Freeze-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. 7
Published by: UNESCO / WFCC - Education Committee 1990
Introduction
The advantages of freeze-drying are obvious. It is a convenient method for the
preservation and long-term storage of a wide variety of microorganisms. However,
special precautions are needed for the preservation of microorganisms sensitive
to desiccation, light, oxygen, osmotic pressure, surface tension and other
factors. During several years of experimentation I have developed various new
methods and have optimised freeze-drying conditions to achieve successful
Iyophilization of a large collection of difficult and fragile microorganisms.
Normally specialized equipment is required to create the conditions-conducive to
the freeze-drying process. The costs of such specialised equipment required for
freeze-drying can be substantial, and thus the process may appear to be
expensive in spite of its many advantages. A freeze-drying process is described
here using a simple apparatus.
This method is based on a new freeze-drying method which has recently been
described (Malik, 1988). Some effective protective agents (Malik, 1976 & 1988)
for example skim milk and meso-inositol or honey or glutamate or raffinose, are
used to suspend cells to be freeze-dried in order to protect these against known
freezing and drying injuries.
Several anaerobic bacteria which are sensitive to aerobic freeze-drying, can
successfully be preserved using activated charcoal (5 % w/v) in the suspending
media along with the above protective agents (Malik, 1990).
With this simple method various yeasts, sporulating fungi, and bacteria can
successfully be preserved. Many delicate microorganisms such as nitrogen-fixing
bacteria like Azospirillum, Azotobacteraceae, Rhizobium, Xanthobacters,
Spirillaceae, Vibrios, and others like Alcaligenes, Ancylobacter,
Flavobacterium, Pseudomonads and few Rhodospirillaceae resulted in
a fairly good viability and stability after Iyophilization and during storage.
However, relatively low viability results in the case of various yeasts such as
Leucosporidium, Sporobolomyces, Rhodosporidium, Zygosaccharomyces and
fragile bacteria like Aquaspirillum, Spirosoma, Flectobacillus, and
others, as compared to the Iyophilization done under standard conditions (Malik,
1988 & 1990). The method is simple and the equipment described here can be
easily constructed in most laboratories.
Material and Methods
Preparation of freeze-dried skim milk ampoules
Ampoules of neutral glass (45 x 10 mm) are washed in a detergent, then rinsed in
distilled water and are air dried. For double vial preparation the ampoules are
labelled and filled with 0,5 ml of 20 % (w/v) skim milk (Bacto-Skim milk Difco
0032) containing 5 % meso-inositol or 5 % raffinose or 10% honey. The ampoules
are loosely plugged with non-absorbent cotton wool and sterilized at 115' C for
13 minutes. These are frozen at about -30 lo -40' C for a few hours and are
freeze-dried for about 6 hours, as described under the freeze-drying procedure
(primary freeze-drying).
Large batches of ampoules should be avoided but this depends upon the capacity
of the freeze-drying system ( vacuum pump, condensing temperature of the cold
trap, etc.).
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 use these are
selected according to the storage temperature available for storage of
freezedried ampoules. For more details see Mali, 1988.
Preparation of cell suspension for freeze-drying
The cultures are grown on appropriate media until the late logarithmic phase of
growth. 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 ready ceil suspensions are kept in an ice bath before filling
the ampoules.
Filling of ampoules and slow freezing of cell suspension
The already freeze-dried skim milk ampoules are cooled to about -30' C for 1-2
hours. To each ampoule about 0,03 ml (1 drop with a Pasteur pipette) of ice cold
cell suspension is added aseptically on to the skim milk plug with care so as
not to touch the sides of the ampoules. The ampoules are quickly placed again
for 1-2 hours in a deep-freeze and are frozen at approximately 1-2'C/min to
about -30' C. This is easily achieved if ice cold samples are placed in
commercially available deep-freezers.
The freeze-drying procedure
Freeze-drying involves the removal of water from frozen cell suspension by
sublimation under reduced pressure. The outline of the freeze-drying procedure
and the major steps involved are shown in Fig. 1.
The cold trap tube (a U-shaped thick glass or preferably a metal tube of about
2-3 cm diam and about 30 cm length), is connected with the vacuum hose
(preferably the U-tube 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 Iyophilization run 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 alternate 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, to cool down the cold
bath and is added to the cold trap periodically throughout the experiment.
