15 Preservation of ThermophilicMicroorganisms

Stefan SpringDSMZ-Deutsche Sammlung vonMikroorganismenund Zellkulturen GmbH, MascheroderWeg1b,D-38124 Braunschweig,Germany

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CONTENTS

IntroductionMaintenance by subculturing and preservationMethods for the long-term preservation of thermophilesVaccum dryingDeep freezing

^^^^^ INTRODUCTION

Thermophilic and hyperthermophilic microorganisms are highly diversein respect of their metabolism and phylogeny. Besides high temperatures,most thermophiles have also adapted their lifestyle also to other environ-mental conditions which can be termed extreme from an anthropocentricpoint of view, e.g. acidic pH values, highly reduced conditions orhypersalinity. This versatility, among other reasons, may have causedan increasing interest in this group of prokaryotes. The mounting researchactivity in this field in many laboratories resulted in a need for reliablemaintenance methods for thermophilic strains. Especially for compara-tive genetic or biochemical studies it is often desirable to work with stocksof preserved cultures, or it could be necessary to maintain a large numberof mutant strains over longer periods of time. On the other hand, standardmethods of preservation often fail in the case of thermophilic micro-organisms, especially if they are also strictly anaerobic or acidophilic.Several methods which can be used for the long-term preservation ofthese fastidious microorganisms are discussed in this chapter. Most ofthese techniques were originally developed for the reliable preservationof extremophiles at culture collections, but with some expertise, it shouldbe possible to adapt these procedures to the technical facilities availablein most research or industrial laboratories.

Resources for material and equipment useful for the describedmethods are listed at the end of this chapter.

METHODS IN MICROBIOLOGY, VOLUME 35 Copyright � 2006 Elsevier Ltd.0580-9517 DOI:10.1016/S0580-9517(05)35015-X All rights reserved.

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^^^^^ MAINTENANCE BYSUBCULTURINGANDPRESERVATION

In several laboratories studying thermophilic prokaryotes, importantstrains are only maintained by frequent subculturing. Subculturingmeans serial transfer of strains from media with depleted nutrientsources to fresh media. After inoculation into fresh media, cultures areincubated to obtain growth and then eventually stored. To preventfrequent subculturing, the metabolic rate of the organism during storageshould be kept at a minimum. Many thermophilic strains stop growingalready below 40�C, so that they can be stored easily without refrigera-tion at room temperature. Refrigeration to 4–8�C can be used to extendthe interval of subculturing of some strains, but on the other hand mayhave a negative effect on the long-term stability of other thermophilicstrains. Viability of distinct cultures over time may vary considerablyand depends largely on the reached growth phase, storage conditionsand quality of the used medium. Hence, time of storage betweentransfers is normally kept at a minimum to ensure survival of importantstrains. Subculturing is inexpensive in terms of equipment, applicable toall cultivable strains and avoids problems associated with the resuscita-tion of preserved stock cultures. However, it can be time-consumingif organisms are handled that require frequent transfers or a wholecollection of strains has to be maintained. Besides the time necessaryfor preparation of media and manipulation of strains, subculturingimplicates several risks and disadvantages. Contamination of purecultures is a permanent threat and the mislabelling or transposition ofvials can lead to an interchange of strains. The risk of mishaps increaseswith the frequency of the manipulation of a strain and can be onlyminimized by the consequent use of sound microbial techniques.Frequent subculturing can also have a negative effect on the geneticintegrity of a strain. Genetic changes or the loss of plasmids may result inthe selection of mutant strains and thereby, to a strain drift. A pro-gressive genetic drift in a distinct culture may remain unrecognized ifinconspicuous traits of the organism are affected.

Only by using reliable long-term preservation techniques, most of theserisks can be efficiently avoided. The investment in laboratory equipmentand training of technical staff, which is necessary for the establishmentof most preservation techniques, appears to be only a small effort incomparison to the damage caused by the potential loss of importantreference strains.

