTENANCE BY SUBCULTURING
AND PRESERVATION In several laboratories studying thermophilic prokaryotes, important strains are only maintained by frequent subculturing. Subculturing means serial transfer of strains from media with depleted nutrient sources to fresh media. After inoculation into fresh media, cultures are incubated to obtain growth and then eventually stored. To prevent frequent subculturing, the metabolic rate of the organism during storage should be kept at a minimum. Many thermophilic strains stop growing already below 40 C, so that they can be stored easily without refrigeration at room temperature. Refrigeration to 4–8 C can be used to extend the interval of subculturing of some strains, but on the other hand may have a negative effect on the long-term stability of other thermophilic strains. Viability of distinct cultures over time may vary considerably and depends largely on the reached growth phase, storage conditions and quality of the used medium. Hence, time of storage between transfers is normally kept at a minimum to ensure survival of important strains. Subculturing is inexpensive in terms of equipment, applicable to all cultivable strains and avoids problems associated with the resuscitation of preserved stock cultures. However, it can be time-consuming if organisms are handled that require frequent transfers or a whole collection of strains has to be maintained. Besides the time necessary for preparation of media and manipulation of strains, subculturing implicates several risks and disadvantages. Contamination of pure cultures is a permanent threat and the mislabelling or transposition of vials can lead to an interchange of strains. The risk of mishaps increases with the frequency of the manipulation of a strain and can be only minimized by the consequent use of sound microbial techniques. Frequent subculturing can also have a negative effect on the genetic integrity of a strain. Genetic changes or the loss of plasmids may result in the selection of mutant strains and thereby, to a strain drift. A progressive genetic drift in a distinct culture may remain unrecognized if inconspicuous traits of the organism are affected. Only by using reliable long-term preservation techniques, most of these risks can be efficiently avoided. The investment in laboratory equipment and training of technical staff, which is necessary for the establishment of most preservation techniques, appears to be only a small effort in comparison to the damage caused by the potential loss of important reference strains.
^^^^^ METHODS FOR THE LONG-TERM
PRESERVATION OF THERMOPHILES Several well-established methods are available for the long-term preservation of thermophiles. The basic principle common to all of
350
these techniques is the reduction of the amount of freely available water to a value where metabolism is suspended. A decrease in water activity can be achieved either by dehydration or freezing. Numerous procedures have been developed for the gentle cryopreservation or drying of cultures in order to minimize cell damage during preservation. Nevertheless, survival rates of some cultures can be rather low. In general, survival rates depend largely on the sensitivity of a strain against the harmful effects caused by the used preservation method and can vary considerably among closely related strains. Consequently, not all of the techniques described in this chapter are applicable to the whole range of thermophilic microorganisms. The main assets and drawbacks of the most common techniques are discussed below.
Freeze-drying or lyophilization is a process which extracts water from a sample by sublimation. Sublimation is the transition of a substance from the solid to the vapour state, without passing through an intermediate liquid phase. Lyophilization involves freezing of the cell pellet so that the water becomes ice, application of vacuum in order to sublimate the ice directly into water vapour and drawing off the water vapour. Cultures preserved by freeze-drying are relatively stable over time and can be stored without further attention. However, due to the low survival rates of susceptible strains, a selection of more resistant subpopulations with different genetic characteristics may take place during freeze-drying. To avoid a genetic drift by the continuous selection of subpopulations, subsequent batches should be always prepared from the same seed stock. Ampoules with lyophilized samples can be transported easily without the risk of damage caused by lyses of sensitive cultures. However, the resuscitation of vacuumdried samples can be labour-intensive, especially if anaerobic strains were preserved. In addition, most strains show a prolonged lag-phase after resuscitation and need two or three transfers until normal growth takes place. This preservation method requires an investment in special equipment, which is mainly useful for the purpose of freeze-drying. Some training of technical staff will also be necessary to ensure a safe and smooth flow of lyophilization procedures. Thus this method is only cost-effective for laboratories which have to prepare larger batches of preserved samples from thermophiles or intend to store a large collection of strains without further attention. Thermophiles that are suitable for preservation by lyophilization represent mainly aerobic or aerotolerant, heterotrophic microorganisms, which are relatively robust and show a good growth yield. In contrast, strains that are extremely sensitive to oxygen or reach only very low cell densities in most cases, hardly survive the harsh
351
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Freeze-drying
conditions during freeze-drying and should be better preserved by cryopreservation.
