Cryopreservation of basidiomycete strains using perlite.

A new alternative method using perlite as a particulate solid carrier in the growth medium with a cryoprotectant was successfully tested for cryopreservation of several basidiomycete species from different genera (Armillaria, Pleurotus, Pluteus, Polyporus) which failed to survive or retain their properties in cryopreservation procedures routinely used in our laboratory. Frozen basidiomycete strains were kept in cryovials submerged in liquid nitrogen and were either immediately after the freezing process or after a 6-month storage thawed and checked for viability, purity and changes in growth, morphology and biochemical characteristics. All cultures survived the cryopreservation procedure and no negative effects of cryopreservation by this method have been observed after 6 months of storage in liquid nitrogen.     

Long-Term Preservation of Fungus Cultures with Liquid Nitrogen Refrigeration

A preservation technique was tested on 162 strains of culturally fastidious fungi sensitive to lyophilization, representing five classes. The results indicated that liquid nitrogen storage of frozen specimens may be used as an alternative to lyophilization for long-term preservation of stock cultures of fungi. The fungus was frozen in 10% (v/v) glycerol-water menstruum in heat-sealed ampoules. The cooling from ambient temperatures to -35 C was controlled at a rate of approximately 1 C per minute. Further cooling to the storage temperature of -165 to -196 C was uncontrolled and took place at an accelerated rate. Frozen ampoules were thawed in a water bath at 38 to 40 C. Viable and unmutated cultures were developed from reactivated specimens after storage for as long as 5 years.     

Functional studies after storage at - 190 degrees C of isolated and cultivated pituitary cells from pig and rat

For the first time, it is shown here that enzymatically dispersed pituitary cells of animals survive freezing and storage at -190 degrees C in liquid nitrogen. Frozen/thawed pituitary cells from both rat and pig are able to form monolayer aggregates in culture, and to produce hormones similar to that observed with unfrozen cells. The production of both basal and LHRH (luteinising hormone releasing-hormone)-induced bioassayable LH (luteinising hormone) were measured before and after cry-opreservation. Though after cryopreservation the number of cells was reduced by about 50%, a highly significant amount of both basal and LHRH stimulation-induced release of LH was measured in cultures from frozen/thawed pituitary cells from both species.     

Long-term preservation of yeast cultures in liquid nitrogen

Nineteen strains of taxonomieally diverse yeast species tested survived freezing and subsequent five-year storage in liquid nitrogen at ™196 °C, using a medium M 2 composed of malt extract, yeast extract, peptone, calf serum and dimethyl sulfoxide. Viability of the yeast cultures after long-term storage ranged from 5 to 97 % (average 62 %) compared with the viability of the cultures prior to freezing. The use of liquid nitrogen refrigeration for preserving yeast cultures is strongly advocated.     

液氮冻结法保藏毛霉目菌种的效果

本文报告了用液氮冻结法保藏毛霉目菌种的效果。对14属26种32株进行了液氮保藏试验,其结果慢速冻结孢子成活率高于快速冻结。用10%的甘油、10%的二甲基亚砜和蒸馏水作保护剂制成的孢子悬液,经慢速冻结后储存在液氮罐气态中二年,以甘油作保护剂的孢子成活率为90%,二甲基亚砜的为85%,蒸馏水的为79%。并检测了某些菌株的反丁烯二酸、蛋白酶、α-半乳糖苷酶、脂肪酶、果胶酶、葡萄糖苷酶等生理活性,结果冻结后的活性与冻结前相比,无明显差异。     

Survival of rapidly frozen hatched mouse blastocysts

The objective of the present study was to examine the effect of rapid freezing on the in vitro and in vivo survival of zona-pellucida-free hatched mouse blastocysts. Hatched blastocysts were rapidly frozen in a freezing medium containing either ethylene glycol (EG) or glycerol (G) in 1.5 M or 3 M concentration. Prior to freezing, embryos were equilibrated in the freezing medium for 2 min, 10 min, 20 min or 30 min at room temperature. To freeze them, embryos were held in liquid nitrogen vapour [approximately 1 cm above the surface of the liquid nitrogen (LN2)] for 2 minutes and then immersed into LN2. After thawing, embryos were transferred either to rehydration medium (DPBS + 10% foetal calf serum +0.5 M sucrose) for 10 minutes or rehydrated directly in DPBS supplemented with foetal calf serum. In vitro survival of embryos frozen with EG was higher than those frozen with G. The highest survival was obtained with 3 M EG and 2 min or 10 min equilibration prior to freezing, combined with direct rehydration after thawing. Frozen blastocysts developed into normal foetuses as well as unfrozen control ones did, with averages of 30% (control), 26% (EG) and 15% (G). The results show that hatching and hatched mouse blastocysts can be cryopreserved by a simple rapid freezing protocol in EG without significant loss of viability. Our data indicate that the mechanical protection of the zona pellucida is not needed during freezing in these stages.     

