ASTM E1342-97(2002)

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Designation:E1342–97 (Reapproved 2002)
Standard Practice for
Preservation by Freezing, Freeze-Drying, and Low
Temperature Maintenance of Bacteria, Fungi, Protista,
Viruses, Genetic Elements, and Animal and Plant Tissues
This standard is issued under the fixed designation E1342; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
Methods used for low temperature preservation of living biological systems include both freezing
and freeze-drying. While in many cases other methods of preservation can be used, these low
temperature methods provide the only real assurance of genetic stability. However, there are reports
of damage to DNA as a result of freeze-drying (1).
This practice assumes a basic knowledge of freezing and freeze-drying methods, and does not
include specific methods used for freezing and freeze-drying.
1. Scope 2. Referenced Documents
1.1 This practice covers the handling of microorganisms 2.1 ASTM Standards:
(bacteria, fungi, and protista), viruses, genetic elements E1564 Guide for Design and Maintenance of Low-
(nucleic acids and plasmids), and animal and plant cell tissues Temperature Storage Facilities for Maintaining Cryopre-
(cell lines), during and after freezing and storage at cryogenic served Biological Materials
temperatures. E1565 Guide for Inventory Control and Handling of Bio-
1.2 This practice also covers the handling of microorgan- logical Material Maintained at Low Temperatures
isms, viruses, and genetic elements in the host cell during and E1566 Guide for Handling Hazardous Biological Materials
after freeze-drying. in Liquid Nitrogen
1.3 While this practice does not cover the specific method-
3. Terminology
ology used to freeze and freeze-dry microorganisms and cell
3.1 Definitions of Terms Specific to This Standard:
lines, the safety aspects of handling microorganisms during
freezing and freeze-drying procedures, and during storage at 3.1.1 cryogenic temperatures—temperaturesbeloworequal
to−100°C.
cryogenic temperatures, are covered. Other guidelines must
also be adhered to regarding the handling of hazardous 3.1.2 cryoprotectant—a chemical substance used to protect
cells during freezing and rewarming.
materials (2).
1.4 This standard does not purport to address all of the 3.1.3 eutectic temperature—the temperature below which
all liquid portions of an aqueous suspension have entered the
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro- solid phase.
priate safety and health practices and determine the applica- 3.1.4 freeze-drying—sublimation of water from a frozen
aqueous suspension.
bility of regulatory limitations prior to use. For specific hazard
statements see Section 6. 3.1.5 freezing—lowering the temperature of an aqueous
suspension to a point at or below the temperature of ice crystal
formation.
3.1.6 low temperature preservation—stabilizing viable or
This practice is under the jurisdiction of ASTM Committee E48 on Biotech-
biologically active material by freezing or freeze-drying.
nology and is the direct responsibility of Subcommittee E48.02 on Characterization
and Identification of Biological Systems.
Current edition approved Oct. 10, 1997. Published December 1997. Originally
published as E1342–90. Last previous edition E1342–90. DOI: 10.1520/E1342- For referenced ASTM standards, visit the ASTM website, www.astm.org, or
97R02. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof Standards volume information, refer to the standard’s Document Summary page on
this practice. the ASTM website.
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E1342–97 (2002)
3.1.7 vitrification—solidification of an aqueous suspension 5.4 Minimizehandlingoffrozenspecimensduringretrieval.
at low temperatures without the formation of ice crystals. To limit the working time in a low temperature freezer, design
an inventory system that allows pinpointing of the exact
4. Significance and Use location of a single ampoule or vial. During retrieval of an
ampoule or vial, make sure that all material remains at a
4.1 The staiblity of cell populations and genetic elements at
relatively constant temperature. Handling procedures require
low temperatures can be affected by the methods used to
thatboxesorothercontainersremainatthecoldesttemperature
preserve the material, and by procedures used in handling the
of the working area of the freezer during retrieval of a single
material during storage (3).
vial.Whilethismaynotalwaysbepossible,takeprecautionsto
4.2 The intent of this practice is to outline procedures that
ensure that only the vial being retrieved is exposed to warmer
can minimize the adverse effects of handling biological mate-
temperatures as it is transferred to a portable container. When
rials during low temperature preservation and maintenance.
transferring material from a low temperature freezer to a
portable container for transporting, all operations must be
5. Freezing
made quickly to minimize exposure of the retrieved vial to
5.1 To successfully freeze living cells, a chemical agent
ambient temperatures.
(that is, a cryoprotectant) is often used to protect the cells
5.5 Several factors can affect the recovery of cells during
during cooling and rewarming.The cells are harvested and the
and after freezing. The type of cells, age of the cells, growth
cryoprotectant is added just prior to freezing. Exposure to the
conditions, cell concentration, rate of cooling, type and con-
cryoprotectant is limited to no more than 1 to 2 h before
centrationofthecryoprotectantandstoragetemperature,areall
beginning cooling of the cells. The optimal exposure time
factors that must be considered when freezing living cells.
varies depending on the material being preserved.
6. Freeze-Drying
5.2 For most microorganisms and cell lines, the optimum
cooling rate when using a single cryoprotectant is a uniform 1
6.1 During dispensing of microbial suspensions for freeze-
to 10°C/min to at least−35°C. Cooling must continue to at
drying, minimize exposure to ambient temperatures to limit
least−35° at this rate before exposing cells to lower tempera-
changes in population size and production of extracellular
tures. Below−35°C more rapid cooling (that is, 50 to 100°C/
productsthatmayaffectthefreeze-dryingprocessandrecovery
mintocryogenictemperatures)ispreferred.Withaggregatesof
of the microorganisms. Dispensing should immediately follow
cells, a mixture of cryoprotectants that solidifies at low
harvesting, and when large volumes of material are dispensed,
temperatureswithouttheformationofice(vitrification)maybe
make sure that the cell suspensions are constantly stirred and
more optimal (4,5). When moving frozen material from one
maintained at 2 to 8°C during the dispensing operation to
container to another, such as from a freezing unit to a liquid
ensure uniformity of the product.
nitrogen freezer, make sure that exposure to ambient tempera-
6.2 Maintenance During Freeze-Drying:
turesisnomorethan5to10s.Ifmaterialmustbemovedmore
6.2.1 Optimum temperatures for freeze-drying are at or just
than a few ft, use a styrofoam carrier containing dry ice or
below the eutectic temperature of the suspension to be freeze-
liquid nitrogen to maintain the temperature of the frozen
dried. For most microbial suspensions this temperature is
material during transport.Makesurethatthecontainerholding
generally warmer than−40°C, a temperature suitable for ice
thefrozenmaterialisindirectcontactwiththedryiceorliquid
crystal formation that is destructive to the viability of most
nitrogen.
microbes. Therefore, accomplish drying in as short a time as
5.3 During storage at cryogenic temperatures the frozen
possible. To ensure adequate heat transfer, use freeze-drying
material must be maintained below a critical predetermined
trays with removable bottoms to allow direct vial contact with
temperature suitable for the material being stored.An increase
theshelf.Thetypeofglassvialcanalsoaffecttheheattransfer,
in temperature above the critical temperature can result in ice
as molded vials may not uniformly contact the shelf.
crystal formation and a consequent loss of viability.
6.2.2 During the freeze-drying process a temperature differ-
5.3.1 When using mechanical freezers, care must be taken ential of at least 20°C between the product and condenser
...