Liquid-drying Unlike lyophilization, liquid-drying (L-drying; Annear, 1956) involves vacuum-drying of samples from the liquid state without freezing. Liquiddrying has, in general, the same benefits and drawbacks as freeze-drying, but the procedure is less elaborate and may have advantages in the preservation of microorganisms that are particularly sensitive to the initial freezing step involved in lyophilization.
Deep Freezing The only technique which seems to be applicable to all thermophilic prokaryotes studied so far is the deep freezing of concentrated cell suspensions in fresh media supplemented with a suitable cryoprotectant. The survival rates of cultures preserved by this method are usually quite high and the long-term stability almost unlimited. The resuscitation of cultures preserved by deep freezing is uncomplicated and growth normally takes place without an extended lag phase. However, it is important that the temperature of storage should be kept below 139 C, because only then all physical or chemical reactions are suspended (Morris, 1981) and a satisfactory stability of the preserved culture can be achieved. In contrast, the storage of cryopreserved stocks in normal freezers at temperatures above 70 C is not recommended, because free water is still available enabling residual physical or chemical activity, which could damage DNA or other essential cell compounds causing a rapid loss of viability in some strains. Normally, storage in liquid nitrogen (196 C) or the nitrogen vapour phase (140 C) is used to achieve the required low temperatures. With exception of the liquid nitrogen storage tank, the equipment necessary for this method is easy to obtain and inexpensive. A major constraint of this preservation technique may be, however, the continuous need for liquid nitrogen to maintain the required storage temperature. A complete evaporation of liquid nitrogen will cause an inevitable temperature rise in the refrigerator which eventually may kill all sensitive stocks. Hence, care has to be taken to ensure that liquid nitrogen is replenished in regular intervals. Nevertheless, in some geographical regions the adequate supply with liquid nitrogen is problematic. In this case, storage of less sensitive cultures in deep freezers at 80 C may be an alternative. However, it has to be noted that although metabolism is suspended, cells can be damaged by the recrystallization of ice, which still takes place at this temperature. In the following sections, standard protocols for the preservation of thermophilic prokaryotes are exemplified on representatives of various physiological groups. 352
^^^^^ VACUUM-DRYING
Equipment Technical equipment necessary for vacuum-drying methods include a vacuum pump, a freeze-dryer equipped with a centrifuge head and manifold or an evacuation jar connected to a moisture or cold trap, constrictors and diverse glass ware. Vacuum-dried samples of cultures can be prepared either in double vial or single vial ampoules. The Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) delivers dried cell pellets exclusively as double vial preparations, sealed under vacuum (Figure 15.1). Double-vial ampoules are more elaborate to prepare, but have the advantage that a contamination of the atmosphere by aerosols that can be produced by sudden release of the vacuum in single-vial preparations is efficiently prevented. In addition, the cell pellet is protected from contamination, because inflowing air filters through the sterile cotton plug of the inner vial.
Centrifugal Freeze-drying Centrifugal freeze-drying is the preferred method for the preservation of fastidious thermophilic strains that are susceptible to the initial freezing in skim milk intrinsic to standard freeze-drying protocols. This method relies on the freezing of the cell suspension by evaporative cooling caused by the loss of water during evacuation of the freeze-drying chamber. To avoid frothing of the suspension due to removal of dissolved gases before freezing is complete, the suspension is centrifuged during the initial stages of drying. In this way, the initial freezing of the cell suspension, which is a critical step of the normal shelf freeze-drying process, can be omitted. At the DSMZ, a special preservation mixture has been developed 353
Preservation of Thermophilic Microorganisms
Figure 15.1. Double-vial preparation of a vacuum-dried sample, sealed under vacuum.
