10.1134/S0012496618030079. PMID 30009350.
Fungi, notably zygomycetes, ascomycetes and higher basidiomycetes, regardless of sporulation, are able to be stored in liquid nitrogen or deep-frozen. Crypreservation is a hallmark method for fungi that do not sporulate (otherwise other preservation methods for spores can be used at lower costs and ease), sporulate but have delicate spores (large or freeze-dry sensitive), are pathogenic (dangerous to keep metabolically active fungus) or are to be used for genetic stocks (ideally to have identical composition as the original deposit). As with many other organisms, cryoprotectants like DMSO or glycerol (e.g. filamentous fungi 10% glycerol or yeast 20% glycerol) are used. Differences between choosing cryoprotectants are species (or class) dependent, but generally for fungi penetrating cryoprotectants like DMSO, glycerol or polyethylene glycol are most effective (other non-penetrating ones include sugars mannitol, sorbitol, dextran, etc.). Freeze-thaw repetition is not recommended as it can decrease viability. Back-up deep-freezers or liquid nitrogen storage sites are recommended. Multiple protocols for freezing are summarized below (each uses screw-cap polypropylene cryotubes):
Many common culturable laboratory strains are deep-frozen to preserve genetically and phenotypically stable, long-term stocks. Sub-culturing and prolonged refrigerated samples may lead to loss of plasmid(s) or mutations. Common final glycerol percentages are 15, 20 and 25. From a fresh culture plate, one single colony of interest is chosen and liquid culture is made. From the liquid culture, the medium is directly mixed with equal amount of glycerol; the colony should be checked for any defects like mutations. All antibiotics should be washed from the culture before long-term storage. Methods vary, but mixing can be done gently by inversion or rapidly by vortex and cooling can vary by either placing the cryotube directly at −50 to −95 °C, shock-freezing in liquid nitrogen or gradually cooling and then storing at −80 °C or cooler (liquid nitrogen or liquid nitrogen vapor). Recovery of bacteria can also vary, namely if beads are stored within the tube then the few beads can be used to plate or the frozen stock can be scraped with a loop and then plated, however, since only little stock is needed the entire tube should never be completely thawed and repeated freeze-thaw should be avoided. 100% recovery is not feasible regardless of methodology.
The microscopic soil-dwelling nematode roundworms Panagrolaimus detritophagus and Plectus parvus are the only eukaryotic organisms that have been proven to be viable after long-term cryopreservation to date. In this case, the preservation was natural rather than artificial, due to permafrost.
- Pegg DE (January 1, 2007). "Principles of cryopreservation". Methods in Molecular Biology. 368: 39–57. doi:10.1007/978-1-59745-362-2_3. ISBN 978-1-58829-377-0. PMID 18080461.
- Sambu S (June 25, 2015). "A Bayesian approach to optimizing cryopreservation protocols". PeerJ. 3: e1039. doi:10.7717/peerj.1039. PMC 4485240. PMID 26131379.
- Costanzo JP, Lee RE, Wright MF (December 1991). "Glucose loading prevents freezing injury in rapidly cooled wood frogs" (PDF). The American Journal of Physiology. 261 (6 Pt 2): R1549–53. doi:10.1152/ajpregu.1991.261.6.R1549. PMID 1750578.
- Lovelock JE (March 1953). "The haemolysis of human red blood-cells by freezing and thawing". Biochimica et Biophysica Acta. 10 (3): 414–26. doi:10.1016/0006-3002(53)90273-X. PMID 13058999.
- Fuller BJ, Lane N, Benson EE, eds. (2004). Life in the Frozen State. CRC Press. p. 7. ISBN 978-0203647073.
- Mazur P (May 1970). "Cryobiology: the freezing of biological systems". Science. 168 (3934): 939–49. Bibcode:1970Sci...168..939M. doi:10.1126/science.168.3934.939. PMID 5462399.
- "The Cryobiological Case for Cryonics" (PDF). Cryonics. Vol. 9(3) no. 92. Alcor Life Extension Foundation. March 1988. p. 27.
- Andjus RK, Smith AU (June 1955). "Reanimation of adult rats from body temperatures between 0 and + 2 degrees C". The 