^^^^^ METHODS FORTHE LONG-TERMPRESERVATIONOF THERMOPHILES

Several well-established methods are available for the long-termpreservation of thermophiles. The basic principle common to all of

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these techniques is the reduction of the amount of freely availablewater to a value where metabolism is suspended. A decrease inwater activity can be achieved either by dehydration or freezing.Numerous procedures have been developed for the gentle cryopreser-vation or drying of cultures in order to minimize cell damage duringpreservation. Nevertheless, survival rates of some cultures can be ratherlow. In general, survival rates depend largely on the sensitivity of astrain against the harmful effects caused by the used preservationmethod and can vary considerably among closely related strains.Consequently, not all of the techniques described in this chapter areapplicable to the whole range of thermophilic microorganisms. Themain assets and drawbacks of the most common techniques arediscussed below.

Freeze-drying

Freeze-drying or lyophilization is a process which extracts water froma sample by sublimation. Sublimation is the transition of a substancefrom the solid to the vapour state, without passing through anintermediate liquid phase. Lyophilization involves freezing of the cellpellet so that the water becomes ice, application of vacuum in order tosublimate the ice directly into water vapour and drawing off the watervapour. Cultures preserved by freeze-drying are relatively stable overtime and can be stored without further attention. However, due to thelow survival rates of susceptible strains, a selection of more resistantsubpopulations with different genetic characteristics may take placeduring freeze-drying. To avoid a genetic drift by the continuousselection of subpopulations, subsequent batches should be alwaysprepared from the same seed stock. Ampoules with lyophilizedsamples can be transported easily without the risk of damage causedby lyses of sensitive cultures. However, the resuscitation of vacuum-dried samples can be labour-intensive, especially if anaerobic strainswere preserved. In addition, most strains show a prolonged lag-phaseafter resuscitation and need two or three transfers until normal growthtakes place.

This preservation method requires an investment in special equipment,which is mainly useful for the purpose of freeze-drying. Some training oftechnical staff will also be necessary to ensure a safe and smooth flow oflyophilization procedures. Thus this method is only cost-effective forlaboratories which have to prepare larger batches of preserved samplesfrom thermophiles or intend to store a large collection of strains withoutfurther attention. Thermophiles that are suitable for preservation bylyophilization represent mainly aerobic or aerotolerant, heterotrophicmicroorganisms, which are relatively robust and show a good growthyield. In contrast, strains that are extremely sensitive to oxygen or reachonly very low cell densities in most cases, hardly survive the harsh

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conditions during freeze-drying and should be better preserved bycryopreservation.

Liquid-drying

Unlike lyophilization, liquid-drying (L-drying; Annear, 1956) involvesvacuum-drying of samples from the liquid state without freezing. Liquid-drying has, in general, the same benefits and drawbacks as freeze-drying,but the procedure is less elaborate and may have advantages in thepreservation of microorganisms that are particularly sensitive to theinitial freezing step involved in lyophilization.

Deep Freezing

The only technique which seems to be applicable to all thermophilicprokaryotes studied so far is the deep freezing of concentrated cellsuspensions in fresh media supplemented with a suitable cryoprotectant.The survival rates of cultures preserved by this method are usually quitehigh and the long-term stability almost unlimited. The resuscitation ofcultures preserved by deep freezing is uncomplicated and growthnormally takes place without an extended lag phase. However, it isimportant 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 beachieved. In contrast, the storage of cryopreserved stocks in normalfreezers at temperatures above �70�C is not recommended, because freewater is still available enabling residual physical or chemical activity,which could damage DNA or other essential cell compounds causing arapid loss of viability in some strains. Normally, storage in liquid nitrogen(�196�C) or the nitrogen vapour phase (�140�C) is used to achieve therequired low temperatures. With exception of the liquid nitrogen storagetank, the equipment necessary for this method is easy to obtain andinexpensive. A major constraint of this preservation technique may be,however, the continuous need for liquid nitrogen to maintain the requiredstorage temperature. A complete evaporation of liquid nitrogen willcause an inevitable temperature rise in the refrigerator which eventuallymay kill all sensitive stocks. Hence, care has to be taken to ensure thatliquid nitrogen is replenished in regular intervals. Nevertheless, in somegeographical regions the adequate supply with liquid nitrogen isproblematic. In this case, storage of less sensitive cultures in deep freezersat �80�C may be an alternative. However, it has to be noted that althoughmetabolism is suspended, cells can be damaged by the recrystallizationof ice, which still takes place at this temperature.

