Factors determining microbial colonization of liquid nitrogen storage tanks used for archiving biological samples.

The availability of bioresources is a precondition for life science research, medical applications, and diagnostics, but requires a dedicated quality management to guarantee reliable and safe storage. Anecdotal reports of bacterial isolates and sample contamination indicate that organisms may persist in liquid nitrogen (LN) storage tanks. To evaluate the safety status of cryocollections, we systematically screened organisms in the LN phase and in ice layers covering inner surfaces of storage tanks maintained in different biobanking facilities. We applied a culture-independent approach combining cell detection by epifluorescence microscopy with the amplification of group-specific marker genes and high-throughput sequencing of bacterial ribosomal genes. In the LN phase, neither cells nor bacterial 16S rRNA gene copy numbers were detectable (detection limit, 102 cells per ml, 103 gene copies per ml). In several cases, small numbers of bacteria of up to 104 cells per ml and up to 106 gene copies per ml, as well as Mycoplasma, or fungi were detected in the ice phase formed underneath the lids or accumulated at the bottom. The bacteria most likely originated from the stored materials themselves (Elizabethingia, Janthibacterium), the technical environment (Pseudomonas, Acinetobacter, Methylobacterium), or the human microbiome (Bacteroides, Streptococcus, Staphylococcus). In single cases, bacteria, Mycoplasma, fungi, and human cells were detected in the debris at the bottom of the storage tanks. In conclusion, the limited microbial load of the ice phase and in the debris of storage tanks can be effectively avoided by minimizing ice formation and by employing hermetically sealed sample containers.

An Improved Cryopreservation Protocol for Mentha spp. Based on Pvs3 as the Cryoprotectant.

BACKGROUND: Vitrification approaches are widely used to cryopreserve Mentha spp. genetic resources. OBJECTIVE: Here, we compared the response of 20 different Mentha species and hybrids during cryopreservation and elucidated the efficacy of two cryoprotectants. MATERIALS AND METHODS: One hundred and fifty three Mentha spp. accessions were cryopreserved using in vitro plants maintained under slow-growth storage and PVS2 or PVS3 as cryoprotectants. RESULTS: The cryoprotectant PVS2 was effective for all species, except M. requienii and M. villosanervata. The use of PVS3 increased the proportion of explants able to regrow after rewarming. The outbreak of endophytes upon rewarming was both less frequent and less severe when PVS3 replaced PVS2. CONCLUSION: Both PVS2 and PVS3 can be used as cryoprotectant for all the species and accessions of Mentha spp. surveyed. Since higher regenerations were achieved using PVS3, and since the risk of an endophyte outbreak was reduced, this cryoprotectant should be preferred in future for cryopreserving Mentha spp.

Ultrastructural changes associated with cryopreservation of potato (Solanum tuberosum l.) shoot tips.

The ultrastructure of cells within shoot tips of S. tuberosum 'Désirée' was studied after different steps of the DMSO droplet cryopreservation method. After 2 h of DMSO treatment, cells contained numerous small vesicles, while at the same time mitochondria and chloroplasts had increased in size and vacuoles had assumed an irregular shape. After rapid cooling in liquid nitrogen, subsequent rewarming, and 1 h incubation there were no apparent changes in the ultrastructural organization of the cells, suggesting that they might be still intact. However, two days after rewarming, the meristematic dome area and part of the epidermis showed signs of extensive damage. Rupture of plasmalemma, plasmolysis and destruction of cell organelles as well as strong heterochromatisation of nuclei were observed. Survival and regeneration of cells were found mainly in leaf primordial regions. Here cells were very active, containing many mitochondria and intact or regenerating chloroplasts. Alternating temperature preculture of donor plants before shoot tip isolation improved the cryopreservation results (plant regeneration 46.5 percent) as compared to constantly warm precultured shoot tips (plant regeneration 20.0 percent), which showed slightly stronger damage after rewarming from liquid nitrogen.

ATP Content and Cell Viability as Indicators for Cryostress Across the Diversity of Life.

