The preservation of salt marsh algae by controlled liquid nitrogen freezing.

Five species of benthic marine algae were preserved by controlled liquid nitrogen freezing and storage over periods extending to 1 year. Only a small percent of the algae survived without cryoprotectant. Nannochloris adamsii was an exception; 67% survived after 12 months of storage. Nitzschia acicularis was the best preserved with 5 glycerol as a cryoprotectant, Dimethylsulfoxide was a better cryoprotectant for N. adamsii and Dunaliella quartolecta. Reducing normal brackish salinity (28‰) of the culture medium to one half (14‰) increased the survival percentages for N. acicularis, Cylindrotheca closterium and Phaeodactylum tricornutum. The morphology and physiology of the species tested were unchanged by long storage time in liquid nitrogen.     

Cryopreservation of four valuable strains of microalgae, including viability and characteristics during 15?years of cryostorage

Recent developments in the technology of cryopreservation have permitted the long-term storage of many strains of microalgae with reliable rates of survival. However, many strains still cannot be recovered from storage in liquid nitrogen. Here, we investigated the effects of various cryoprotectants in achieving comparatively high survival rate around 50%. The strains tested included two freshwater algae, Chlorella vulgaris C-27 and M-207A7, and two marine algae, Nannochloropsis oculata ST-4 and Tetraselmis tetrathele T-501. Cells of these strains were suspended in various cryoprotective solutions and slowly cooled to ?40°C prior to immersion in liquid nitrogen. Little or no cryoprotection was seen with dimethyl sulfoxide (DMSO) alone or in combination with sorbitol or proline; with glycerol alone; or with ethylene glycol (EG) alone. However, survival rates of approximately 50% were observed using a cryoprotectant mixture of 5% DMSO, 5% EG, and 5% proline. Viability persisted during a storage period of 15?years. Similarly, chlorophyll content was not significantly changed during this 15-year interval. Thus, the present study demonstrates the advantage of cryopreservation using liquid nitrogen. We expect that this method will contribute to both basic and applied biology through the establishment of cryopreserved microalgal culture collections.     

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.     

Routine Cryopreservation of Isolates of Steinernema and Heterorhabditis spp.

Infective-stage juveniles of Steinernema and Heterorhabditis spp. were cryopreserved using two-stage incubation in glycerol and 70% methanol before storage in cryotubes in liquid nitrogen. Optimal glycerol concentrations and incubation times for survival were determined for different species, but acceptable survival of all species and isolates of entomopathogenic nematodes can be obtained using 15% (w/w) glycerol and incubation for 48 hours. Mean survival was 69% for isolates of Steinernema and 68% for isolates of Heterorhabditis (n = 84). The maximum survival recorded was 97% for S. feltiae K254 stored in liquid nitrogen for 12 months.     

Cryopreservation of Pratylenchys spp.

Abstract: Optimal conditions for cryopreserving of populations of root lesion nematode (Pratylenchus spp.) were determined. Nematode survival was achieved using glycerol pre-treatments in the range of 14-17% (w/w). Increasing duration of the incubation in glycerol (up to 5 days) before immersion in liquid nitrogen significantly influenced nematode survival The highest mean survival for P. thornei was 76% after incubation in glycerol for 5 days. Nematodes were able to reproduce and infect carrot disc cultures after cryopreservation. This technique has valuable applications for long-term germplasm storage and maintenance of genetic lines.     

In vitro postwarming viability of vitrified porcine embryos: Effect of cryostorage length

Porcine embryos, which had been vitrified and stored in liquid nitrogen for up to three yr, were retrospectively analyzed to evaluate the influence of duration of storage on their in vitro viability post-warming. All embryos were vitrified (OPS or SOPS) and warmed (three-step or direct warming) using procedures that resulted in the same in vitro survival, hatching rates, and numbers of cells. Therefore, embryo data obtained using the different procedures were pooled according to their developmental stage as morulae (n = 571) or blastocysts (n = 797) and to the length of their storage in liquid nitrogen: a) 1-9 d; b) 10-30 d; c) 31-90 d; d) 1-3 yr. Non-vitrified embryos of corresponding developmental stages were used as a fresh control group (n = 280). Survival and hatching rates were evaluated after in vitro culture to assess embryo viability. The total number of cells was counted in the resulting viable blastocysts as an indicator of quality. A total of 1,648 fresh and vitrified embryos were analyzed. In vitro survival and hatching rates, but not the number of cells, differed significantly between vitrified morulae and their fresh counterparts irrespective of the duration of cryostorage. Length of storage in liquid nitrogen (LN₂) did not influence in vitro viability among different groups of vitrified/warmed morulae nor embryos at the blastocyst stage. In conclusion, duration of storage in LN₂ has no effect on the post-warming viability of porcine embryos vitrified at morula or blastocyst stage.     

