极端嗜盐菌的保藏研究

研究了在极端嗜盐菌的液氮超低温冻结保藏和真空冷冻干燥保藏中,不同种类的保护剂和保护剂的不同盐浓度对存活性的影响．结果表明：在极端嗜盐菌的保藏中,一定浓度的NaCl是保护剂中不可缺少的组成成分之一,但保护剂的盐浓度没有必要达到或接近生长需要的最适盐浓度（20％—25％NaCl）;在真空冷冻干燥保藏中,用6％的海藻糖代替脱脂牛奶作为保护剂,可以得到较高的细胞存活率。77株嗜盐古细菌保藏12个月后检测,全部存活,其中19株菌保藏24个月,检测结果为全部存活．     

去甲基万古霉素菌种低温冷冻、冷干保藏条件优化及10 年菌种保藏效果

【目的】针对去甲基万古霉素产生菌不耐保藏的问题,改进菌种保藏方法,对超低温液氮保藏、-80°C低温冷冻保藏、冷干保藏方法跟踪考察10年保藏稳定性,评价不同保藏方法对去甲基万古霉素产生菌的保藏适用性。【方法】采用甘油作基础保护剂进行超低温液氮保藏和-80°C低温冷冻保藏,采用脱脂牛奶作基础保护剂进行冷干保藏,针对超低温液氮保藏进行降温速率考察,研究非渗透性冷冻保护剂海藻糖、聚乙烯吡咯烷酮(PVP)等对3种保藏方法的冻存影响,对优选出的保藏方法进行10年跟踪考察。【结果】3种保藏方法冻后菌种存活率依次为:-80°C低温冷冻保藏超低温液氮保藏冷干保藏。液氮保藏最适降温速率为快速冷冻。优选出最佳保护剂配方:超低温液氮保藏为甘油8.0%,海藻糖3.5%;-80°C低温冷冻保藏为甘油6.0%,PVP 5.0%;冷干保藏为脱脂牛奶,6.0%海藻糖。采用优化保藏条件,液氮保藏10年存活率稳定在70.6%,菌种发酵水平为入藏水平的92.9%。【结论】在优化条件下,尤以超低温液氮保藏适合于去甲基万古霉素产生菌长期保藏。     

液氮超低温冻结保藏细菌十年效果的检测

采用液氮超低温冻结保藏法保藏３９属、１２８种、１０６０株细菌。储藏１０年（部分７年）后,检测存活率为９７．８３％。     

液氮超低温冻结保藏酵母菌十年的检测报告

采用液氮超低温冻结保藏酵母菌48属129种1630株,保藏十年后检测其存活率为96．5％。     

细菌超低温冻结保藏的研究

本文报道10属19种19株细菌超低温冻结保藏试验的结果。从细胞存活率看,冻结保藏8个月,10％甘油、10%二甲基亚砜保护剂保藏效果优于蒸馏水作保护剂,少数菌株三种保护剂保藏效果相近。快速冻结与慢速冻结对细咆存活率影响不显著。恶臭醋杆菌混浊变种(Acelobacter rancens var. turbidans) AS 1.41,产氨短杆菌 (Brevibacterium ammoniagenes) AS1．844,细胞悬液浓度大,细胞存活率有增高趋势。电镜观查,超低温冻结细胞死亡率高的产气气杆菌(Aerobacter aerogenes) AS 1.489有胞壁破裂、胞质溢出现象,是细胞死亡原因之一。冻结融化后直接测定,植物乳杆菌(Lactobacillus plantarum) AS 1.557乳酸生成力下降3．4—13.8％,溶壁小球菌(Micrococcus lysodeikticus) AS 1.634对溶菌酶敏感性下降14—23％。冻结融化后移接2代测定,钝齿棒杆菌(Corynebactertum crenatum) AS 1.998 产 L-异亮氨酸,大肠埃希氏菌(Escherichia coli) AS 1.76产青霉素酰化酶酶活力,铜绿假单胞菌(Pseudomonas aeruginosa) AS 1．647产2-酮基-L-古龙酸,植物乳杆菌产乳酸均与冻结前相近。     

