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Appendix
M/1998/3.00 Appendix 5.01
PRESERVATION OF BACTERIA BY FREEZING AND LOW TEMPERATURE STORAGE IN GLASS CAPILLARY TUBES
INTRODUCTION
Cryopreservation of bacteria at temperatures between -196°C and -150°C (in or above liquid nitrogen) is a reliable method for long-term storage. To save space in the liquid nitrogen (LN2) container, bacteria can be preserved in glass capillaries which are placed in capillary holders (aluminium tubes, 58 x 12 mm, or PP tubes of suitable size). Three to five aluminum tubes are fixed to a single aluminium cane depending on its length. Several canes (numbered and/or colour coded) are collected in a single canister which is placed in a nitrogen container. Colour coding of preserved cultures can be done by inserting glass capillaries into differently coloured PVC straws. In a single aluminium tube about 22 capillaries in PVC straws or 40 capillaries without straws may be stored. Aluminum tubes, PP tubes or PVC straws are labelled with the collection no. of the strain preserved. For recovery, a single capillary may be removed without thawing or contaminating the parallel samples. Glass capillaries can be stored in the gas phase of liquid nitrogen as well as in the liquid phase, and are heavy enough not to rise to the surface when immersed. However, a tight seal is very important if capillaries are to be stored in the liquid phase. 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. Figure 1 shows the procedure for the capillary method.
Glass capillary tubes have several advantages compared with other methods such as freezing in ampoules, or in plastic straws or the glass-bead technique. They can be sealed hermetically and, because of their impermeability to gases, anaerobes are well protected against oxidation. In combination with the ‘Hungate technique', the glass capillary method has proved to be convenient for preserving strict anaerobes like the methanogenic bacteria.
The described method of low temperature storage of bacteria using glass capillaries is based on the publication of H. Hippe, Maintenance of methanogenic bacteria, published in Kirsop/Doyle (eds.), Maintenance of Microorganisms and Cultured Cells, 2nd edition, Academic Press, London, 1991).
For recording the preservation and viability checks use form
M/1998/3.00 Appendix 5.01.1, M/1998/3.00 Appendix 5.01.2 and M/1998/3.00 Appendix 5.01.3.PROCEDURE
1 Preparation of glass capillary tubes
Glass capillary tubes (Hilgenberg GmbH,D-34321 Malsfeld; article no. 1400290, "Sodaklarglas", length 90 mm, O. D. 1,4 mm, wall 0.261 mm) are rinsed several times in demineralized water, dried, stored in test tubes and autoclaved at 121°C for 20 min, but may be used also without prior washing.
2 Preparation of capillary holders and plastic straws
For storing the capillaries aluminium tubes or polypropylene tubes are used. They are labelled with the number of the strain preserved by special ink markers (Nalgene Cryo-Marker). If capillaries of more than one strain are to be stored in the same capillary holder, the capillaries are colour coded by putting them into PVC straws cut to length and squeezed together at one end. The accession no. of the strain(s) is written on the straws and the capillary holder. The straws (2 mm in diameter) are available in different colours from A. Albrecht GmbH & Co. KG, D-88323 Aulendorf.
3 Suspending medium and cryoprotectant
Fresh culture medium containing 10 % (v/v) glycerol or 5 % (v/v) dimethyl sulphoxide (DMSO) is used as suspending medium. It is prepared just before use by adding 0.55 ml of sterile glycerol or 0.27 ml of sterile DMSO to 5 ml of sterile, fresh, aerobic or anaeroanaerobic medium in a culture tube. The cryoprotectant is sterilized separately by autoclaving (DMSO: 10 min, 115°C) aerobically or in anaerobic test tubes under nitrogen atmosphere.
4 Preparation of cultures
Cultures are grown aerobically or anaerobically in culture media as listed in the Catalogue of Strains, in the Accession Form or in any other medium suitable to produce freeze-resistant cells. Cells are harvested when actively growing (mid logarithmic phase). In most cases, aerobic bacteria are grown on agar slants. About 0.5 ml of protective medium is added to a slant and the bacterial growth is suspended.
Anaerobic bacteria and archaea may be grown in screw-capped bottles, serum bottles, serum tubes (Balch type) or Hungate tubes and have to be transferred under anaerobic gas to centrifuge tubes to collect the cells. Anaerobic bacteria may be grown also in heavy-walled, round-bottomed bottles (c. 70 ml volume) (made by glass blowers, e.g. Ochs GmbH, D-37120 Bovenden-Lenglern) with necks that can be closed with a butyl rubber septum and a screw-cap as with Hungate anaerobe tubes (Bellco Glass Inc., 2047-16125). They fit into a normal laboratory centrifuge and are used both for growing the cells and for centrifugation.