which is now routinely used for the protection of thermophilic strains during the centrifugal freeze-drying process. The use of this protective solution allows the lyophilization of most aerobic thermophiles, with the exception of strains that grow only to a very low cell density. The supplementation of this solution with amorphous ferrous sulfide also allows the lyophilization of some anaerobic thermophiles without using an oxygen-free gas atmosphere during the processing of samples. Preparation of vials 1. Plug clean glass vials (44 11 mm, flat bottom) loosely with nonabsorbent cotton wool plugs (dental rolls size no. 2, 40 mm in length) and sterilize at 121 C for 20 min. 2. Prepare outer glass tubes (135 215 mm, soft glass) by placing a few pieces of self-indicating silica gel (e.g. silicagel rubin) into the tubes which are covered with a small amount of cotton wool (see Figure 15.1). Preparation of the suspending medium 1. Preservation mixture: Dissolve 2% (w/v) gelatine, 2% (w/v) yeast extract and 24% (w/v) sucrose in distilled water, bubble with N2 gas for at least 30 min to remove oxygen and fill in a suitable container, which can be tightly closed, e.g. a serum bottle with butyl rubber stopper and aluminium crimp. Autoclave the protective solution at 121 C for 20 min. The suspending medium is prepared by mixing an equal ratio of freshly prepared sterile cultivation medium and preservation mixture. 2. Amorphous ferrous sulfide for anaerobic strains: The ferrous sulfide is prepared according to Brock and O’Dea (1977). Briefly, equimolar amounts of ferrous ammonium sulfate and sodium sulfide react in solution to form amorphous FeS, which precipitates. After settling of the FeS precipitate, the supernatant is discarded. This procedure is repeated several times with an anoxic solution of 0.9% (w/v) NaCl until the supernatant no longer contains detectable amounts of ferrous or sulfide ions. Finally, the purified aqueous suspension of FeS is dispensed under nitrogen gas atmosphere in suitable vessels and autoclaved. The final concentration of amorphous ferrous sulfide in the suspending medium should be approximately 1 mg ml1. Preparation of the cell suspension Depending on the oxygen relationship and acid tolerance of the strain to be preserved, different procedures are necessary for the preparation of the cell suspension. Ideally cells are harvested when they are most active and have reached the mid exponential growth phase. The processing of aerobes is exemplified on representatives of the genera Aeropyrum and Sulfolobus, whereas the lyophilization of anaerobes is illustrated by a protocol suitable for hyperthermophilic Pyrococcus species. 354
Aerobic heterotrophic thermophiles
Strictly anaerobic thermophiles Special care has to be taken to avoid exposure to oxygen during harvesting of Pyrococcus spp. and other obligate anaerobic thermophiles and hyperthermophiles. Either anaerobically grown cultures have to be transferred under an oxygen-free gas atmosphere to anoxic and gas-tight centrifuge bottles or in a more straightforward approach are cultured in heavy-walled, round-bottomed glass bottles, which can be used for growing of cells and centrifugation. Suitable glass bottles (50–70 ml volume) can be ordered from most glass blowers as custom-made product and should have necks that can be closed with a butyl rubber septum and screw cap as with Hungate-type anaerobe tubes. After centrifugation the centrifuge bottle is opened, a gassing cannula inserted and the supernatant is removed aseptically under a flow of oxygen-free gas. Normally, the gas-mixture used corresponds to the gas atmosphere is used to cultivate the respective strain. However, a mixture of N2 and CO2 instead of a H2 containing gas mixture should be used to avoid risks caused by the generation of flammable gas mixtures in the laboratory. Cell pellets of one or more bottles are collected using suspending medium that has been supplemented with 1 mg ml1 amorphous FeS and transferred to a tube continuously flushed with oxygen-free N2 gas. Filling of vials is carried out under oxic conditions using for instance an Eppendorf Multipipette with 2.5 ml Combitip. This procedure should be done quickly because the ferrous sulfide provides protection of cells against oxygen only for a limited time.
Drying procedure Drying is carried out in two stages. Primary drying is achieved by centrifugal freeze-drying, whereas standard shelf freeze-drying is used to 355
Preservation of Thermophilic Microorganisms
Aeropyrum spp. and other oxygen-tolerant hyperthermophiles can be harvested as usual by centrifugation of liquid medium in sterile centrifuge tubes or bottles. The cell pellet is resuspended in approximately 5 ml of suspending medium containing the preservation mixture and aseptically distributed in aliquots of 0.15 ml to each single vial. Several thermoacidophilic species, e.g. Sulfolobus solfataricus, are sensitive to low pH values at the end of the growth phase and easily lose viability. Therefore, the growth medium is neutralized prior to harvesting of the cells by adding a small amount of solid, sterilized calcium carbonate. After 10–15 min, the undissolved carbonate forms a deposit which then can be removed from the culture supernatant. In addition, the pH value of the thermoacidophiles growth medium is adjusted to a moderate value (pH 4.0–4.5) prior to mixing with the preservation mixture in order to prevent damage of cells during lyophilization.
