Liquid-drying of micro-organism using a simple apparatus by Webmaster Online protocols 2016-02-06 18:32:45

Liquid-drying of micro-organism using a simple apparatus

Khursheed A. Malik, Ph.D.

DSM-Deutsche Sammlung von MIkroorganismen und Zellkulturen GmbH,
Mascheroder Weg 1B, D-3300 Braunschweig, Federal Republic of Germany


World Federation for Culture Collections
Technical information sheet No 8

Published by: UNESCO/WFCC - Education Committee 1990


Liquid-drying (L-Drying) involves vacuum-drying of samples from the liquid state without freezing. It is one of the sophisticated techniques used for the long-term preservation of microorganisms.

Several microorganisms, which are sensitive to freezing or freeze- drying, can successfully be preserved by liquid drying. Liquid-drying has several advantages over freeze-drying and has been effectively used for preserving large collections of fragile microorganisms in various culture collections. However, specialized equipment is required for L-drying. Recently a simple and effective liquid-drying method has been described for the successful preservation of sensitive microorganisms (including various anaerobes) which fail to survive freezing or freeze-drying (Malik, 1990).

Based on this process a liquid-drying method using a simple apparatus is described. The method is simple and the equipment described here can be easily constructed in most laboratories.

Material and Methods

Preparation of thin discs of carrier material

Ampoules of neutral glass (45 x 10 mm) are filled with 0,1 ml of 20 % (w/v) skim milk (Bacto, Difco 0032) containing 10% (w/v) neutral activated charcoal and one of the effective protective agents such as 5 % (w/v) meso-inositol, 5% (w/v) glutamate, 5 % (w/v) raffinose or 10% (w/v) honey. The activated charcoal used is of medicinal grade ( available from Caelo, 4010 Hilden, FRG) but any other bacteriological activated charcoal of comparable quality can also be used. The ampoules are loosely plugged with non-absorbent cotton wool and sterilized at 115 ’C for 13 minutes. These are frozen at about -30 ’C for a few hours and then freeze-dried in bulk for about 6 hours using a standard freezedrying technique (Malik,1988 or for freeze-drying with a simple apparatus see TIS No.7. Malik, 1990).

Preparation of protective agents

Solutions of most effective protective agents like meso-inositol (5 % w/v), honey (10 % w/v), sodium glutamate (5 % w/v), raffinose (5 % w/v) are prepared in distilled water, filter sterilized and stored at 4’ C. For oxygen sensitive microorganisms 10% (w/v) neutral activated charcoal with 10% (w/v) meso-inositol is prepared in a screw cap bottle (Malik, 1990). The solution is boiled, bubbled with nitrogen gas and the bottle is closed tightly and autoclaved at 115 ’C for 13 minutes.

Preparation of cell suspension for L-dryinq

A thick cell suspension (at least 10(8) cells per ml) is prepared in an appropriate protective medium. In the case of liquid cultures, the cells are harvested by aseptic centrifugation for 30 minutes at 4000 xg in screw-cap bottles and the pellet is suspended in a protective medium to yield a heavy cell suspension. The thick cell suspensions under anaerobic conditions are obtained as described previously [ see TIS No.4, Malik,1989 ].

Filling of ampoules with cell suspensions

The ready ampoules (containing a thin disc of carrier material) are equilibrated at 2†-25’ C for few minutes. To each ampoule about 0,025 ml (1 drop with a Pasteur pipette) of cell suspension are added aseptically onto the thin-disc with care so as not to touch the sides of the ampoules. The ampoules are quickly placed in metallic lids and transferred into a metallic jar maintained at 20 ’C in a water bath (see Fig.1 A).

After 20 to 30 minutes of equilibration these are subjected to drying under vacuum. For strains very sensitive to oxygen, the ready ampoules are first placed for few hours in anaerobic Bio-bags (Type A, Marion Scientific Corporation, Kansas City, Ml) or Anaerocult P bags (E. Merck, Postfach 4119, D-6100 Darmstadt). These are carefully taken out one by one and after the addition of reduced cell suspension the ampoules are immediately transferred again to such fresh anaerobic bags, sealed and kept for about one hour at room temperature for equilibration and removal of oxygen. Thereafter, these are subjected to drying under vacuum.

The liquid-dryinq procedure

The outline of the L-drying procedure and the major steps involved are shown in Fig.1. For the double vial preparation, liquid-drying is done in two stages involving primary-drying and secondary-drying.

The primary drying is achieved in two stages. The cold trap is chilled to about -35’C and is connected to the vacuum pump and the metallic evacuation jar (maintained at 20’C in a water bath see Fig.1 A). A vacuum controller is attached between the cold trap and the vacuum pump to control the vacuum.

The cold trap tube is a U-shaped thick glass or preferably a metal tube of about 2-3 cm diam and about 30 cm length. It is filled with blue/dry silica gel and is placed in a metallic beaker as shown in Fig.1 A. It is chilled to about -35’C. A cooling mixture of ethylene glycol: water (1: 1) is placed for few hours in a deep-freezer and is cooled down to about -30’C (preferably to -40’C). This is poured into the cold trap to give a maximum depth. The cooling mixture is also sealed in deep-freeze plastic bags and is cooled down or frozen in a deep-freezer or over liquid- nitrogen. This super cooled or frozen coolant in the bags is added to the cold trap and the bags are changed periodically (preferably after every 20 25 min) throughout the drying process in order to maintain the temperature at a minimum level. Commercially available anti-freeze liquids used in car radiators as coolants are also satisfactory as an alternative for the low-temperature bath and cold trap. A double jacketed straight tube (exterior about 6x 30 cm and interior about 3x25 cm with outlet and inlet tubes of about 2x5 cm, as shown in Fig.1A) can also be used as a cold trap. If available, dry ice is used for freezing the ampoules and is added to the cold trap periodically throughout the experiment.