A new method for liquid nitrogen storage of phototrophic bacteria under anaerobic conditions

A simple, effective and economical method for the long-term preservation of bacteria in liquid nitrogen under anaerobic conditions is described. As a case example anaerobic photosynthetic bacteria were successfully preserved. Gas tight small screw-cap glass ampoules with butyl rubber septa were used for freezing the specimen anaerobically. During experimental manipulations no anaerobic chamber or glove boxes were required. All teste cultures yielded high recoveries after repeated thawing and during storage. After freezing, survival recoveries of Rhodospirillaceae range from 70–100%, whereas with strict anaerobic strains of Chlorobiaceae and Chromatiaceae a maximum loss of 1–2 log₁₀ counts was observed. No further loss in viability occurred after 1–2 years of storage.The small size of the ampoules and the use of single ampoule for 15–20 repeated retrievals proved economical with respect to storage space and costs.The system is compact and suitable for the preservation of anaerobic phototropic bacteria and other fragile anaerobic microorganisms.     

Survival and genetic stability of shoot tips of <Emphasis Type="Italic">Hedeoma todsenii</Emphasis> R.S.Irving after long-term cryostorage

Endangered and rare species for which seed banking is not possible require alternative methods of ex situ conservation for long-term preservation. These methods depend primarily on cryopreservation methods, such as shoot tip cryopreservation, but there are few datasets with information on the long-term survival of shoot tips stored in liquid nitrogen. In this study, survival and genetic stability of shoot tips of the endangered species, Hedeoma todsenii, banked over multiple years were examined. In vitro cultures cryopreserved with both the encapsulation dehydration and the encapsulation vitrification methods showed good average survival after up to 13 yr of storage in liquid nitrogen. The application of droplet vitrification to this species increased survival significantly, with an average of 72%, compared with 24–45% survival obtained with other methods. As measured with microsatellite and sequence-related amplified polymorphism (SRAP) markers, the genetic stability of the same genotypes stored over different periods of time typically did not change. However, there was an average of 10.4% band loss between replicate samples that did indicate a potential change in DNA composition. These results demonstrate the use of shoot tip cryopreservation as an effective ex situ conservation tool for this species, but genetic stability of the cryopreserved tissues should be closely monitored.     

Survival of Entamoeba and related amoebae at low temperature-II. Viability of amoebae and cysts stored in liquid nitrogen

It was found that E. histolytica, E. histolytica-like, E. hartmanni, E. invadens, E. terrapinae, E. moshkovskii grown with a mixed bacterial flora, could be recovered after prolonged storage in liquid nitrogen. The longest period yet tested for E. histolytica is 382 weeks (7·3 years). Storage of amoebae of E. ranarum and E. coli was less successful. E. histolytica amoebae grown axenically or monoxenically were less easily stored than those amoebae grown with a mixed bacterial flora. Cysts were non-viable after freezing.E. histolytica amoebae showed the same virulence to rats and sensitivity to emetine after storage in liquid nitrogen, as was observed before freezing.A summary of a recommended procedure for freezing Entamoeba and related amoebae is given.     

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.     