obtain double-vial preparations in the second step. 1. Primary drying 1.1. Place vials in the centrifuge head of a freeze-drying machine, switch on the centrifuge and thereafter apply vacuum. 1.2. Centrifuge at approximately 750 rpm for 2–3 h. 1.3. Switch off the centrifuge and continue primary drying until the vacuum has dropped to 1–10 mbar. 1.4. Switch off refrigerator and vacuum pump, allow air to slowly enter the vacuum chamber and remove vials from the centrifuge head. 2. Secondary drying 2.1. Cut off parts of the cotton-wool plugs that are projecting from the vials and place vials in outer glass tubes containing self-indicating silica gel. 2.2. To protect the cotton-wool plugs from heat during constriction cover the vials with glass wool (Tempstran 475–106) slightly compressed to a layer of 1–2 cm thickness (see Figure 15.1). The outer tubes are constricted just above the glass wool either by hand or by using a semiautomatic ampoule constrictor. 2.3. After cooling, attach the double-vial preparations to the manifold of a freeze-drying machine for secondary drying for at least 2 h or overnight (see Figure 15.2). 2.4. At a vacuum of at least 0.1 mbar, the tubes are flame-sealed at the middle of the constriction.
Liquid-drying The L-drying method has been successfully applied at the DSMZ to several thermophilic, obligate chemolithoautotrophic bacteria, which are difficult to preserve using one of the standard freeze-drying protocols. Examples of successfully preserved thermophiles include Aquifex pyrophilus, Thermocrinis albus, Hydrogenobacter thermophilus and Sulfurihydrogenibium azorense. In general, members of these genera and related hydrogen-oxidizing thermophiles are microaerophilic and sensitive to elevated levels of oxygen. Thus, during handling of these cultures care has to be taken to ensure that exposure to atmospheric levels of oxygen is limited to a minimum. The following outline of an L-drying preservation method suitable for fastidious thermophilic strains is based on the description given by Malik (1990). A setup of the equipment necessary for primary L-drying is shown in Figure 15.3. Alternatively, this method can be carried out without a freeze-drying machine following the simplified procedure described by Malik (1991).
356
Preservation of Thermophilic Microorganisms Figure 15.2. Secondary vacuum-drying. Constricted outer tubes containing inner vials are attached to a manifold and mounted on a freeze-drying machine. Adapted from Malik (1990) with permission.
Preparation of the carrier material A thin disc of carrier material is prepared in order to protect the cell suspension from freezing during the evacuation period. 1. Fill glass vials (44 11 mm, flat bottom) with 0.1 ml of 20% (w/v) skim milk containing 1% (w/v) neutral activated charcoal and 5% (w/v) meso-inositol. 2. The vials are loosely plugged with non-absorbent cotton wool and sterilized at 115 C for 13 min.
357
Figure 15.3. L-drying procedure. Setup of the necessary equipment: (a) freeze-drying machine; (b) vacuum pump; (c) water bath kept at a constant temperature of 20 C; (d) metallic evacuation jar equipped with vacuum valve; (e) aluminium Erlenmeyer cap; (f) inner vial with sample and cotton plug. Adapted from Malik (1990) with permission.