In the following sections, standard protocols for the preservation ofthermophilic prokaryotes are exemplified on representatives of variousphysiological groups.

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^^^^^ VACUUM-DRYING

Equipment

Technical equipment necessary for vacuum-drying methods include avacuum pump, a freeze-dryer equipped with a centrifuge head andmanifold or an evacuation jar connected to a moisture or cold trap,constrictors and diverse glass ware. Vacuum-dried samples of culturescan be prepared either in double vial or single vial ampoules. TheDeutsche 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 moreelaborate to prepare, but have the advantage that a contamination of theatmosphere by aerosols that can be produced by sudden release of thevacuum in single-vial preparations is efficiently prevented. In addition,the cell pellet is protected from contamination, because inflowing airfilters through the sterile cotton plug of the inner vial.

Centrifugal Freeze-drying

Centrifugal freeze-drying is the preferred method for the preservation offastidious thermophilic strains that are susceptible to the initial freezingin skim milk intrinsic to standard freeze-drying protocols. This methodrelies on the freezing of the cell suspension by evaporative cooling causedby the loss of water during evacuation of the freeze-drying chamber. Toavoid frothing of the suspension due to removal of dissolved gases beforefreezing is complete, the suspension is centrifuged during the initialstages 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 beomitted. At the DSMZ, a special preservation mixture has been developed

Figure 15.1. Double-vial preparation of a vacuum-dried sample, sealed under vacuum.

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which is now routinely used for the protection of thermophilic strainsduring the centrifugal freeze-drying process. The use of this protectivesolution allows the lyophilization of most aerobic thermophiles, withthe exception of strains that grow only to a very low cell density. Thesupplementation of this solution with amorphous ferrous sulfide alsoallows the lyophilization of some anaerobic thermophiles without usingan oxygen-free gas atmosphere during the processing of samples.

Preparation of vials

1. Plug clean glass vials (44� 11 mm, flat bottom) loosely with non-absorbent 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 afew pieces of self-indicating silica gel (e.g. silicagel rubin) into thetubes which are covered with a small amount of cotton wool (seeFigure 15.1).

Preparation of the suspending medium

1. Preservation mixture: Dissolve 2% (w/v) gelatine, 2% (w/v) yeastextract and 24% (w/v) sucrose in distilled water, bubble with N2 gasfor 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 rubberstopper and aluminium crimp. Autoclave the protective solution at121�C for 20 min. The suspending medium is prepared by mixingan equal ratio of freshly prepared sterile cultivation medium andpreservation mixture.

2. Amorphous ferrous sulfide for anaerobic strains: The ferrous sulfide isprepared according to Brock and O’Dea (1977). Briefly, equimolaramounts of ferrous ammonium sulfate and sodium sulfide react insolution to form amorphous FeS, which precipitates. After settling ofthe FeS precipitate, the supernatant is discarded. This procedureis repeated several times with an anoxic solution of 0.9% (w/v) NaCluntil the supernatant no longer contains detectable amounts of ferrousor sulfide ions. Finally, the purified aqueous suspension of FeS isdispensed under nitrogen gas atmosphere in suitable vessels andautoclaved. The final concentration of amorphous ferrous sulfide inthe suspending medium should be approximately 1 mg ml�1.

Preparation of the cell suspension

Depending on the oxygen relationship and acid tolerance of the strainto be preserved, different procedures are necessary for the preparation ofthe cell suspension. Ideally cells are harvested when they are most activeand have reached the mid exponential growth phase. The processingof aerobes is exemplified on representatives of the genera Aeropyrumand Sulfolobus, whereas the lyophilization of anaerobes is illustrated bya protocol suitable for hyperthermophilic Pyrococcus species.