雨生红球藻FACHB-712营养细胞和厚壁孢子低温保藏效果及优化

以高产虾青素的雨生红球藻（Haematococcus pluvialis Flotow）FACHB-712藻株为材料,研究2种细胞形态（营养细胞和厚壁孢子）在低温保藏下的复苏率及其差异原因。结果显示,采用两步法（先预冻降温后再投入液氮中）冻存其营养细胞,在不同冻存条件下,其存活率均低于5%,以10%甘油作为保护剂、冻存速率为0.5℃/min、预冻温度为-40℃、保留30 min,然后再投入液氮罐（-196℃）中保藏,其存活率可达到13.3%。采用两步法冻存厚壁孢子,其复苏存活率高达66.13%,复苏萌发后细胞的生长特性、虾青素含量与液氮保藏前无明显差异（P > 0.05）。对液氮保藏前后藻细胞形态和超微结构观察结果表明,超低温保藏后,营养细胞的结构受到较大损伤,而厚壁孢子受到的损伤相对较小。当添加不同保护剂后,直接将厚壁孢子分别冻存在-20℃、-80℃低温及液氮中,发现-80℃低温冻存处理组的复苏存活率相对较高,可达27%。研究表明采用两步法先预冻降温后再投入液氮中冻存厚壁孢子,是长期保藏雨生红球藻FACHB-712的最佳方法,也可采用一步法将厚壁孢子冻存于-80℃冰箱中。     

基于电容测定的罗汉果细胞超低温保藏快速方法

建立了一种基于活细胞电容值定量测定的植物细胞超低温保藏的快速评价方法,优化了罗汉果细胞超低温保藏方法。通过采用活细胞传感仪测定冻存后细胞的存活率并结合细胞生活力(细胞线粒体活性/TTC)对罗汉果细胞的低温保藏过程进行优化,确定了罗汉果细胞较为适宜的冷冻保护剂组分为基本培养基中添加10%的蔗糖和10%的DMSO。预处理剂的考察实验表明,采用0.2 mol/L蔗糖的预处理剂处理细胞时冻存后细胞存活率和细胞活力较高;采用0.2 mol/L蔗糖预处理剂处理细胞时,随着预处理时间的增加,细胞存活率先增加后降低,预处理时间为9 h时,细胞存活率和细胞活力最高。保藏后的细胞复苏实验结果表明:细胞存活率与采用活细胞电容值得到的细胞存活率具有很好的一致性,同时经过冻存的细胞复苏培养后,仍保留了原始细胞的形态和合成甜苷V的特性,说明该冻存方法适用于罗汉果细胞的超低温保藏。因此基于活细胞传感仪测得的电容值进行细胞冻存过程细胞活性的快速评价方法具有较好的可行性和可靠性。     

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。     

菌种保藏方法对裂殖壶菌生长及发酵性能的影响

考察保护剂、保藏温度及预冷冻方法对Schizochytrium sp.HX-308菌种存活率及发酵性能保持的影响。结果显示:在-80℃低温保藏6个月后,渗透性保护剂的细胞存活率均比非渗透性保护剂高了5%,其中用60%(质量分数)海藻糖的保护剂最终的株细胞存活率达到80.02%,明显优于其他保护剂。采用液氮-196℃保藏菌种(两步预冷冻法、60%海藻糖保护剂),存储6个月后存活率高达90.70%,生物量、油产量和二十二碳六烯酸(DHA)产量分别达到了61.65、26.41和11.10 g/L,为最优的保藏方法,为裂殖壶菌的实验室研究及工业化生产提供了一种长期安全的保藏法。     

酒酒球菌液氮超低温保存

【目地】为安全、长期的保藏酒酒球菌,本文研究了菌体生长时间、冷冻方法、解冻温度、菌密度以及保护剂等对酒酒球菌细胞冷冻存活率的影响,找到最优液氮超低温保存方法。【方法】采用平板计数法测定冷冻存活率。【结果】实验结果表明酒酒球菌的最佳保存方法为:首先在稳定期前期离心收集菌体;其次加入保护剂(20 g/L酵母浸提物,40V/V甘油,20 g/L蔗糖,30 g/L谷氨酸钠)稀释菌体,使菌密度为109CFU/mL;然后直接投入液氮冷冻;最后在37℃温水浴中迅速解冻。保存6个月后,其中21株酒酒球菌的冷冻存活率达到99%以上。【结论】初步研究表明酵母浸提物,甘油,蔗糖,谷氨酸钠复合保护剂对酒酒球菌的保护效果较好,液氮超低温保存可用于酒酒球菌的长期保存。     

Use of a protective solution in the inoculation of bacterial cultures

The influence of the composition of solutions used for growing the bacterial cultures on the survival of microorganisms has been studied. The use of a protective solution, consisting of sodium chloride, orthophosphates, magnium sulfate, and gelatin, for suspensions of bacterial cultures has provided a higher isolation rate of enteric microorganisms, vs. cultivation with 0.9% sodium chloride solution. In evaluating the sensitivity of thioglycol medium test cultures are recommended to be inoculated with the above protective solution.     