液氮保存曲霉菌种的效果

本文报告了以蒸馏水、10%甘油和5%二甲基亚砜(DMSO)为保护剂,用液氮冻结法保存5种8株曲霉的效果,并检测了这些菌分别产生的亚甲基丁二酸、柠檬酸、蛋白酶和糖化酶的生理活性。这些菌在液氮气相(接近-150℃)中保存180天全部保持着生活能力,它们的培养特征和形态特征保留原来的形状。所测定的液氮保存8株菌种的生理活性,除两株糖化酶活力稍有降低外,其它菌株没有明显的变化。     

The use of freeze-preserved treponemes in the Treponema pallidum immobilization test

Treponema pallidum extracted from rabbit testes and quick frozen in liquid nitrogen were vigorously motile upon thawing, when cryoprotected with dimethyl sulfoxide (DMSO). However, this chemical was toxic to unfrozen treponemes unless normal serum, 10–20% final concentration, was added to suspensions before freezing, or immediately after thawing. Such organisms survived the in vitro incubation of the T. pallidum immobilization (TPI) test, but DMSO partially inhibited immune immobilization. This inhibition was concentration dependent and was not observed in treponeme preparations diluted fourfold with fresh medium after 10% DMSO storage. Frozen treponeme suspensions used in this fashion yielded TPI test results comparable to those of fresh suspensions, and were still satisfactory after 12 months storage.     

Long-term cryogenic storage of Neurospora crassa spores

Neurospora crassa on agar slants in ampules were frozen by direct immersion into liquid nitrogen and stored at −196 °C for 0–5.5 years. The survival, estimated as percentage germination of conidia, after warming, varied between 81.1 and 97.6%. Although there was a slight decline in survival immediately after the freezing treatment, no further significant decline has been detected during 5.5 years storage. The regression coefficients, (0.43, control, and 0.97, liquid nitrogen storage for 5.5 years), do not predict extinction of viability.     

Preservation of some extremely thermophilic chemolithoautotrophic bacteria by deep-freezing and liquid-drying methods

Long-term preservation methods for extreme thermophilic chemolithoautotrophic bacteria representing various species are described. The cultures were cryopreserved in liquid nitrogen under anaerobic conditions using 5% dimethylsulfoxide as a cryoprotectant. For easy storage and transport, the cultures were successfully liquid-dried, directly from the liquid phase without involving freezing under semiaerobic conditions using effective protective agents such as ethylenediamine and meso-inositol. The tested cultures showed good stability and survival rates after drying, after cryopreservation and on long-term storage. All tested strains were successfully preserved and reactivated within relatively short time. The viability, stability and ability of chemolithoautotrophic growth was not affected. Cryopreservation, liquid-drying and reactivation under microaerobic conditions proved very effective for these oxygen sensitive cultures.     

Long-term viability of preserved eukaryotic algae

Levels of viability of Chlorella emersonii after storage of dried material for one year were 0.1% on rehydration, all other dried organisms examined in this study failed to recover after prolonged storage. In addition, no detectable recovery was observed in any of the algae tested after storage of freeze-dried cultures. Methods have also been developed to cryopreserve a range of microalgae, but no single protocol has been found to be universally satisfactory. Some strains are apparently not able to withstand cryopreservation using known methods, whilst others may be frozen successfully in the absence of cryoprotectant by plunging directly into liquid nitrogen. A two-step protocol (cooling to an intermediate subzero temperature prior to plunging into liquid nitrogen) has been used to cryopreserve the majority of strains. Where this has proven successful, post-thaw viability levels of over 95% have been attained for some algae. This paper demonstrates that, where applicable, cryopreservation allows the long-term preservation of frozen algae with no significant reduction in viability up to 22 years storage. (Previous location of Culture Collection of Algae and Protozoa) This revised version was published online in September 2006 with corrections to the Cover Date.     