The cultures are centrifuged directly in the unopened bottle, after which the bottle is opened, a gassing cannula inserted and the supernatant aseptically removed as completely as possible using a 20-50 ml hypodermic syringe and a long needle of 2 mm diameter. The gas corresponds to the gas required for preparing the anaerobic medium, except for media under CO2 were N2 + CO2 should be used (otherwise, capillaries may explode when opened!). To the cell pellet, 0.5-1.5 ml of suspending medium is added and cells are suspended by means of a Pasteur pipette, bent and heat-sealed at the tip. Depending on the number of capillaries that have to be prepared, cell suspensions from several culture vessels are collected in one bottle by transferring with a sterile Pasteur pipette.
5 Filling and sealing of glass capillary tubes
0.5 to 1 ml of the dense cell suspension is transferred into a small sterile vial (for preserving anaerobes the vial is kept anaerobic by gassing with the appropriate gas mixture). The vial is placed in an ice bath. One glass capillary is taken from the sterile stock by fitting it to the tip of a micropipetter (micro-classic, Brand GmbH & Co., D-97861 Wertheim, order no. 259 00) and enough of the cell suspension is aspirated to fill one-third of the length of the capillary. The volume taken up is ca. 0.025 ml. The suspension within the capillary is further aspirated until it is 1 cm from the free end which is sealed in a fine, hot gas flame. The second seal is made at ca. 2.5 cm from the other end (which had been marked before sterilizing the capillaries) by heating and drawing out. As capillaries are prepared, they are placed in 75 % ethanol or a disinfectant solution.
All capillaries are examined for correct seals under a stereomicroscope. Because moist capillaries will stick together when frozen, they are dried by placing them between absorbent paper and gently pressing and rolling with the flat of the hand. The time lapse between preparation of the suspension and final
6 Freezing of glass capillaries
The capillaries are placed in an open capillary holder that is labelled with the accession no. of the strain(s) on the outside. This is placed in the vapour phase above liquid nitrogen (highest level possible) for freezing. Shock freezing by immersing capillaries directly in liquid nitrogen should be avoided because not all capillaries may withstand rapid cooling and may break.
7 Storage of glass capillaries
For storage, the capillary holder is immersed into liquid nitrogen or kept in the gas phase above liquid nitrogen.
8 Recovery
A capillary is removed from the liquid nitrogen container and thawed rapidly in a screw-cap bottle1 containing warm water of 37°C. In most cases water of room temperature will work equally well. The thawing rate has been calculated to be over 1000°C per minute. The capillary is opened at one end as shown in Fig.1b. 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. A little experience is needed to avoid, in case of anaerobes, the uptake of air bubbles along with the cell suspension, which is then transferred to a culture tube containing 5 ml of medium.
An alternative method for removing the cell suspension from a capillary is to use a 1ml hypodermic syringe with a 0.50 or 0.65 mm needle. In this case, the capillary is opened at both ends, the contents aspirated with a syringe and transferred to a tube containing 5 ml culture medium.
In case of anaerobes, the syringe is flushed with oxygen-free gas. After aspirating the cell suspension from the capillary the needle is inserted through the rubber closure of an unopened fresh culture tube. The tube is inverted and the suspension along with some medium is drawn into the syringe. Still in an inverted position, the contents of the syringe are injected into the tube. This may be repeated once, but care should be taken not to expel gas bubbles into the tube.
1
9 Viability testing
The viability or the number of colony forming units of the strain is determined before and after the preservation step and, depending from the strain, in certain intervals during the storage period. For documentation of the viability protocol form
M/1998/3.00 Appendix 5.01.1 is used.
SAFETY PRECAUTIONS
General safety precautions for handling liquid nitrogen (see
M/1998/1.06 Appendix 1) have to be followed. Protective clothing (coat, gloves, glasses) should be used while handling cryogenic equipment.Fig. 1a. Glass capillary tube method for low-temperature preservation of microorganisms:
Filling and sealing of capillary tubes (copied from the article of H. Hippe)
OFG marks the steps in the preservation of anaerobic bacteria where vials are
gassed with oxygen-free gas.
Fig. 1b. Glass capillary tube method for low-temperature preservation of microorganisms:
Removal of tubes from storage unit, opening, and recovery of cell suspension
(copied from the article of H. Hippe)
OFG marks the steps in the preservation of anaerobic bacteria where vials are
gassed with oxygen-free gas.
Guidelines prepared for CABRI by DSMZ, CBS and BCCM, 17 May 1998
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