Localization of α-Galactosidase in an Alkalophilic Strain of Micrococcus†

Localization of α-galactosidase in an alkalophilic strain of Micrococcus was investigated in relation to the cell membrane as a permeability barrier. The most α-galactosidase appered to be intracellular; only about 4% of α-galactosidase was released by lysozyme or freeze-thaw treatments of the whole cells. The enzyme activity was not inhibited by treatment of the whole cells with diazo-7-amino-1,3-naphthalene disulfonic acid (NDS) which penetrated the cell wall but not the cytoplasmic membrane. The enzyme activity of the whole cells increased about four-fold by toluene-acetone treatment which caused an alteration in the membrane permeability. The enzyme in such cells became to be relatively sensitive to pH. These results showed that cell membrane played a protective role as a permeability barrier against alkaline environment.     

Buffering capacity of bacilli that grow at different pH ranges.

Cytoplasmic buffering capacities and buffering by whole cells were examined in six bacterial species: Bacillus acidocaldarius, Bacillus stearothermophilus, Escherichia coli, Bacillus subtilis, Bacillus alcalophilus, and Bacillus firmus RAB. Acid-base titrations were conducted on whole cells and cells permeabilized with Triton X-100 or n-butanol. In all of the species examined, the buffering capacity of intact cells was generally a significant proportion of the total buffering capacity, but the magnitude of the buffering capacity varied from species to species. Over the entire range of pH values from 4 to 9.5, B. subtilis exhibited a cytoplasmic buffering capacity that was much higher than that of B. stearothermophilus, B. acidocaldarius, or E. coli. The latter three species had comparable cytoplasmic buffering capacities at pH 4 to 9.5, as long as optimal conditions for cell permeabilization were employed. All of the nonalkalophiles exhibited a decrease in cytoplasmic buffering capacity as the external pH increased from pH 5 to 7. At alkaline pH values, the two thermophiles in the study had particularly low cytoplasmic buffering capacities, and the two alkalophilic bacteria had appreciably higher cytoplasmic buffering capacities than any of the other species studied. Cytoplasmic buffering capacities as high as 1,100 nmol of H+ per pH unit per mg of protein were observed in alkalophilic B. firmus RAB. Since previous studies have shown that immediate cytoplasmic alkalinization occurs upon loss of the active mechanisms for pH homeostasis in the alkalophiles, the very high buffering capacities apparently offer no global protection of internal pH. Perhaps, the high buffering capacities reflect protective mechanisms for specific macromolecules or process rather than part of the mechanisms for bulk pH homeostasis.     

High motility reduces grazing mortality of planktonic bacteria

We tested the impact of bacterial swimming speed on the survival of planktonic bacteria in the presence of protozoan grazers. Grazing experiments with three common bacterivorous nanoflagellates revealed low clearance rates for highly motile bacteria. High-resolution video microscopy demonstrated that the number of predator-prey contacts increased with bacterial swimming speed, but ingestion rates dropped at speeds of >25 microm s(-1) as a result of handling problems with highly motile cells. Comparative studies of a moderately motile strain (<25 microm s(-1)) and a highly motile strain (>45 microm s(-1)) further revealed changes in the bacterial swimming speed distribution due to speed-selective flagellate grazing. Better long-term survival of the highly motile strain was indicated by fourfold-higher bacterial numbers in the presence of grazing compared to the moderately motile strain. Putative constraints of maintaining high swimming speeds were tested at high growth rates and under starvation with the following results: (i) for two out of three strains increased growth rate resulted in larger and slower bacterial cells, and (ii) starved cells became smaller but maintained their swimming speeds. Combined data sets for bacterial swimming speed and cell size revealed highest grazing losses for moderately motile bacteria with a cell size between 0.2 and 0.4 microm(3). Grazing mortality was lowest for cells of >0.5 microm(3) and small, highly motile bacteria. Survival efficiencies of >95% for the ultramicrobacterial isolate CP-1 (< or =0.1 microm(3), >50 microm s(-1)) illustrated the combined protective action of small cell size and high motility. Our findings suggest that motility has an important adaptive function in the survival of planktonic bacteria during protozoan grazing.     

Specific inhibition of the Na(+)-driven flagellar motors of alkalophilic Bacillus strains by the amiloride analog phenamil.

Amiloride, a specific inhibitor for the Na(+)-driven flagellar motors of alkalophilic Bacillus strains, was found to cause growth inhibition; therefore, the use of amiloride for the isolation of motility mutants was difficult. On the other hand, phenamil, an amiloride analog, inhibited motor rotation without affecting cell growth. A concentration of 50 microM phenamil completely inhibited the motility of strain RA-1 but showed no effect on the membrane potential, the intracellular pH, or Na(+)-coupled amino acid transport, which was consistent with the fact that there was no effect on cell growth. Kinetic analysis of the inhibition of motility by phenamil indicated that the inhibition was noncompetitive with Na+ in the medium. A motility mutant was isolated as a swarmer on a swarm agar plate containing 50 microM phenamil. The motility of the mutant showed an increased resistance to phenamil but normal sensitivity to amiloride. These results suggest that phenamil and amiloride interact at different sites on the motor. By examining various bacterial species, phenamil was found to be a specific and potent inhibitor for the Na(+)-driven flaggellar motors not only in various strains of alkalophilic Bacillus spp. but also in a marine Vibrio sp.     