3. Freeze the vials at 20 C for several hours and thereafter transfer them to the drying chamber of a freeze-drying machine. Apply vacuum and freeze-dry overnight until vacuum has dropped to 0.1 mbar or less. Preparation of the cell suspension Cells are concentrated by harvesting and suspension in a protective medium. A mixture of activated charcoal and meso-inositol has proven most effective in preventing harmful effects on the cells during liquid-drying. 1. Suspend 1% (w/v) activated charcoal and dissolve 3% (w/v) mesoinositol in distilled water (pH adjusted to 7.0), bubble with N2 gas for at least 30 min to remove oxygen and fill in a suitable container, e.g. a serum bottle with butyl rubber stopper and aluminium crimp, which is tightly closed. Autoclave the protective solution at 115 C for 13 min. 2. Harvest cells as described in the section on centrifugal freeze-drying by aseptic centrifugation and suspend cell pellet in the protective solution, while maintaining anoxic conditions. The obtained cell suspension should have a concentration of about 108–109 cells ml1. Drying procedure L-drying is carried out in two stages. In the following L-drying procedure, the primary drying is achieved in two steps. 1. To the thin carrier disc of each vial, one drop (25–30 ml) of the cell suspension is transferred aseptically with care so as not to touch the side of the vial. 358
Storage and Recovery It was shown that the viability of vacuum-dried samples depends on the storage temperature (Banno and Sakane, 1981). As a rule of thumb, it was found that survival values were comparable when ampoules were stored at 5–9 C for a number of years or at 30–37 C for the same number of weeks (Malik, 1999). Thus ampoules should be kept in a dark cold place, or even better, a refrigerator. Resuscitation Please observe general safety precautions and wear protective goggles when opening ampoules. To open double-vial ampoules, the pointed part of the outer tube is heated in a Bunsen flame. Place two or three drops of water onto the hot tip to crack the glass. Strike off the glass tip with an appropriate tool (e.g. forceps). The inner vial is taken out and about 0.2–0.5 ml of fresh medium is added to the dried sample in order to dissolve and resuspend it. After about 2–5 min the content of the inner vial is usually rehydrated and can be transferred to a tube with the appropriate cultivation medium. In several cases it was observed that the ingredients of the protective medium can inhibit growth of fastidious strains in the first culture tube. Hence, a serial dilution of at least three or four tubes should be prepared. If the preserved culture can be grown easily on solid media, a few drops of the rehydrated sample should also be tranferred to an agar plate or slant to obtain single colonies in order to check the purity of the strain. In the case of obligate anaerobic strains, it is important to retain anoxic conditions during all steps after opening of the ampoule. This can be achieved in several ways depending on the used anaerobic technique and available equipment in the laboratory. If the Hungate technique is used, it is recommended to keep the inner 359
Preservation of Thermophilic Microorganisms
2. Place ready vials quickly in aluminium Erlenmeyer caps and then transfer into a metallic jar maintained at 20 C in a water bath. 3. After 20–30 min of equilibration, apply a vacuum of 30–80 mbar and dry for 4–6 h. 4. For further drying, adjust vacuum to approx. 20–25 mbar and dry overnight while maintaining the temperature at 20 C. 5. Replace vacuum in the metallic jar with N2 gas and transfer the vials to soft glass tubes (135 15 mm) containing silica gel and cotton plugs. Add glass wool, constrict outer tubes, attach to the manifold of a freeze-drying machine and vacuum-dry for further 2–3 h at 0.01–1 mbar as described in the section on centrifugal freeze-drying. 6. At a vacuum of at least 0.1 mbar, the outer tubes are flame-sealed at the middle of the constriction.
vial under a flow of oxygen-free gas by inserting a gassing cannula until the cell pellet is completely resuspended. If the ampoule should be opened in an anaerobic gas chamber, it is necessary to score the ampoule with a sharp file at the middle of its shoulder about one cm from the tip. Transfer the ampoule with the file mark in the anaerobic chamber and strike the ampoule with a file or large forceps to remove the tip. If necessary, wrap the ampoule in tissue paper and enlarge the open end by striking with a file or pencil, then remove the glass wool insulation and the inner vial. Gently raise the cotton plug and sterilize the upper part of the inner vial by wiping it with tissue paper soaked in 70% ethanol. After opening of the ampoule, add approximately 0.5 ml of anoxic medium to resuspend the cell pellet and transfer the suspension to a vial with the recommended cultivation medium (5–10 ml).
Viability testing It is advisable to determine the survival rate of a preserved culture in order to determine the success of the vacuum-drying procedure. Sometimes, if no living cells can be recovered from lyophilized samples, it has turned out that cultures were incubated too long prior to preservation and thus were already inactive. For determination of the viability before the preservation process, an aliquot of the suspension used to fill the inner vials is inoculated in the appropriate culture medium and serially diluted to extinction in order to determine the approximate number of living cells. The same procedure is then repeated with a vacuum-dried sample. Although the stability of most dried cultures is satisfactory, there are also examples of fastidious strains that lose viability within several years of storage. Therefore, the survival rate of important vacuum-dried stock cultures should be checked in intervals of at least 5 years.