Piglets produced from in vivo blastocysts vitrified using the Cryologic Vitrification Method (solid surface vitrification) and a sealed storage container

The objective was to develop a simple successful porcine cryopreservation protocol that prevented contact between embryos and liquid nitrogen, avoiding potential contamination risks. In vivo-derived blastocysts were collected surgically from donor pigs, and two porcine embryo vitrification protocols (one used centrifugation to polarize intracytoplasmic lipids, whereas the other did not) were compared using the Cryologic Vitrification Method (CVM), which used solid surface vitrification. The CVM allowed embryos to be vitrified, without any contact between embryos and liquid nitrogen. Both protocols resulted in similar in vitro survival rates (90% and 94%) and cell number (89 ± 5 and 99 ± 5) after 48 h in vitro culture of vitrified and warmed blastocysts. The protocol that did not use centrifugation was selected for continued use. To protect vitrified embryos from contact with liquid nitrogen and potential contamination during storage, a sealed outer container was developed. Use of this sealed outer container did not affect in vitro survival of cryopreserved blastocysts. In vivo blastocysts (n = 151) were collected, vitrified, and stored using the selected protocol and sealed container. These embryos were subsequently warmed and transferred to six recipients; five became pregnant and farrowed a total of 26 piglets. This embryo vitrification method allowed porcine embryos to be successfully vitrified and stored without any contact with liquid nitrogen.     

无细胞百日咳疫苗效价检定用攻击菌液氮保存的稳定性

为了考察无细胞百日咳疫苗效价检定用攻击菌在液氮中保存的稳定性,对液氮保存1~7年的4~9代百日咳攻击菌随机抽取7~11批次样品,采用LD50检测并经统计学处理,分析其样品的毒力变化。试验结果显示,4~9代的百日咳攻击菌液氮保存5年,毒力仍能达到《中华人民共和国药典》三部(2005版)的要求;保存6年和7年,4~7代的百日咳攻击菌毒力仍符合上述要求,8、9代的攻击菌毒力的合格率分别为84%和40%。试验证实,无细胞百日咳效价检定用攻击菌液氮保存5年具有良好的稳定性,可用于无细胞百日咳疫苗的效价检定。     

Freeze-preservation of buds from Scots pine trees

A procedure has been developed for freeze-preservation of buds of the Scots pine (Pinus sylvestris L.). Instead of liquid nitrogen, cold storage in –80°C was used. The partly dormant material used in the experiments was obtained directly from a natural stand in Northern Finland and no prefreezing or cryoprotectants for preconditioning were used. Cooling velocity was 1°C/min up to a terminal freezing temperature of –39°C, after which the buds were immersed in liquid nitrogen at –196°C for 10 minutes. The material was then transferred to a deepfreezer at –80°C and stored up to 6 months. After rapid thawing, the buds were sterilized and their viability was tested by FDA staining and by culturing meristems on 1/2 MS medium for at least two weeks. All the freezing experiments were performed during March and April. The best survival of buds (90–100%) was achieved at the beginning of April, after which a pronounced decline in survival occurred obviously due to a rise in the water content of the buds.     

Frankia菌种保藏

对用4种方法保藏的Frankia菌,进行了培养物存活、形态及其固氮活性的检测.发现在无氮液体培养基中保藏6年的Frankia.菌丝断裂,孢囊不完整.同期经有氮液体保藏的Frankia菌孢囊较完整.冷冻干燥保藏3.5年和砂管保藏8年,孢囊和菌丝均较完整.上述方法保藏的菌种,经活化后均能生长,且具有典型的Frankia菌形态特征和固氮活性.4种方法比较,无氮液体保藏法的菌体细胞生长速度快,固氮活性强,有侵染结瘤能力.     

Survival and growth of embryo axes of temperate trees after two decades of cryo-storage

Liquid nitrogen (LN) storage is recommended for conserving plants with seeds for which standard seed banking is not feasible, but there are few empirical reports confirming its long-term effectiveness. In this study we evaluated in vitro viability and ex vitro growth of embryo axes of a species that is short-lived in seed bank conditions (Juglans nigra) and two recalcitrant seeded species (Aesculus hippocastanum and A. glabra) that were stored 11–23 years in LN. Viability of J. nigra axes did not decrease significantly after 23 years and produced normal-appearing plantlets. Similarly, viability of A. hippocastanum axes did not decrease after 23 years. However, A. glabra axes showed a significant decline in viability after 23 years, from 80% when freshly harvested to 33%. These results demonstrate that LN storage can preserve embryo axes of J. nigra and Aesculus sp. for over two decades, providing a workable conservation tool for these and similar species.     

