Research Concerning Use of Long-Term Preservation Techniques for Microorganisms (original) (raw)
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Chapter 18 Collection and Preservation of Frozen Microorganisms
The storage of the different microorganisms over long periods is necessary to ensure reproducible results and continuity in research and in biomedical processes and also for commercial purposes. Effective storage means that a microorganism is maintained in a viable state free of contamination or genetic drift and must be easily restored without genotypic or phenotypic alterations to its original characteristics and properties. To this end, different techniques have been described and advances in cryopreservation technology have led to methods that allow low-temperature maintenance of a variety of cell types, minimizing the risks of genetic change and are now recommended for long-term storage of most microorganisms. This chapter summarizes the most important steps and components in the process of low-and ultra-low temperatures freezing of bacteria, parasites, yeasts and fungi, viruses, and recombinant microorganisms. Cryogenic preservation is the act of freezing and storing cells at very low temperatures. The effects of the freezing and thawing process on living cells are not fully understood: when water changes from liquid to solid state, cellular metabolism ceases and, when cells are warmed and water returns to liquid, cellular function resumes. Cell cryopreservation process remains the main method of cell preservation to date, and the high survival rates achieved by this method are of interest from both the biophysical and the practical points of view. Storing over long periods of bacterial or fungal strains, parasites, and viruses allows future research study, and it is essential for clinical, epidemiological, educational, microbiological, and commercial reasons. A special approach is required for the proper storage of recombinant microorganisms.
The analysis of viability evaluation method for microorganism cellular forms after cryopreservation
2000
SUMMARY The review of existing evaluation methods of viability of microorganisms is presented. The feasibility to apply these methods for the estimation of viability of microorganisms after cryopreservation was analysed. KEY WORDS: microorganism, cryopreservation, viability evaluation method To develop immune biological preparations, methods of diagnostics of the infectional dis-eases, infections of alive microorganisms in the environment, conducting of epidemiological studies and providing the works of microbiologi-cal productions, the collections of different mi-croorganisms [17] are used. Cryopreservation is one of the most reliable and widely applied ways of a long-term storage of microorganisms [4,13]. When creating the efficient regimens of microor-ganism cryopreservation it is necessary to imply the most distinct and adequate assessment meth-ods for microorganism viability state after a low temperature effect. More frequently the cellular forms of microorganisms are used by r...
Viability and Antagonism of Cryopreserved Lactic Acid Bacteria
HERALD OF SCIENCE OF S SEIFULLIN KAZAKH AGRO TECHNICAL RESEARCH UNIVERSITY Veterinary sciences, 2023
Preserving and advancing bioresources involving industrial microorganisms is of paramount importance for every nation. However, long-term storage of these strains often leads to diminished viability and biological activity. Thus, it is crucial to investigate the properties of cryopreserved strains stored at-80°C in a 10% glycerol solution within low-temperature refrigerators. This study aimed to comparatively analyze the viability of 129 lactic acid bacteria strains, including Lactobacillus sp.,
Applied Microbiology and Biotechnology, 2008
The effects of slow chilling (2°C min-1) and rapid chilling (2,000°C min-1) were investigated on the survival and membrane fluidity of Escherichia coli, of Bacillus subtilis, and of Saccharomyces cerevisiae. Cell death was found to be dependent on the physiological state of cell cultures and on the rate of temperature downshift. Slow temperature decrease allowed cell stabilization, whereas the rapid chilling induced an immediate loss of viability of up to more than 90 and 70% for the exponentially growing cells of E. coli and B. subtilis, respectively. To relate the results of viability with changes in membrane physical state, membrane anisotropy variation was monitored during thermal stress using the fluorescence probe 1,6diphenyl-1,3,5-hexatriene. No variation in the membrane fluidity of all the three microorganisms was found after the slow chilling. It is interesting to note that fluorescence measurements showed an irreversible rigidification of the membrane of exponentially growing cells of E. coli and B. subtilis after the instantaneous cold shock, which was not observed with S. cerevisiae. This irreversible effect of the rapid cold shock on the membrane correlated well with high rates of cell inactivation. Thus, membrane alteration seems to be the principal cause of the cold shock injury.