^^^^^ DEEP FREEZING Freezing and storage of cultures in liquid nitrogen has the advantage that the survival rates of the more susceptible microorganisms are usually higher as with methods based on vacuum-drying and that the procedures can be carried out easily under an oxygen-free atmosphere. Therefore, cryopreservation is the most effective method for the maintenance of fastidious thermophiles which grow only to a very low cell density or strains that are extremely sensitive to oxygen, e.g. most representatives of the hyperthermophilic methanogens (Methanocaldococcus spp. etc.). Moreover, this procedure is in general applicable to all known prokaryotes and hence has been established as standard preservation method for most seed stocks of prokaryotes held at the DSMZ culture collection. 360
Equipment The only major investment which is necessary to establish this method is a cryogenic storage tank for liquid nitrogen or alternatively a mechanical deep freezer, which can cool below 70 C. However, deep-freezers are only second quality and should only be used if a regular supply with liquid nitrogen cannot be guaranteed. The liquid nitrogen container has to be equipped with storage canes and canisters for the storage of glass capillaries in the liquid phase of nitrogen or racks with dividers for the storage of plastic cryotubes in the vapour phase. Further useful equipment includes standard laboratory accessories and diverse glass ware which can be obtained easily at low costs.
Preparation of Cell Suspension
Fresh culture medium containing 10% (v/v) glycerol or 5% (v/v) dimethyl sulfoxide (DMSO) is used as a suspending medium. DMSO is often more satisfactory than glycerol, because it requires less time to penetrate the organism. The cryoprotectant is sterilized separately by autoclaving in anoxic test tubes under nitrogen gas atmosphere (DMSO: 10 min, 115 C). Add cryoprotectant aseptically just before use to the appropriate sterile, aerobic or anaerobic cultivation medium. Preparation of cultures The cultivation and harvesting of aerobic and anaerobic thermophiles can be done as described in the corresponding paragraphs of the section on centrifugal freeze-drying. Again, the survival rates of aerobic thermoacidophiles like Metallosphaera or Sulfolobus species can be increased by neutralizing the growth medium prior to harvesting with some solid calcium carbonate.
Distribution in Aliquots and Freezing Cell suspensions can be distributed for freezing and subsequent storage either in screw cap plastic ampoules (cryotubes) or glass capillaries. Cryotubes have the advantage that the filling with suspension and the recovery of cultures is easy and needs no special training or equipment. However, there is always the risk of an imperfect sealing that could cause seepage of liquid nitrogen into the screw cap vial with subsequent explosion upon thawing. Therefore, it is safer to store cryotubes in the vapour phase, instead of submersing them in liquid nitrogen. In addition, plastic cryotubes can not be used for strictly anaerobic thermophiles. The material of the ampoules is not gas tight so that oxygen can penetrate the vial and irreversibly damage the cells. In contrast, glass capillary tubes can be hermetically sealed and are then absolutely impermeable to liquid 361
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Preparation of suspending medium
nitrogen and gases. They can be stored in the vapour phase as well as in the liquid phase of nitrogen and need very little storage place due to their small size. It has been found that for the cryopreservation of various filamentous fungi cooling rates in the range of 0.5 to 200 C min1 over the critical period from þ5 to 50 C were optimal (Smith and Thomas, 1998). However, it seems that controlled cooling rates may not have a significant effect on the survival of the much smaller cells of prokaryotes and hence this technique is not applied in the protocols given below. Plastic ampoules Polypropylene screw cap ampoules (1.8 ml vol.) can be obtained sterilized from the manufacturer. Aliquots of the concentrated cell suspension (approx. 0.5–1.0 ml) are distributed aseptically in the cryovials, which are then secured by tightly screwing down the lids. Place the cryotubes in suitable racks and then transfer in the vapour phase of liquid nitrogen for freezing. Glass capillary tubes The procedure for the freezing of microorganisms in glass capillary tubes is based on the description given by Hippe (1991) and illustrated in Figure 15.4A. Preparation of glass capillary tubes Glass capillary tubes (length 90 mm, outer diameter 1.4 mm, wall thickness 0.26 mm) are rinsed several times in distilled water and then dried. Mark capillaries at approx. 2.5 cm from one end with a permanent marker (water resistant ink), place capillaries in test tubes that are closed with aluminium caps and autoclave at 121 C for 20 min. Filling and sealing ofcapillaries 1. Transfer 0.5–1.0 ml of the concentrated cell suspension into a small sterile vial, which is placed in an ice bath. For the preservation of anaerobes, the vial is kept anoxic by gassing with an oxygen-free gas mixture that complies with the gas atmosphere used to cultivate the respective strain. However, CO2 gas should be replaced with a mixture of 80% N2 and 20% CO2, in order to avoid problems related to the high solubility of CO2 in water, which eventually could lead to the explosion of frozen capillaries upon thawing. 2. One glass capillary is taken from the sterile stock by fitting it to the tip of a micropipettor (e.g. micro-classic pipette controller; Brand GmbH, Wertheim) and enough of the cell suspension is aspirated to fill one-third of the length of the capillary. The volume taken up is approximately 25 ml. The suspension within the capillary is further aspirated until it is about 1 cm from the free end, which is sealed in a fine, hot gas flame. The second seal is made at 2.5 cm from the other end of the capillary (at the mark, which was made prior to sterilizing) 362
Preservation of Thermophilic Microorganisms
A.