In many natural environments, organisms get exposed to low temperature and/or to strong temperature shifts. Also, standard preservation protocols for live cells or tissues involve ultradeep freezing in or above liquid nitrogen (-196°C or -150°C, respectively). To which extent these conditions cause cold- or cryostress has rarely been investigated systematically. Using ATP content as an indicator of the physiological state of cells, we found that representatives of bacteria, fungi, algae, plant tissue, as well as plant and human cell lines exhibited similar responses during freezing and thawing. Compared to optimum growth conditions, the cellular ATP content of most model organisms decreased significantly upon treatment with cryoprotectant and cooling to up to -196°C. After thawing and a longer period of regeneration, the initial ATP content was restored or even exceeded the initial ATP levels. To assess the implications of cellular ATP concentration for the physiology of cryostress, cell viability was determined in parallel using independent approaches. A significantly positive correlation of ATP content and viability was detected only in the cryosensitive algae Chlamydomonas reinhardtii SAG 11-32b and Chlorella variabilis NC64A, and in plant cell lines of Solanum tuberosum. When comparing mesophilic with psychrophilic bacteria of the same genera, and cryosensitive with cryotolerant algae, ATP levels of actively growing cells were generally higher in the psychrophilic and cryotolerant representatives. During exposure to ultralow temperatures, however, psychrophilic and cryotolerant species showed a decline in ATP content similar to their mesophilic or cryosensitive counterparts. Nevertheless, psychrophilic and cryotolerant species attained better culturability after freezing. Cellular ATP concentrations and viability measurements thus monitor different features of live cells during their exposure to ultralow temperatures and cryostress.

Cryopreservation of yams using vitrification modified by including droplet method: effects of cold acclimation and sucrose.

Cryopreservation of yams (Dioscorea spp.) is important for the preservation of valuable genotypes for food, medicine and breeding purposes. This study on four species of yams was conducted to evaluate the influence of cold acclimation in an alternating 5 degree C and 28 degree C, 12 h thermo-photo-period and of two sucrose concentrations in the preculture medium using a modified droplet method. Acclimation of a 3-week period provided the best preconditioning treatment averaged over four genotypes. Effect of sucrose concentration in the preculture medium depended on the genotype (significant genotype x sucrose interaction; P = 0.036). High survival (67 to 70%) with 30% to 50% shoot recovery was obtained for D. bulbifera, D. polystachya and D. cayenensis, compared to 20 percent survival without shoot recovery for D. alata.

Formation of onion bulblets in vitro and viability during medium-term storage.

The influences of light conditions, sucrose and ethylene on in vitro formation and storability of onion (Allium cepa L.) bulblets were studied in various accessions. Light, sucrose and ethylene influenced bulb formation. Storability was primarily enhanced by a high sucrose concentration (100 g/l) in the culture medium. The bulbing process was characterised by changes in bulbing ratio, leaf length, number of leaves and leaf development time. The viability of bulbs after 1 year of in vitro storage at low temperatures was determined by their growth reaction in subsequent subcultures, growth after transfer into the greenhouse and tetrazolium staining. Sufficient sprouting of bulblets previously stored at -1  °C demonstrated the possibility of storing them in a low-temperature, slow-growth culture.

Investigating a new cryopreservation protocol for yams ( Dioscorea spp.).

Experiments were initiated to develop protocols for four species of Dioscorea, namely D. bulbifera L., D. oppositifolia L., D. alata L. and D. cayenensis Lam., and to assess the effects of various factors on the survival and regrowth of explants after rewarming. The treatments consisted of three different methods-vitrification, droplet and a modified droplet method-as well as the following variables-cryoprotective solution, sucrose concentration in the preadaptation medium and cold acclimation. While most of the protocols resulted in low to zero survival and regrowth rates, maximum survival rates of 100% and 75% were obtained for D. oppositifolia L. and D. cayenensis Lam., respectively, using two different protocols of the modified droplet method. A higher average survival rate was obtained using the droplet and modified droplet techniques than for the original vitrification methods. When the average of all the experiments was taken, more than one-half (52%) of the surviving explants developed further within 1 month with the modified droplet method compared to zero regrowth with the two other methods. The optimum protocol for cryopreservation is specific for each genotype.