Viability of basidiomycete strains after cryopreservation: comparison of two different freezing protocols

The viability of 250 basidiomycete strains was determined after a 2-d and then after a 2-year storage under liquid nitrogen using two different freezing protocols. Using an original agar plug protocol (OP), 162 strains (65%) of the 250 strains survived a 2-d storage and 158 strains (63%) survived a 2-year storage in liquid nitrogen. Using a straw protocol (CP), 246 strains (98%) of the 250 strains survived a 2-d storage and 243 strains (97%) a 2-year storage in liquid nitrogen. In addition, other 106 strains were newly estimated using the CP protocol; 104 (98%) of them survived successfully a 2-d storage and 101 (95%) of them survived a 2-year storage in liquid nitrogen. The results indicate that the protocol used for cryopreservation can significantly influence strain survival. Markedly better results were obtained using the CP protocol.     

嗜碱细菌的液氮超低温冻结保藏

本文报道7株嗜碱细菌的液氮超低温快速冻结保藏的试验结果。从细胞存活率看,冻结保藏3个月,自然pH的10%甘油、5%二甲基亚砜保护剂保藏嗜碱细菌的效果相似于该方法用于一般细菌保藏的保存结果,说明液氮超低温冻结保藏法用于嗜碱细菌的保藏是安全有效的。如选择pH值接近嗜碱细菌的最适生长pH值的保护剂,则可以提高细胞存活率。     

Survival of Nippostrongylus brasiliensis larvae after freezing over liquid nitrogen

Survival of Nippostrongylus brasiliensis larvae after freezing over liquid nitrogen. International Journal for Parasitology4: 173–176. Third stage larvae of N. brasiliensis were frozen over liquid nitrogen and after storage for 7 days were thawed rapidly and inoculated subcutaneously into rats. When ensheathed larvae were used, none survived freezing as judged by motility and infectivity trials. Separate vials of exsheathed larvae survived freezing in proportions ranging from 10 to 64 per cent. Female worms, derived from frozen exsheathed larvae, had a normal complement of eggs in the uterus and both male and female worms had a normal histological appearance. Exsheathed larvae frozen in the presence of 10 per cent dimethylsulphoxide had the same survival rate as those frozen without the addition of cryoprotectant. The addition of 10 per cent glycerol adversely effected the survival of frozen exsheathed larvae.     

Vermiculite as a culture substrate greatly improves the viability of frozen cultures of ectomycorrhizal basidiomycetes

We assessed a new cryopreservation protocol that uses vermiculite as a culture substrate, called the vermiculite protocol (VP), by assessing the viability, recovery time of hyphae after revival, and colony diameter of cryosensitive ectomycorrhizal basidiomycete strains after storage for 2 weeks or 1 year in a vapour-phase liquid nitrogen tank. Twelve difficult-to-preserve strains of nine species (Amanita citrina, A. pantherina, A. rubescens, A. spissa, Kobayasia nipponica, Lactarius akahatsu, L. hatsudake, Sarcodon aspratus, and Tricholoma flavovirens) that did not achieve good revival after cryopreservation with our previous Homolka’s perlite protocol and modified perlite protocol (MPP) experiments were used to assess the new methodology. Vermiculite and liquid medium were put into a cryotube and inoculated with an agar plug containing mycelia. The cryotube was cultured for various incubation times. After adequate mycelial growth, a mixture of cryoprotectants (5% dimethyl sulfoxide and 10% trehalose [5D10T] or 5% glycerol and 10% trehalose [5G10T]) was placed into the cryotube. The cryotube was frozen in a freezing container in a –80 °C freezer and then stored in vapour-phase liquid nitrogen. In the recovery test, 10 of 12 strains showed 100% revival after 2 weeks of storage in the 5G10T cryoprotectant, and all 12 strains showed 100% revival after 2 weeks of storage in the 5D10T cryoprotectant. Furthermore, all strains were viable after 1 year of storage in a vapour-phase liquid nitrogen tank. Thus, the VP is applicable to a wide range of ectomycorrhizal basidiomycete cultures, including highly cryosensitive strains.     

Death of Lactobacillus bulgaricus Resulting from Liquid Nitrogen Freezing

Concentrated cell suspensions of Lactobacillus bulgaricus prepared from cells grown in semisynthetic media were frozen in liquid nitrogen. After storage for 24 hr, the cell suspensions were found to have decreased colony counts and acid-producing capacity in milk. The amount of loss varied among the different strains tested. The addition of known cryoprotective agents to cell suspensions of the most labile strain before freezing provided little or no protection to the cells. However, storage stability of all strains investigated was improved by supplementing the growth medium with Tween 80 (polyoxyethylene sorbitan monooleate). The concentration of Tween 80 necessary for maximal storage stability varied among strains.     