Figure 15.4. Glass capillary tube method for the low-temperature preservation of microorganisms. From Hippe (1991) with permission. (A) Filling, sealing and storage of capillary tubes. (B) Removal of capillaries from freezing storage, opening, and recovery of cell suspension.
by heating and subsequent tearing of the softened glass. The making of the second seal is a critical step of this method and needs some training in order to achieve capillaries that are hermetically sealed. 3. As capillaries are prepared, they are placed in a vial with 75% ethanol for disinfection. The vial is placed in an ice/water bath in order to cool down capillaries immediately after the sealing procedure. 4. All capillaries are examined for correct seals under a stereomicroscope. As with ampoules, improperly sealed glass capillaries will take up nitrogen and will explode on removal from the cold. Nevertheless, a perfect seal is easier to achieve with capillaries. To avoid that moist 363
B.
Figure 15.4. Continued.
capillaries stick together when frozen, they are dried by placing them between absorbent paper and gently pressing and rolling with the flat hand. 5. The capillaries are stored in a capillary holder (aluminium or polypropylene tube), which is labelled with the strain designation on the outside and then placed in the vapour phase above liquid nitrogen for freezing. The time lapse between preparation of the suspension and freezing should be kept as short as possible. Rapid freezing by immersing capillaries directly in liquid nitrogen should be avoided, because not all capillaries may withstand the developing pressure and could break. 364
Storage and Recovery Cryotubes can be easily labelled with special cryo-markers prior to freezing and subsequent storage in racks in the vapour phase above liquid nitrogen. In contrast, the permanent labelling of capillaries is more difficult, but may be sometimes desirable if samples of different strains should be stored in the same capillary holder. In this case, capillaries can be colour coded by putting them into PVC straws (outer diameter 2 mm) of different colours, which are cut to length and squeezed together at one end. For storage, the capillary holder can be placed into an aluminium cane that is immersed into liquid nitrogen. Usually, several canes are collocated into one canister.
General safety precautions should be observed for exposure to liquid nitrogen or cryogenic equipment. Wear protective clothing and goggles while handling frozen samples! It has been found that slow warming may cause damage of cryopreserved samples due to the recrystallization of ice, therefore rapid thawing is recommended. To achieve the required fast thawing rates, frozen samples are immersed in warm water (30–37 C) immediately upon removal from the liquid nitrogen container. It has been observed in several cases that, strictly chemolithoautotrophic strains are inhibited by a remaining amount of the cryoprotectant in the first inoculated culture tube. Therefore, it is recommended to prepare a serial dilution of two or three tubes for the resuscitation of sensitive autotrophic strains. Plastic cryotubes Thaw the cryotube in a suitable container filled with warm water until the last visible ice has melted. Unscrew the cryotube, remove sample aseptically by using a Pasteur pipette and inoculate a suitable growth medium. Glass capillary tubes For the recovery of a strain, one capillary is removed from the liquid nitrogen tank and thawed rapidly in a container with warm water. The capillary is removed from the water bath, dried and opened at one end as shown in Figure 15.4B. In the case of aerobic thermophiles, the small volume of cell suspension is aspirated with a sterile Pasteur pipette that has been drawn out very finely to a length of 4 cm. While aspirating the suspension, the tip of the pipette is slowly moved further into the capillary. The suspension is then transferred to 5 ml of the freshly prepared appropriate medium. For the recovery of anaerobic strains, it is recommended to use a 1 ml disposable (tuberculin) syringe with a 25G hypodermic needle. Prior to use, the syringe is flushed with oxygen-free gas. The capillary is opened at both ends and the contents aspirated into 365
Preservation of Thermophilic Microorganisms
Resuscitation
the syringe while avoiding to uptake air bubbles along with the cell suspension. After aspirating the cell suspension from the capillary, the needle is inserted through the rubber closure of an unopened tube with 5–10 ml of anoxic medium. The tube is inverted and the suspension along with some medium is drawn into the syringe. Then, the tube is still in an inverted position, the contents of the syringe is injected into the tube. Viability testing The determination of survival rates after cryopreservation can be done according to the description given above in the section on vacuum drying. However, the testing of preserved cultures in regular intervals is not so important than with vacuum-dried samples, because the stability of frozen cultures stored in liquid nitrogen is practically unlimited. Further detailed information on preservation methods is available from the CABRI consortium, which has developed several guidelines for the maintenance of microorganisms (URL: http://www.cabri.org).