The protective properties of myelopeptides in the development of infectious processes caused by bacteria of the genus Salmonella

The protective properties of myelopeptides in the development of bacterial infection in mice and young pigs, caused by S. typhimurium 415, S. cholerae-suis 1422 and 370, have been studied. Myelopeptides have been found to possess protective properties when injected into animals infected with S. typhimurium and S. cholerae-suis in lethal doses. The best protective effect (survival rate of 100%) has been achieved by the injection of myelopeptides 24 hours before challenge. Myelopeptides have also been found to promote the weight gain of young pigs infected with S. cholerae-suis.     

Use of alginate and cryo-protective sugars to improve the viability of lactic acid bacteria after freezing and freeze-drying

Summary In the present paper, the effect of cryo-protective sugars on the survival rate of different strains of Lactic Acid Bacteria (LAB, Lactobacillus acidophilus, Lactobacillus delbrueckii subspbulgaricus, Streptococcus salivarius subsp.thermophilus), after freezing or freeze-drying procedures, was compared. The cells were incubated at 4 °C in 32% final concentration sugar solutions (trehalose, maltose, sucrose, glucose and lactose), and viability was evaluated by the enumeration of colony-forming units. All sugars tested showed a protective effect on cell viability as compared to isotonic solution, especially after freeze-drying procedures (log c.f.u./ml ranging between 1.16 and 2.08, P < 0.001). Furthermore, the resistance to different stress agents (lysozyme, pepsin, bile salts) was estimated. Trehalose was the most effective sugar in preserving bacterial viability [% (log c.f.u. trehalose/log c.f.u. isotonic solution) ranging between 124 and 175, P < 0.001] although each strain showed a different sensitivity. Finally, the protective effect of immobilization of LAB in Ca-alginate beads was compared to that exercised by trehalose. The immobilization induced a good survival rate but lower as compared to the trehalose effect, mainly after freeze-drying in the presence of the selective agents [% (log c.f.u. alginate/log c.f.u. trehalose ranging between 81.1 and 94.5, P < 0.0001]. The protective effect of trehalose was evident in particular for Lactobacillus delbrueckii subsp. bulgaricus in presence of lysozyme. Therefore, because of its chemical inertness and low cost, trehalose could be easily utilized as excellent bacterial preservative, both to improve the viability of starter cultures and to obtain probiotic formulations more resistant to a variety of stressful conditions.     

Na+ modulates the K+ permeability and the membrane potential of alkalophilic Bacillus

In the absence of Na+ in the medium, the membrane potential of obligately alkalophilic Bacillus cells was found to be decreased by the addition of K+ to the medium, whereas K+ addition in the presence of Na+ had no effect. Rb+ showed essentially the same effect as K+. The decreased membrane potential was quickly restored by lowering the K+ concentration in the medium or by adding Na+ or Li+ to the medium. Thus, in the absence of Na+, the membrane potential of alkalophilic Bacillus seems to be affected by the concentration difference of K+ between inside and outside of the cell, and Na+ or Li+ in the medium suppresses the K+ effect. An exchange between extracellular Rb+ and intracellular K+ was observed in the absence of Na+. However, the exchange was greatly suppressed by the addition of Na+ or Li+ to the medium, indicating that Na+ in the medium modulates the K+ permeability of the alkalophilic Bacillus cell membrane. The K+-induced decrease in the membrane potential of alkalophilic Bacillus in the absence of Na+ is accounted for by the increased K+-permeability of the cell membrane.     

Feasibility of prophylaxis and therapy against Gram-negative infections by human monoclonal antibodies

Abstract In a murine model of Gram-negative sepsis, we have shown that the prophylactic application of human monoclonal antibodies (HmAbs) with specificity for lipopolysaccharides (LPS) of Pseudomonas aeruginosa protected against bacterial infection. In this paper we show that the therapeutical application of 5 μg of these HmAbs up to 6 h after challenge with a lethal dose of live P. aeruginosa results in a protection rate of 70–90%. Administration 18 h after bacterial challenge, diminished the protection to 43% survival rate. Furthermore, using a mixture of HmAbs recognizing a total of six different P. aeruginosa serotypes, no interference in their protective capacities was found. Finally, these HmAbs also protected galactosamine-sensitized mice against lethal challenge with LPS. Our data show that the described HmAbs confer bactericidal activity as well as anti-endotoxic activity in vivo.