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Aerobic heterotrophic thermophiles

Aeropyrum spp. and other oxygen-tolerant hyperthermophiles can beharvested as usual by centrifugation of liquid medium in sterilecentrifuge tubes or bottles. The cell pellet is resuspended in approxi-mately 5 ml of suspending medium containing the preservationmixture and aseptically distributed in aliquots of 0.15 ml to each singlevial. Several thermoacidophilic species, e.g. Sulfolobus solfataricus, aresensitive to low pH values at the end of the growth phase and easilylose viability. Therefore, the growth medium is neutralized prior toharvesting of the cells by adding a small amount of solid, sterilizedcalcium carbonate. After 10–15 min, the undissolved carbonate forms adeposit which then can be removed from the culture supernatant. Inaddition, the pH value of the thermoacidophiles growth medium isadjusted to a moderate value (pH 4.0–4.5) prior to mixing with thepreservation mixture in order to prevent damage of cells duringlyophilization.

Strictlyanaerobic thermophiles

Special care has to be taken to avoid exposure to oxygen duringharvesting of Pyrococcus spp. and other obligate anaerobic thermophilesand hyperthermophiles. Either anaerobically grown cultures have to betransferred under an oxygen-free gas atmosphere to anoxic and gas-tightcentrifuge bottles or in a more straightforward approach are cultured inheavy-walled, round-bottomed glass bottles, which can be used forgrowing of cells and centrifugation. Suitable glass bottles (50–70 mlvolume) can be ordered from most glass blowers as custom-made productand should have necks that can be closed with a butyl rubber septum andscrew cap as with Hungate-type anaerobe tubes. After centrifugation thecentrifuge bottle is opened, a gassing cannula inserted and the super-natant is removed aseptically under a flow of oxygen-free gas. Normally,the gas-mixture used corresponds to the gas atmosphere is used tocultivate the respective strain. However, a mixture of N2 and CO2 insteadof a H2 containing gas mixture should be used to avoid risks caused bythe generation of flammable gas mixtures in the laboratory. Cell pellets ofone or more bottles are collected using suspending medium that has beensupplemented with 1 mg ml�1 amorphous FeS and transferred to a tubecontinuously flushed with oxygen-free N2 gas. Filling of vials is carriedout under oxic conditions using for instance an Eppendorf Multipipettewith 2.5 ml Combitip. This procedure should be done quickly becausethe ferrous sulfide provides protection of cells against oxygen only fora limited time.

Drying procedure

Drying is carried out in two stages. Primary drying is achieved bycentrifugal freeze-drying, whereas standard shelf freeze-drying is used to

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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 thecentrifuge head.

2. Secondary drying

2.1. Cut off parts of the cotton-wool plugs that are projecting fromthe vials and place vials in outer glass tubes containingself-indicating silica gel.

2.2. To protect the cotton-wool plugs from heat during constric-tion cover the vials with glass wool (Tempstran 475–106)slightly compressed to a layer of 1–2 cm thickness (seeFigure 15.1). The outer tubes are constricted just above the glasswool either by hand or by using a semiautomatic ampouleconstrictor.

2.3. After cooling, attach the double-vial preparations to the manifoldof a freeze-drying machine for secondary drying for at least 2 hor overnight (see Figure 15.2).

2.4. At a vacuum of at least 0.1 mbar, the tubes are flame-sealed at themiddle of the constriction.

Liquid-drying

The L-drying method has been successfully applied at the DSMZ toseveral thermophilic, obligate chemolithoautotrophic bacteria, whichare difficult to preserve using one of the standard freeze-dryingprotocols. Examples of successfully preserved thermophiles includeAquifex pyrophilus, Thermocrinis albus, Hydrogenobacter thermophilus andSulfurihydrogenibium azorense. In general, members of these genera andrelated hydrogen-oxidizing thermophiles are microaerophilic and sensi-tive to elevated levels of oxygen. Thus, during handling of these culturescare has to be taken to ensure that exposure to atmospheric levels ofoxygen is limited to a minimum.

The following outline of an L-drying preservation method suitable forfastidious thermophilic strains is based on the description given by Malik(1990). A setup of the equipment necessary for primary L-drying is shownin Figure 15.3. Alternatively, this method can be carried out withouta freeze-drying machine following the simplified procedure described byMalik (1991).

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Preparation of the carrier material

A thin disc of carrier material is prepared in order to protect the cellsuspension from freezing during the evacuation period.

1. Fill glass vials (44� 11 mm, flat bottom) with 0.1 ml of 20% (w/v) skimmilk containing 1% (w/v) neutral activated charcoal and 5% (w/v)meso-inositol.