List of suppliers Air Liquide, Division Mate´riel Cryoge´nique 75 Quai d’Orsay 75321 Paris cedex 07, France http://www.airliquide.com Cryogenic storage vessels A. Albrecht GmbH & Co. KG Hauptstrasse 6-8 D-88326 Aulendorf, Germany PVC straws Chart Industries, Inc., MVE Bio-Medical Division 1800 Sandy Plains Industrial Parkway Marietta, GA 30066, USA http://www.chartbiomed.com Cryogenic storage vessels FGT Feingera¨tetechnik GbR Ernst-Tha¨lmann-Str. 27 D-99510 Apolda, Germany http://www.fgt.de Ampoule constrictor Hilgenberg GmbH Strauchgraben 2 366
D-34323 Malsfeld, Germany http://www.hilgenberg-gmbh.de Glass capillary tubes Lehmann & Voss & Co. Alsterufer 19 D-20354 Hamburg, Germany http://www.lehvoss.de Glass wool Martin Christ Gefriertrocknungsanlagen GmbH P.O. Box 1713 D-37507 Osterode am Harz, Germany http://www.martinchrist.de
Nalge Nunc International Corp. 75 Panorama Creek Drive Rochester, New York 14625-2385, USA http://www.nuncbrand.com Cryotubes Ochs Glasgera¨tebau GmbH Pappelweg 26 D-37120 Bovenden/Lenglern, Germany http://www.labor-ochs.de Glassware Paul Hartmann AG P.O. Box 1420 D-89504 Heidenheim, Germany http://www.hartmann-online.com Cotton plugs Gebr. Rettberg GmbH Rudolf-Wissell-Strasse 17 D-37079 Go¨ttingen, Germany http://www.rettberg.biz Glassware Sigma-Aldrich Chemie GmbH Eschenstrasse 5 D-82024 Taufkirchen bei Mu¨nchen, Germany http://www.sigmaaldrich.com 367
Preservation of Thermophilic Microorganisms
Freeze-drying machines
Chemicals, Silicagel rubin Vacuubrand GmbH & Co. KG Alfred-Zippe-Str.4 D-97877 Wertheim, Germany http://www.vacuubrand.de Vacuum pumps
References Annear, D. I. (1956). The preservation of bacteria by drying in peptone plugs. J. Hyg. 54, 487–508. Banno, I. and Sakane, T. (1981). Prediction of prospective viability of L-dried cultures of bacteria after long-term preservation. Inst. Ferment. Osaka Res. Commun. 10, 33–38. Brock, T. D. and O’Dea, K. (1977). Amorphous ferrous sulfide as a reducing agent for culture of anaerobes. Appl. Environ. Microbiol. 33, 254–256. Hippe, H. (1991). Maintenance of methanogenic bacteria. In Maintenance of Microorganisms and Cultured Cells (B. E. Kirsop and A. Doyle, eds), 2nd edn., pp. 101–113. Academic Press, London. Malik, K. A. (1990). A simplified liquid-drying method for the preservation of microorganisms sensitive to freezing and freeze-drying. J. Microbiol. Meth. 12, 125–132. Malik, K. A. (1991). Maintenance of microorganisms by simple methods. In Maintenance of Microorganisms and Cultured Cells (B. E. Kirsop and A. Doyle, eds), 2nd edn., pp. 121–132. Academic Press, London. Malik, K. A. (1999). Preservation of some extremely thermophilic chemolithoautotrophic bacteria by deep-freezing and liquid-drying methods. J. Microbiol. Meth. 35, 177–182. Morris, G. J. (1981). Cryopreservation. Institute of Terrestrial Ecology, Cambridge. Smith, D. and Thomas, V. E. (1998). Cryogenic light microscopy and the development of cooling protocols for the cryopreservation of filamentous fungi. World J. Microbiol. Biotechnol. 14, 49–57.
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