Two-Step Freezing Procedure for Cryopreservation of Rumen Ciliates, an Effective Tool for Creation of a Frozen Rumen Protozoa Bank

The present study aimed at the long-term storage of rumen protozoa as living cells in liquid nitrogen. The two-step or interrupted slow freezing procedure was used to cryopreserve six of the dominant species of rumen ciliates isolated from monofaunated animals, Dasytricha ruminantium, Entodinium caudatum, Epidinium ecaudatum caudatum, Eudiplodinium maggii, Isotricha prostoma, and Polyplastron multivesiculatum. We optimized the first step in the interrupted slow freezing procedure, from the extracellular ice nucleation temperature to the holding temperature, and studied the effects of the cooling rates on survival. In addition to the nature of the cryoprotectant (dimethyl sulfoxide), the equilibration temperature and equilibration time (25°C and 5 min, respectively), and the holding time at subzero temperature (45 min) recommended previously (S. Kišidayová, J. Microbiol. Methods 22:185-192, 1995), we found that a holding temperature of −30°C, a cooling rate from extracellular ice nucleation temperature to holding temperature of between 1.2°C/min and 2.5°C/min, depending on the ciliate, and rumen juice as the freezing and thawing medium markedly improved the survival rate. Survival rates determined after 2 weeks in liquid nitrogen were 100% for Isotricha, 98% for Dasytricha, 85% for Epidinium, 79% for Polyplastron, 63% for Eudiplodinium, and 60% for Entodinium. They were not significantly modified after a period of 1 year in liquid nitrogen. Four of the five ciliate species cryopreserved for 8 months in liquid nitrogen successfully colonized the rumen when inoculated into defaunated animals. These results have made it possible to set up a bank of cryopreserved rumen protozoa.     

In vitro response of encapsulated and non-encapsulated somatic embryos of Kinnow mandarin (Citrus nobilis Lour × C. deliciosa Tenora)

The aim of present investigation was to study the effect of storage conditions on percentage germination of encapsulated and non-encapsulated somatic embryos of Kinnow mandarin (Citrus nobilis Lour × C. deliciosa Tenora). Different batches of encapsulated and non-encapsulated embryos were preserved at room temperature, 4°C, in liquid nitrogen as such and by embedding in liquid paraffin. In the encapsulated somatic embryos stored at room temperature in sealed Petri plates, percentage of germination was 24.99%, but 5.55% in non-encapsulated embryos after 3 days of storage. Encapsulated embryos stored in vials containing liquid paraffin at room temperature were germinated at 18.05% after 60 days of storage, while it was 13.88% in non-encapsulated embryos after 45 days of storage. Encapsulated somatic embryos stored at 4°C in sealed Petri plates remained viable for up to 75 days with 6.94% germination, whereas non-encapsulated embryos remained viable for up to 45 days with 24.99% germination. Encapsulated embryos stored at 4°C in vials filled with paraffin germinated at 11.11% after 120 days of storage, but 5.55% in non-encapsulated embryos after 90 days of storage. Encapsulated and non-encapsulated embryos stored in liquid nitrogen showed 58.33 and 51.38% survival, respectively, after 7 months of storage. The plantlets developed from these embryos were transplanted after acclimatization and are growing normal.     

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.     

Effects of storage temperature on viable bioprosthetic heart valves.

Long-term in vivo success of bioprosthetic allografts is dependent upon retention of cellular functions, such as protein synthesis. The purpose of the experiments presented in this report was to determine the storage conditions necessary for retention of protein synthetic functions in human allograft heart valve leaflets. Tissue viability was assessed by measurement of tritiated-glycine incorporation into proteins. Comparison of short-term (less than 3 month)- and long-term (1 and 2 years)-cryopreserved heart valve leaflet storage in a liquid nitrogen freezer below -135 degrees C demonstrated preservation of fibroblast protein synthesis. In contrast, storage in a mechanical freezer at -80 degrees C resulted in a time-dependent loss of fibroblast protein synthesis. There was no statistically significant effect on protein synthesis in leaflets stored for 1 week at 4 degrees C compared to control cryopreserved liquid nitrogen-stored leaflets. After 2 weeks of 4 degrees C storage leaflet protein synthesis declined significantly to 15% that of cryopreserved controls. These results demonstrate that liquid nitrogen storage of valve bioprostheses is required for long-term preservation of cellular functions.     

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.