2. The vials are loosely plugged with non-absorbent cotton wool andsterilized at 115�C for 13 min.

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.

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3. Freeze the vials at�20�C for several hours and thereafter transfer themto the drying chamber of a freeze-drying machine. Apply vacuum andfreeze-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 protectivemedium. A mixture of activated charcoal and meso-inositol hasproven most effective in preventing harmful effects on the cells duringliquid-drying.

1. Suspend 1% (w/v) activated charcoal and dissolve 3% (w/v) meso-inositol in distilled water (pH adjusted to 7.0), bubble with N2 gas forat least 30 min to remove oxygen and fill in a suitable container, e.g. aserum bottle with butyl rubber stopper and aluminium crimp, which istightly closed. Autoclave the protective solution at 115�C for 13 min.

2. Harvest cells as described in the section on centrifugal freeze-dryingby aseptic centrifugation and suspend cell pellet in the protectivesolution, while maintaining anoxic conditions. The obtained cellsuspension should have a concentration of about 108–109 cells ml�1.

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 cellsuspension is transferred aseptically with care so as not to touch theside of the vial.

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.

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2. Place ready vials quickly in aluminium Erlenmeyer caps and thentransfer 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 anddry for 4–6 h.

4. For further drying, adjust vacuum to approx. 20–25 mbar and dryovernight while maintaining the temperature at 20�C.

5. Replace vacuum in the metallic jar with N2 gas and transfer thevials to soft glass tubes (135� 15 mm) containing silica gel andcotton plugs. Add glass wool, constrict outer tubes, attach tothe manifold of a freeze-drying machine and vacuum-dry for further2–3 h at 0.01–1 mbar as described in the section on centrifugalfreeze-drying.

6. At a vacuum of at least 0.1 mbar, the outer tubes are flame-sealed atthe middle of the constriction.

Storage and Recovery

It was shown that the viability of vacuum-dried samples depends on thestorage temperature (Banno and Sakane, 1981). As a rule of thumb, it wasfound that survival values were comparable when ampoules were storedat 5–9�C for a number of years or at 30–37�C for the same number ofweeks (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 protectivegoggles 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 mediumis 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 andcan be transferred to a tube with the appropriate cultivation medium.In several cases it was observed that the ingredients of the protectivemedium can inhibit growth of fastidious strains in the first culturetube. Hence, a serial dilution of at least three or four tubes should beprepared. If the preserved culture can be grown easily on solid media,a few drops of the rehydrated sample should also be tranferred to anagar plate or slant to obtain single colonies in order to check the purityof the strain.

In the case of obligate anaerobic strains, it is important to retainanoxic conditions during all steps after opening of the ampoule.This can be achieved in several ways depending on the usedanaerobic technique and available equipment in the laboratory. If theHungate technique is used, it is recommended to keep the inner

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vial under a flow of oxygen-free gas by inserting a gassing cannulauntil the cell pellet is completely resuspended. If the ampoule shouldbe opened in an anaerobic gas chamber, it is necessary to scorethe ampoule with a sharp file at the middle of its shoulder aboutone cm from the tip. Transfer the ampoule with the file mark in theanaerobic chamber and strike the ampoule with a file or large forceps toremove the tip. If necessary, wrap the ampoule in tissue paper andenlarge the open end by striking with a file or pencil, then remove theglass wool insulation and the inner vial. Gently raise the cotton plugand sterilize the upper part of the inner vial by wiping it with tissue papersoaked in 70% ethanol. After opening of the ampoule, add approximately0.5 ml of anoxic medium to resuspend the cell pellet and transferthe 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 culturein order to determine the success of the vacuum-drying procedure.Sometimes, if no living cells can be recovered from lyophilizedsamples, it has turned out that cultures were incubated too long priorto preservation and thus were already inactive. For determination of theviability before the preservation process, an aliquot of the suspensionused to fill the inner vials is inoculated in the appropriate culture mediumand serially diluted to extinction in order to determine the approximatenumber of living cells. The same procedure is then repeated with avacuum-dried sample. Although the stability of most dried cultures issatisfactory, there are also examples of fastidious strains that lose viabilitywithin several years of storage. Therefore, the survival rate of importantvacuum-dried stock cultures should be checked in intervals of at least5 years.