The vacuum is switched on and the temperature of the water bath is maintained at 20’C. First step drying (Drying-l) is continued for about 30 minutes at about 5-10 mbar after which the vacuum controller is readjusted for 1,0 to 0,1 mbar vacuum and second step drying (Drying-ll) is conducted for about one hour.

At the end, the vacuum is replaced with nitrogen gas (especially for strict anaerobes and for the ampules which are not to be sealed under vacuum). For secondary drying and sealing, the ampoules are transferred to soft glass tubes (130 x 15 mm) containing silica gel and cotton plugs. The outer tubes (outer vials) are then constricted by hand or by using Edward Ampoule Constrictor), attached to the manifold and mounted on an evacuation jar. This operation is illustrated in Fig.1 B. The vacuum is switched on and secondary drying is conducted for 1-2 hours (at 0,1 to 0,001 mbar). The pink silica gel in the outer tubes will turn again to blue at this stage.

The constricted outer tubes are carefully sealed, by hand or by using a Flaminaire blow torch, one by one maintaining vacuum (Fig.1 C). For more details see TIS No.7.

Revival of cultures from liquid-dried ampoules

Improvement in viability can be achieved through the use of different culture media. Thus during reactivation of preserved microorganisms it is recommended to use the most favourable media and growth conditions. During several years of experience I have observed that in the case of sensitive microorganisms when the preserved (dried or cryogenically stored) cultures are revived, the counts on agar media are usually lower than in liquid media and similarly agar media of higher surface tension (such as nutrient agar) usually results in lower viable counts than mineral media of relatively lower surface tension.

The contents of the liquid-dried ampoule are thus dissolved in sterile (prereduced in the case of anaerobes) liquid growth media and added or injected (for anaerobes) into 15-20 ml of growth medium. Freshly inoculated phototrophic cultures are placed for a few hours in the dark at appropriate incubation temperatures and later under normal growth conditions in the light. The dried cultures are incubated at a relatively lower temperature than the optimum growth temperature. A few cultures may exhibit a prolonged lag period and thus are incubated for relatively longer periods. Normal growth usually appears after a second transfer into fresh medium. The use of activated charcoal in suspending media for reactivation is recommended due to its various advantages (Malik.1990).

Estimation of viability and stability

Survival recoveries are checked before L-drying, immediately after L-drying and after storage. For the estimation of viability counts serial dilutions are prepared in appropriate liquid media. From each serial dilution 0,1 ml volumes are plated on agar media plates. The number of colonies are counted from the plates and average colony forming units per sample are calculated. The revived cultures are also observed for mutation, change in colony morphology or other characters. For cultures which were difficult to grow in or on agar, only liquid dilutions series are made. In such cases the number of cells is determined using the most probable number method (MPN).

Long-term storage

Stability of L-dried cultures during storage is very important. A high level of residual moisture content or exposure to oxygen have detrimental effects on the dried product. Liquid-dried material is hygroscopic and its exposure to moisture during storage can destabilise the product.

The higher the storage temperature, the faster a product will degrade. Thus, the storage of L-dried cultures at lower temperatures will extend their shelf life. The unsealed L-dried ampoules can safely be stored for several years at about -30’C. It has been observed by the author that similar viability counts were obtained after 4-5 years of storage when unsealed freeze-dried culture were maintained at -30’C as compared to the freeze-dried cultures which were sealed under vacuum and were stored at 9’C (Malik, 1976).

Selected references

Annear, D.l. (1956). The preservation of bacteria by drying in peptone plugs. Journal of Hygiene 54: 487.

Banno, T. and Sakane, T. (1979). Viability of various bacteria after L-drying. IFO Res. Comm. 9, 3545.

Hieda, K. (1981). Induction of genetic changes in Saccharomyces cerevisiae by partial drying in air of constant relative humidity and by UV. Mutation Res. 84: 17- 27

Malik, K.A. (1976). Preservation of Knallgas bacteria. In Proceedings of Fifth International Fermentation Symposium (H.Dellway, Ed.) p. 180. Westkreuz Druckerei and Veriag, Bonn and Berlin.

Malik, K.A. (1985) . Modern Methods of Gene Conservation. A Laboratory Manual. PASTIC Press Pakistan Science and Technology Information Centre, Islamabad, Pakistan.

Malik, K.A. (1987). The role of culture collections in the stability and preservation of microorganisms (J.Amen and P. Tesson, Eds.) pp 118-150. Societe Francaise de Microbiologie. Paris

Malik, K.A. (1988). A new freeze-drying method for the preservation of nitrogen-fixing and other fragile bacteria. J. Microbiol. Methods 8: 259-271

Malik, K.A. (1989). Cryopreservation of bacteria with special reference to anaerobes. Publication No.4. UNESCO/WFCC Technical Information Sheets (TIS). DSM, Braunschweig

Malik, K.A. (1990). Freeze-drying of microorganisms using a simple apparatus. Publication No.7. UNESCO/WFCC Technical Information Sheets (TIS). DSM, Braunschweig

Malik, K.A. (1990). Use of activated charcoal for the preservation of anaerobic phototrophic and other sensitive bacteria by freeze-drying. J. Microbiol Methods

Malik, K.A. (1990). A simplified liquid-drying method for the preservation of microorganisms sensitive to freezing and freeze-drying. J. Microbiol Methods

Sakane, T, I. Banno and T. Iijima (1983). Compounds protecting L-dried cultures from mutation. IFO Res. Comm. 11: 14-24