A contribution to the quantitative evaluation of yeast cultures stored in liquid nitrogen

Different ways of the evaluation of viability of cultures stored in liquid nitrogen are compared. The streaking technique is recommended for a rapid evaluation. It is sufficient to determine the suitability of the medium, of cryopreservatives and of the whole procedure. On storage of melted cultures for 1–7 d at 4 °C it was found that the cultures could be transported directly in a suitable package maintaining the low temperature on the condition that the transportation would not last longer than 5 d.     

Liquid nitrogen storage of yeast cultures I. Survival,and literature review of the preservation of fungi at ultralow temperatures

All 20 yeast strains of 17 species tested survived 75 days (the length of the experimental period) in liquid nitrogen at-196 C. The components of the more protective of the two freezing media used were (w/v) malt extract 2.5 %, yeast extract 0.25 %, peptone 0.5 %, calf serum 15 % (v/v) and dimethyl sulfoxide 10 % (v/v). Viability of the cells in this medium after rapid uncontrolled freezing and thawing in sealed plastic ampoules ranged from 2 % to 98 % (average 67 %) compared with the viability of the cultures before freezing. In only 4 strains was survival lower than 50 %. (90 references).     

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.     

The influence of nitrogen availability on carbon and nitrogen storage in the biennial Cirsium vulgare (Savi) Ten. II. The cost of nitrogen storage

The cost of nitrogen storage to current growth was examined in relation to N availability in the biennial Cirsium vulgare. Plants were grown outdoors, in sand culture, with continuous diel drip irrigation of fertilization medium containing one of five different N concentrations. Plants grown at the highest N concentration stored twice as much N in their tap roots as did plants grown at the lowest N concentration. In high-N-grown plants, the storage of N reserves occurred during the period of maximum growth, at the same time as tap-root production. At the time of maximum biomass, stored N was also at a maximum. During the period following maximum biomass, no additional storage of N occurred. This pattern was observed despite frequent late-season leaf senescence which resulted in a large pool of potentially mobile N which could have been stored at no cost to growth. In low-N-grown plants, the production of tap-root storage tissue and the filling of that tissue with stored N were staggered. Tap-root production and growth occurred during the period of maximum growth, as in the high-N-grown plants. However, filling of the storage tissue with N occurred late in the growing season, when the pool of mobile N from senescent leaves was large. The utilization of this late-season N source occurred with little or no cost to growth, and this N is labelled, according to previous definitions, as ‘accumulated’. The costs of storing N in plants of the different N treatments were calculated using two models based on different growth constraints. In one model, the cost of N storage was represented as lost growth due to allocation of N to storage, rather than to the photosynthetic shoot (i.e. growth was assumed to be limited by carbon acquisition). In the second model, the storage cost was calculated as lost growth due to allocation of N to storage, rather than to the nitrogen-acquiring fine-root system (i.e. growth was assumed to be limited by nitrogen acquisition). In both models, the total cost of N storage was predicted to decrease as N availability decreased due to smaller storage pool sizes in plants of the low-N treatments. The cost of filling the tap root with stored N as a percentage of the total storage cost was also reduced as N availability decreased due to the occurrence of late-season accumulation. By relying, at least in part, on late-season accumulation, plants grown at the lowest three levels of N availability reduced total storage costs by 15 to 22%. The results demonstrate that plants are capable of adjusting their storage patterns in response to low nitrogen availability such that the costs of storage are reduced.     

Stability of mutant type II dihydrofolate reductase proteins in suppressor strains.

In the course of study of a (Glu-58 to Gln-58) mutant type II dihydrofolate reductase (DHFR), it was found that the altered DHFR was poorly produced in vivo. Investigations with several common laboratory Escherichia coli strains including htpR and lon strains bearing plasmids expressing the Gln-58 DHFR indicated a correlation of rapid degradation with the presence of a sup+ phenotype. The supo strain MC1061(p3) was transformed with a series of plasmids containing the Gln-58 DHFR gene with and without an additional supF gene, and expression levels were compared. The supF+ constructs exhibited little accumulation of the Gln-58 DHFR, while reasonable levels were found in the supo cases. Experiments with extracts of plasmid-free sup+ and supo strains showed rapid degradation by certain strains compared to MC1061(p3) and this degradation was not dependent upon ATP. In another route to increasing the stability of labile DHFR derivatives, mutagenesis of a strain bearing a N-terminally shortened Gln-58 DHFR was performed. Selection and analysis of a trimethoprim-resistant stable mutant showed that this DHFR gene contained a triple repeat of leu-pro-ser in the enzymatically non-essential N-terminal portion of the protein.     