^^^^^ DEEP FREEZING

Freezing and storage of cultures in liquid nitrogen has the advantage thatthe survival rates of the more susceptible microorganisms are usuallyhigher as with methods based on vacuum-drying and that the procedurescan be carried out easily under an oxygen-free atmosphere. Therefore,cryopreservation is the most effective method for the maintenance offastidious thermophiles which grow only to a very low cell density orstrains that are extremely sensitive to oxygen, e.g. most representativesof the hyperthermophilic methanogens (Methanocaldococcus spp.etc.). Moreover, this procedure is in general applicable to all knownprokaryotes and hence has been established as standard preservationmethod for most seed stocks of prokaryotes held at the DSMZ culturecollection.

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Equipment

The only major investment which is necessary to establish this method isa cryogenic storage tank for liquid nitrogen or alternatively a mechanicaldeep freezer, which can cool below �70�C. However, deep-freezers areonly second quality and should only be used if a regular supply withliquid nitrogen cannot be guaranteed. The liquid nitrogen container hasto be equipped with storage canes and canisters for the storage of glasscapillaries in the liquid phase of nitrogen or racks with dividers for thestorage of plastic cryotubes in the vapour phase. Further useful equip-ment includes standard laboratory accessories and diverse glass warewhich can be obtained easily at low costs.

Preparation of Cell Suspension

Preparation of suspending medium

Fresh culture medium containing 10% (v/v) glycerol or 5% (v/v)dimethyl sulfoxide (DMSO) is used as a suspending medium. DMSOis often more satisfactory than glycerol, because it requires less time topenetrate the organism. The cryoprotectant is sterilized separately byautoclaving in anoxic test tubes under nitrogen gas atmosphere (DMSO:10 min, 115�C). Add cryoprotectant aseptically just before use to theappropriate sterile, aerobic or anaerobic cultivation medium.

Preparation of cultures

The cultivation and harvesting of aerobic and anaerobic thermophiles canbe done as described in the corresponding paragraphs of the section oncentrifugal freeze-drying. Again, the survival rates of aerobic thermo-acidophiles like Metallosphaera or Sulfolobus species can be increased byneutralizing the growth medium prior to harvesting with some solidcalcium carbonate.

Distribution in Aliquots and Freezing

Cell suspensions can be distributed for freezing and subsequent storageeither in screw cap plastic ampoules (cryotubes) or glass capillaries.Cryotubes have the advantage that the filling with suspension and therecovery of cultures is easy and needs no special training or equipment.However, there is always the risk of an imperfect sealing that could causeseepage of liquid nitrogen into the screw cap vial with subsequentexplosion upon thawing. Therefore, it is safer to store cryotubes in thevapour phase, instead of submersing them in liquid nitrogen. In addition,plastic cryotubes can not be used for strictly anaerobic thermophiles. Thematerial of the ampoules is not gas tight so that oxygen can penetrate thevial and irreversibly damage the cells. In contrast, glass capillary tubescan be hermetically sealed and are then absolutely impermeable to liquid

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nitrogen and gases. They can be stored in the vapour phase as well asin the liquid phase of nitrogen and need very little storage place due totheir small size.

It has been found that for the cryopreservation of various filamentousfungi cooling rates in the range of �0.5 to �200�C min�1 over the criticalperiod from þ5 to �50�C were optimal (Smith and Thomas, 1998).However, it seems that controlled cooling rates may not have a significanteffect on the survival of the much smaller cells of prokaryotes and hencethis technique is not applied in the protocols given below.

Plastic ampoules

Polypropylene screw cap ampoules (1.8 ml vol.) can be obtained sterilizedfrom the manufacturer. Aliquots of the concentrated cell suspension(approx. 0.5–1.0 ml) are distributed aseptically in the cryovials, which arethen secured by tightly screwing down the lids. Place the cryotubes insuitable racks and then transfer in the vapour phase of liquid nitrogenfor freezing.

Glass capillary tubes

The procedure for the freezing of microorganisms in glass capillarytubes is based on the description given by Hippe (1991) and illustratedin Figure 15.4A.