Influence of freezing with liquid nitrogen on whole-knee joint grafts and protection of cartilage from cryoinjury in rabbits

Improving survival rates for sarcoma patients are necessitating more functional and durable methods of reconstruction after tumor resection. Frozen osteoarticular grafts are utilized for joint reconstruction, but the joint may develop osteoarthritic change. We used a frozen autologous whole-rabbit knee joint graft model to investigate the influence of freezing on joint components. Thirty rabbit knee joints that had been directly immersed into liquid nitrogen (L) or saline (C) without use of cryoprotectants were re-implanted. Histological observations were made after 4, 8, and 12 weeks. Both groups had bone healing. In group L, despite restoration of cellularity to the menisci and ligaments, no live chondrocytes were observed and cartilage deterioration progressed over time. It was concluded that cryoinjury of chondrocytes caused osteoarthritic change. Then we tested whether a vitrification method could protect cartilage from cryoinjury. Full-thickness articular cartilage of rabbit knee was immersed into liquid nitrogen with and without vitrification. Histology, ultrastructure, and chondrocyte viability were examined before and after 24 h of culture. Vitrified cartilage cell viability was >85% compared with that of fresh cartilage. Transmission electron microscopy revealed preservation of original chondrocyte structure. Our vitrification method was effective for protecting chondrocytes from cryoinjury. Since reconstructing joints with osteoarticular grafts containing living cartilage avert osteoarthritic changes, vitrification method may be useful for storage of living cartilage for allografts or, in Asian countries, for reconstruction with frozen autografts containing tumors.     

Basidiomycete cryopreservation on perlite: evaluation of a new method

A new cryopreservation method using perlite as a carrier was evaluated on a large set of mycelial cultures of basidiomycetes. The viability and some other characteristics--growth, macro- and micromorphology, and laccase production--of 442 strains were tested after 48-h and then after 3-year storage in liquid nitrogen using a perlite protocol (PP). All (100%) of them survived successfully both 48-h storage and 3-year storage in liquid nitrogen without noticeable growth and morphological changes. Also laccase production was unchanged. The viability and laccase production of a part (250) of these strains were compared with those of the strains subjected to an original agar plug protocol (OP). Using OP, 144 strains (57.6%) out of 250 survived a 3-year storage in liquid nitrogen. The results indicate that the cryopreservation protocol used significantly influences survival of the strains. Markedly better results were achieved using the PP.     

Cryopreservation of immature embryos of Theobroma cacao

Immature, white zygotic embryos of Theobroma cacao L. (cacao) retained the ability to produce callus and to undergo somatic embryogenesis after slow hydrated freezing and desiccated fast freezing in liquid nitrogen. The highest rate of somatic embryogenesis occurred in embryos which were precultured on a medium containing 3% sucrose, frozen slowly with cryoprotectants before exposure to liquid nitrogen, and recovered on a medium containing 3 mg/liter NAA. Embryos precultured on media containing sucrose increasing to 21% had a higher rate of survival but were less embryogenic after freezing. These results suggest that immature embryos might be used for long-term germplasm storage of T. cacao germplasm.     

Operating conditions that affect the resistance of lactic acid bacteria to freezing and frozen storage

Thermophilic lactic acid bacteria exhibit different survival rates during freezing and frozen storage, depending on the processing conditions. We used a Plackett and Burman experimental design to study the effects of 13 experimental factors, at two levels, on the resistance of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus to freezing and frozen storage. The resistance was evaluated by quantifying the decrease of acidification activity during freezing and throughout 8 weeks of storage. Acidification activity after freezing and frozen storage was affected by 12 experimental factors. Only the thawing temperature did not show any significant effect. S. thermophilus was more resistant than L. bulgaricus and the cryoprotective effect of glycerol during freezing and storage was confirmed. The temperature and duration of the cryoprotection step influenced acidification activity following the freezing step: the lower the temperature and the shorter the duration, the higher the activity. Acidification activity after storage was affected by several experimental factors involved in the fermentation stage: use of NaOH instead of NH4OH for pH control, addition of Tween 80 in the culture medium, and faster cooling led to better cryotolerance. Resistance to freezing and frozen storage was improved by using a high freezing rate and a low storage temperature. Finally, this study revealed that the conditions under which lactic acid bacteria are prepared should be well controlled to improve their preservation and to limit the variability between batches and between species.