Different rate of chromosome breakage in human fibroblast strains after storage in liquid nitrogen.

Cytogenetic investigation was carried out on fibroblasts stored in liquid nitrogen during a period of 7-99 months. Cell strains were from 9 individuals, 2 of whom were affected by xeroderma pigmentosum group C (XPC), and 2 XPC heterozygotes. In cell samples from 3 normal subjects and from 1 patient, high frequencies of abnormal mitoses were observed at the first passage after thawing, which returned to normal values after a few subcultures. The most frequent lesions were chromosome gaps and breaks. The cells damaged the most were those from one XP patient. These findings indicate that cells from some individuals are hypersensitive to clastogenic factors acting during freezing and thawing procedures. This sensitivity could be related to the genetic constitution, although the XP homozygous condition is not an essential or sufficient factor.     

Stability of biotransformation capacity in Digitalis lanata cell cultures after cryogenic storage

Suspension cultures of Digitalis lanata were stored at the temperature of liquid nitrogen. After thawing, viable cultures were recovered which showed a growth rate equal to that of untreated cells. The capacity of Digitalis cells to transform β-methyldigitoxin to β-methyldigoxin remained unchanged after cryostorage. This was shown in 300-ml shake cultures as well as in 20-1 bioreactors. From these results it is clear that cryostorage is a suitable method for preserving cell lines with specific capacities to produce compounds of medical and industrial importance.     

Liquid storage of miniature boar semen.

The effects of liquid storage at 15 degrees C on the fertilizing ability of miniature pig semen were investigated. Characterization of ejaculated semen from 3 miniature boars was carried out. Semen volume and pH were similar among these boars. In one of the boars, sperm motility was slightly low, and sperm concentration and total number of sperm were significantly lower than in the others (P < 0.01). Seminal plasma of the semen was substituted with various extenders (Kiev, Androhep, BTS and Modena) by centrifugation and semen was stored for 7 days at 15 degrees C. Sperm motility was estimated daily at 37 degrees C. For complete substitution of seminal plasma, Modena was significantly more efficient than the other extenders (P < 0.001) in retaining sperm motility. Semen from each of the 3 miniature boars that had been stored for 5 to 7 days at 15 degrees C in Modena was used for artificial insemination of 15 miniature sows. The farrowing rates were 100, 100 and 60%, and litter sizes were 6.4 +/- 1.5, 5.8 +/- 0.8 and 5.0 +/- 1.0 for each boar semen, respectively. The boar that sired the smallest farrowing rate was the same one that showed lower seminal quality with respect to sperm motility, sperm concentration and total number of sperm. These results suggest that miniature boar semen can be stored for at least 5 days at 15 degrees C by the substitution of seminal plasma with Modena extender.     

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。     

Causes of conductance change in yeast cultures.

The conductance change due to growth of Saccharomyces cerevisiae Y112, Zygosaccharomyces bailii M and Rhodotorula rubra NCYC 63 in culture media containing glucose, tartrate pH buffer and ammonium ions as sole nitrogen source was compared with that in a medium containing L-asparagine as sole nitrogen source. Decreases in conductance were observed in glucose-ammonium cultures of all three yeasts while little change occurred in cultures with L-asparagine as sole nitrogen source. This supports the hypothesis that the metabolic activity primarily responsible for conductance change in yeast cultures is the uptake of charged ammonium ions as nitrogen source and the reaction of protons with pH buffer compounds. Rhodotorula rubra cultures with L-asparagine as sole carbon source caused large increases in conductance with growth. Chemical analyses of culture filtrates showed that this increase in conductance was due to use of L-asparagine as carbon source and the excretion of nitrogen surplus to biosynthetic needs as ammonium. In addition, the production of aspartate, acetate and bicarbonate contributed to the increase in conductance.