Preparation ofglass capillary tubes

Glass capillary tubes (length 90 mm, outer diameter 1.4 mm, wallthickness 0.26 mm) are rinsed several times in distilled water and thendried. Mark capillaries at approx. 2.5 cm from one end with a permanentmarker (water resistant ink), place capillaries in test tubes that are closedwith aluminium caps and autoclave at 121�C for 20 min.

Fillingand sealingofcapillaries

1. Transfer 0.5–1.0 ml of the concentrated cell suspension into a smallsterile vial, which is placed in an ice bath. For the preservation ofanaerobes, the vial is kept anoxic by gassing with an oxygen-free gasmixture that complies with the gas atmosphere used to cultivate therespective strain. However, CO2 gas should be replaced with a mixtureof 80% N2 and 20% CO2, in order to avoid problems related to thehigh solubility of CO2 in water, which eventually could lead tothe explosion of frozen capillaries upon thawing.

2. One glass capillary is taken from the sterile stock by fitting it to the tipof a micropipettor (e.g. micro-classic pipette controller; Brand GmbH,Wertheim) and enough of the cell suspension is aspirated to fillone-third of the length of the capillary. The volume taken up isapproximately 25 ml. The suspension within the capillary is furtheraspirated until it is about 1 cm from the free end, which is sealed ina fine, hot gas flame. The second seal is made at 2.5 cm from the otherend of the capillary (at the mark, which was made prior to sterilizing)

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by heating and subsequent tearing of the softened glass. The making ofthe second seal is a critical step of this method and needs some trainingin order to achieve capillaries that are hermetically sealed.

3. As capillaries are prepared, they are placed in a vial with 75% ethanolfor disinfection. The vial is placed in an ice/water bath in order to cooldown 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 upnitrogen and will explode on removal from the cold. Nevertheless,a perfect seal is easier to achieve with capillaries. To avoid that moist

A.

Figure 15.4. Glass capillary tube method for the low-temperature preservation of micro-

organisms. 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.

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capillaries stick together when frozen, they are dried by placing thembetween absorbent paper and gently pressing and rolling with theflat hand.

5. The capillaries are stored in a capillary holder (aluminium orpolypropylene tube), which is labelled with the strain designation onthe outside and then placed in the vapour phase above liquid nitrogenfor freezing. The time lapse between preparation of the suspensionand freezing should be kept as short as possible. Rapid freezing byimmersing capillaries directly in liquid nitrogen should be avoided,because not all capillaries may withstand the developing pressure andcould break.

B.

Figure 15.4. Continued.

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Storage and Recovery

Cryotubes can be easily labelled with special cryo-markers prior tofreezing and subsequent storage in racks in the vapour phase aboveliquid nitrogen.

In contrast, the permanent labelling of capillaries is more difficult,but may be sometimes desirable if samples of different strains should bestored in the same capillary holder. In this case, capillaries can be colourcoded by putting them into PVC straws (outer diameter 2 mm) ofdifferent colours, which are cut to length and squeezed together at oneend. For storage, the capillary holder can be placed into an aluminiumcane that is immersed into liquid nitrogen. Usually, several canes arecollocated into one canister.

Resuscitation

General safety precautions should be observed for exposure to liquidnitrogen or cryogenic equipment. Wear protective clothing and goggleswhile handling frozen samples! It has been found that slow warming maycause damage of cryopreserved samples due to the recrystallization ofice, therefore rapid thawing is recommended. To achieve the required fastthawing rates, frozen samples are immersed in warm water (30–37�C)immediately upon removal from the liquid nitrogen container. It has beenobserved in several cases that, strictly chemolithoautotrophic strainsare inhibited by a remaining amount of the cryoprotectant in the firstinoculated culture tube. Therefore, it is recommended to prepare a serialdilution of two or three tubes for the resuscitation of sensitive autotrophicstrains.

Plasticcryotubes

Thaw the cryotube in a suitable container filled with warm water untilthe last visible ice has melted. Unscrew the cryotube, remove sampleaseptically by using a Pasteur pipette and inoculate a suitable growthmedium.