Cryobiology, 2001
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 NH 4 OH 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.
Journal of IMAB - Annual Proceeding (Scientific Papers), 2021
The low temperature in the storage processes can vary in the range of +4°C to -80°C and even lower, using liquid nitrogen. Depending on this, the time for which we expect the microbial culture to remain viable also changes. Agar slant culture, covered with oil, stab culture, saline suspension, glycerol and DMSO preservation, drying on silica gel, drying on soil, sterile water, lyophilization, cryopreservation, etc. are methods employed for the preservation of microorganisms. However, the choice of method to be used depends on the type of microorganism, the purpose of storage, and duration of preservation. The aim of the study is to compare storage techniques using semisolid agar and cultivation at 4°C and using BHI broth with glycerol and cultivation at -20°C. These are two commonly used and accessible methods for bacteria and yeast preservation. After performing the storage procedure for a total of 18 strains of S. aureus, E. coli and C. albicans, we re-cultivated them after two an...
NACHEVA, I. and Ts. TSVETKOV, 2007. Mathematical evaluation of factors that influence on the survivability of some prokaryotes and eukaryotes after freeze-drying. Bulg. J. Agric. Sci., 13: 341-347 The mathematical evaluation of the importance of the factors "regime of freezing" and "cryo- protective medium" on the survivability of probiotic microorganisms of prokaryotic and eukary- otic type after freeze-drying is conducted. For the first time, the cryoprotective effect of some natural polymeric compounds types, such as rubbers (complex plant oligo- and polysaccharides) is investigated. It is found that the application of investigated cryoprotectors in optimal concentrations increases the survivability of probiotic microorganisms after freeze-drying. The results obtained, are compared with analytical samples without cryoprotectors and samples including saccharose as traditional cryoprotector.
Archives of Microbiology, 2010
The relationship between membrane permeabilization and loss of viability by chilling depending on the chilling rate was investigated in two bacterial models: one Gram-positive bacterium, Lactobacillus plantarum, and one Gram-negative bacterium, Escherichia coli. Cells were cold shocked slowly (2°C/min) or rapidly (2,000°C/min) from physiological temperature to 0°C and maintained at this temperature for up to 1 week. Loss of membrane integrity was assessed by the uptake of the fluorescent dye propidium iodide (PI). Cell death was found to be strongly dependent on the rate of temperature downshift to 0°C. Prolonged incubation of cells after the chilling emphasized the effect of treatment on the cells, as the amount of cell death increased with the length of exposure to low temperature, particularly when cells were rapidly chilled.
… Stiintifice: Zootehnie si …, 2007
The aim of this work is to analyze the viability of microorganisms from Collection of Industrial Microorganisms from Faculty of Animal Science and Biotechnology -Timisoara, during freezing and thawing as part of cryopreservation technique. The stability in real time of 19 strains cryopreserved in 16% glycerol was evaluated during a 6-months period. The strains studied were: Escherichia coli, Lactobacillus acidophilus, Rhizobium meliloti, Saccharomyces cerevisiae, Aspergillus oryzae, Aspergillus niger, Trichoderma viride, Bacillus globigii, Bacillus licheniformis, and 9 strains of Bacillus subtilis. The strains cryopreserved at -20 o C and -70 o C were activated using the fast thawing protocol. A better cell recovery was achieved with the -70 o C protocol reaching an average viability for E. coli of 86,3%, comparing with 78,6% in -20 o C protocol. The cell recovery percentages for the other strains were: 92,4% for L. acidophilus, 93,9% for A.niger, 89% for A. oryzae, 86,7% for T. viride, 94,2% for R. meliloti, 82,1% for S. cerevisiae, 89,9% for B. licheniformis. Regarding the viability of genetically modified microorganisms, the values shows a good recovering after freezing and thawing, even after 180 days of cryopreservation. With the -20 o C protocol lower viability was observed due probably to the formation of eutectic mixtures and recrystalization processes.