ASTM E1342-97

NOTICE: This standard has either been superseded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
Designation: E 1342 – 97
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 E 1342; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) 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 served Biological Materials
E 1565 Guide for Inventory Control and Handling of Bio-
1.1 This practice covers the handling of microorganisms
logical Materials Maintained at Low Temperatures
(bacteria, fungi, and protista), viruses, genetic elements
E 1566 Guide for Handling Hazardous Biological Materials
(nucleic acids and plasmids), and animal and plant cell tissues
in Liquid Nitrogen
(cell lines), during and after freezing and storage at cryogenic
temperatures.
3. Terminology
1.2 This practice also covers the handling of microorgan-
3.1 Definitions of Terms Specific to This Standard:
isms, viruses, and genetic elements in the host cell during and
3.1.1 cryogenic temperatures—temperatures below or equal
after freeze-drying.
to −100°C.
1.3 While this practice does not cover the specific method-
3.1.2 cryoprotectant—a chemical substance used to protect
ology used to freeze and freeze-dry microorganisms and cell
cells during freezing and rewarming.
lines, the safety aspects of handling microorganisms during
3.1.3 eutectic temperature—the temperature below which
freezing and freeze-drying procedures, and during storage at
all liquid portions of an aqueous suspension have entered the
cryogenic temperatures, are covered. Other guidelines must
solid phase.
also be adhered to regarding the handling of hazardous
3.1.4 freeze-drying—sublimation of water from a frozen
materials (2).
aqueous suspension.
1.4 This standard does not purport to address all of the
3.1.5 freezing—lowering the temperature of an aqueous
safety concerns, if any, associated with its use. It is the
suspension to a point at or below the temperature of ice crystal
responsibility of the user of this standard to establish appro-
formation.
priate safety and health practices and determine the applica-
3.1.6 low temperature preservation—stabilizing viable or
bility of regulatory limitations prior to use. For specific hazard
biologically active material by freezing or freeze-drying.
statements see Section 6.
3.1.7 vitrification—solidification of an aqueous suspension
2. Referenced Documents at low temperatures without the formation of ice crystals.
2.1 ASTM Standards:
4. Significance and Use
E 1564 Guide for Design and Maintenance of Low-
4.1 The staiblity of cell populations and genetic elements at
Temperature Storage Facilities for Maintaining Cryopre-
low temperatures can be affected by the methods used to
preserve the material, and by procedures used in handling the
This practice is under the jurisdiction of ASTM Committee E-48 on Biotech-
material during storage (3).
nology and is the direct responsibility of Subcommittee E48.02 on Characterization
4.2 The intent of this practice is to outline procedures that
and Identification of Biological Systems.
Current edition approved Oct. 10, 1997. Published December 1997. Originally
published as E 1342 – 90. Last previous edition E 1342 – 90.
The boldface numbers in parentheses refer to the list of references at the end of
this practice. Annual Book of ASTM Standards, Vol 11.05.
Copyright © ASTM, 100 Barr Harbor Drive, West Conshohocken, PA 19428-2959, United States.
NOTICE: This standard has either been superseded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
E 1342
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
centration of the cryoprotectant and storage temperature, are all
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/ products that may affect the freeze-drying process and recovery
min to cryogenic temperatures) is preferred. With aggregates of 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,
temperatures without the formation of ice (vitrification) may be 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:
tures is no more than 5 to 10 s. If material must be moved more 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. Make sure that the container holding generally warmer than −40°C, a temperature suitable for ice
the frozen material is in direct contact with the dry ice or liquid 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 the shelf. The type of glass vial can also affect the heat transfer,
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 is
not to warm the internal environment of the unit more than required to ensure an adequate vapor pressure of less than 10
10°C when adding or retrieving material. Minimize the interval μm Hg (0.01 torr). During freeze-drying maintain a pressure of
during which the door to the unit is open. Make sure that 50 μm Hg (0.05 torr) or below. When elevated pressure drying
removal and replacement of frozen material during retrieval can be used, it will accelerate the rate of drying (7). However
operations is minimized. Design an inventory system providing to ensure adequate drying the pressure must be reduced to
ready access to all material in the unit to avoid inadvertent below 50 μm Hg during the final stages of the drying process,
warming of preserved material during retrieval of other items. and the shelf raised to ambient temperature.
5.3.2 When using all vapor storage
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