Glass capillary tubes

For the recovery of a strain, one capillary is removed from the liquidnitrogen tank and thawed rapidly in a container with warm water. Thecapillary is removed from the water bath, dried and opened at one endas shown in Figure 15.4B. In the case of aerobic thermophiles, the smallvolume of cell suspension is aspirated with a sterile Pasteur pipette thathas been drawn out very finely to a length of 4 cm. While aspirating thesuspension, the tip of the pipette is slowly moved further into thecapillary. The suspension is then transferred to 5 ml of the freshly pre-pared appropriate medium. For the recovery of anaerobic strains, it isrecommended to use a 1 ml disposable (tuberculin) syringe with a 25Ghypodermic needle. Prior to use, the syringe is flushed with oxygen-freegas. The capillary is opened at both ends and the contents aspirated into

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the syringe while avoiding to uptake air bubbles along with the cellsuspension. After aspirating the cell suspension from the capillary, theneedle is inserted through the rubber closure of an unopened tube with5–10 ml of anoxic medium. The tube is inverted and the suspension alongwith some medium is drawn into the syringe. Then, the tube is still in aninverted position, the contents of the syringe is injected into the tube.

Viability testing

The determination of survival rates after cryopreservation can be doneaccording to the description given above in the section on vacuum drying.However, the testing of preserved cultures in regular intervals is notso important than with vacuum-dried samples, because the stability offrozen cultures stored in liquid nitrogen is practically unlimited.

Further detailed information on preservation methods is available fromthe CABRI consortium, which has developed several guidelines for themaintenance of microorganisms (URL: http://www.cabri.org).

List of suppliers

Air Liquide, Division Materiel Cryogenique75 Quai d’Orsay75321 Paris cedex 07, Francehttp://www.airliquide.com

Cryogenic storage vessels

A. Albrecht GmbH & Co. KGHauptstrasse 6-8D-88326 Aulendorf, Germany

PVC straws

Chart Industries, Inc., MVE Bio-Medical Division1800 Sandy Plains Industrial ParkwayMarietta, GA 30066, USAhttp://www.chartbiomed.com

Cryogenic storage vessels

FGT Feingeratetechnik GbRErnst-Thalmann-Str. 27D-99510 Apolda, Germanyhttp://www.fgt.de

Ampoule constrictor

Hilgenberg GmbHStrauchgraben 2

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D-34323 Malsfeld, Germanyhttp://www.hilgenberg-gmbh.de

Glass capillary tubes

Lehmann & Voss & Co.Alsterufer 19D-20354 Hamburg, Germanyhttp://www.lehvoss.de

Glass wool

Martin Christ Gefriertrocknungsanlagen GmbHP.O. Box 1713D-37507 Osterode am Harz, Germanyhttp://www.martinchrist.de

Freeze-drying machines

Nalge Nunc International Corp.75 Panorama Creek DriveRochester, New York 14625-2385, USAhttp://www.nuncbrand.com

Cryotubes

Ochs Glasgeratebau GmbHPappelweg 26D-37120 Bovenden/Lenglern, Germanyhttp://www.labor-ochs.de

Glassware

Paul Hartmann AGP.O. Box 1420D-89504 Heidenheim, Germanyhttp://www.hartmann-online.com

Cotton plugs

Gebr. Rettberg GmbHRudolf-Wissell-Strasse 17D-37079 Gottingen, Germanyhttp://www.rettberg.biz

Glassware

Sigma-Aldrich Chemie GmbHEschenstrasse 5D-82024 Taufkirchen bei Munchen, Germanyhttp://www.sigmaaldrich.com

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Chemicals, Silicagel rubin

Vacuubrand GmbH & Co. KGAlfred-Zippe-Str.4D-97877 Wertheim, Germanyhttp://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-driedcultures 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 reducingagent for culture of anaerobes. Appl. Environ. Microbiol. 33, 254–256.

Hippe, H. (1991). Maintenance of methanogenic bacteria. In Maintenance ofMicroorganisms 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 preservationof microorganisms sensitive to freezing and freeze-drying. J. Microbiol.Meth. 12, 125–132.

Malik, K. A. (1991). Maintenance of microorganisms by simple methods. InMaintenance 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 chemo-lithoautotrophic 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 thedevelopment of cooling protocols for the cryopreservation of filamentousfungi. World J. Microbiol. Biotechnol. 14, 49–57.

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