For the double vial preparation freeze-drying is done in two stages involving
primary freeze-drying and secondary drying. When the condenser (cold trap)
temperature has reached about -35' C the frozen (to about -30' C) ampoules are
transferred quickly to the chilled metallic evacuation jar, which is dipped to
about 2 cm depth in the cold bath containing coolant at about -35'C (Fig.1 A).
The vacuum is switched on and the temperature of the cold bath is allowed to
elevate. If available, a vacuum meter or controller can be attached between the
vacuum pump and the evacuation jar to control the vacuum. Primary-drying is
continued for about 3 hours to achieve maximum desiccation ( at about 1 to 0,1
torr or mbar). At the end, the vacuum is replaced with nitrogen gas (especially
for ampoules which are not subjected to secondary drying and are not to be
sealed under vacuum). At the end of the experiment the water collected in the
cold trap is drained out or the silica gel is replaced.
Constriction of ampoules, secondary drying and sealing
In a double vial system the ampoules (containing primary freeze-dried cell
material) are sealed under vacuum in a soft glass tube. After primary
freeze-drying of the ampoules the projecting ends of the cotton plugs are
trimmed in level to the end of the ampoules and these are transferred into
soft-glass large tubes (130 x 15 mm) containing blue silica gel and cotton
plugs. For insulation small amount of glass wool is also pushed down along with
the ampoule (inner vial ) to the bottom of the large tube (outer vial). This
outer tube is then constricted, on a low flame by hand or by using Edward
Ampoule Constrictor, 20-25 mm above the glass wool to avoid burning of the
cotton plug of the inner vial. The constricted tubes are then attached to the
manifold and mounted on a metallic evacuation jar. This operation is illustrated
in Fig.1 B . The vacuum is switched on and secondary drying is conducted for 12
hours (at 0,1 to 0,001 torr). 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). To avoid
cracking of glass a flame containing oxygen should never be applied to such
neutral glass that has not previously been warmed on a normal flame
Revival of cultures from freeze-dried ampoules
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
(Iyophilized 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
freeze-dried ampoule are thus dissolved in sterile liquid growth media and
incubated at a relatively lower temperature than the optimum growth
temperatures. 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. In a few cases growth is inhibited by the
high concentration of protective mixture used during Iyophilization.
During reactivation the presence of activated charcoal in the suspending media
results in higher survival recoveries, and the reactivated cultures grown in the
presence of activated charcoal prove much more stable and could be maintained
relatively longer as living cultures (Malik,1990).
The use of activated charcoal as an adsorbent of harmful radicals in suspending
media for the reactivation of anaerobes is also recommended
Estimation of viability and stability
Survival recoveries are chocked before freeze-drying, immediately after
freeze-drying and after storage. For the estimation of viability counts serial
dilutions are prepared in liquid media. From each serial dilution 0,1 ml volumes
are plated on appropriate growth 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.
Long-term storage
Stability of freeze-dried cultures during storage is very important. A high
level of residual moisture content or exposure to oxygen have detrimental
effects on the freeze-dried product. Freeze-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 freeze-dried cultures at lower temperatures will extend their shelf
life. The unsealed freeze-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
Ellis, J.J. and J.A. Roberson. 1968. Viability of fungus cultures preserved by
Iyophilization. Mycologia 60: 399-405
Heckly, R.J. (1985). Principles of preserving bacteria by freeze-drying.
Developments in Industrial Microbiology 26: 379-395
Mackenzle, A.P. (1977). Comparative Studies on the freeze-drying survival of
various bacteria: Gram type, suspending medium and freezing rate. Develop. biol.
Standard, Vol.36: 263-277 (S. Karger, Basel).
Malik, K.A. (1976) Preservation of Knallgas bacteria. In Proceedings of Vth
Intern. Fermentation Symposium (H.Dellway, Ed.) p. 180 Westkreuz Druckerei and
Verlag, Bonn and Berlin.
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. (1988). Long-term preservation of some Rhodospirillaceae by
freeze-drying. J. Microbiol. Methods 8: 273-280
Malik, K.A. (1990). Use of activated charcoal for the preservation of anaerobic
phototrophic and other sensitive bacteria by freeze-drying. J. Microbiol.
Methods