Spin FreezeDrying
All culture work should be carried out in an appropriate microbiological safety cabinet see Note 1 . 1. Grow cultures under the optimal growth conditions for the species and on suitable media see Subheading 2.2., items 2 and 3 Note 4 . 2. Prepare a spore suspension in sterile 10 w v skimmed milk and 5 w v inositol mixture. 3. Dispense 0.2-mL aliquots of the spore suspension into the sterilized and labeled ampoules ensuring the suspension does not run down the inside of the ampoule see Note 15 ....
Sample Freezing
Regarded as the first step in the process, the formulated product must be frozen before evacuating the chamber to induce sublimation 13,14 . Freezing will 1. Immobilize the components in the solution and prevent foaming as the vacuum is applied. 2. Reduce thermal inactivation of the dispensed product. 3. Induce a specific ice-crystal structure within the frozen mass, which will facilitate or inhibit vapor migration from the drying cake. In short, the ice structure formed during freezing will...
Vitrification Methods
Much of the very early work in cryobiology, notably by Luyet 22 , had been based on the assumption that freezing damaged cells directly and, consequently, Fig. 9. Supplemented phase diagram for glycerol water. The intersection of the melting curve and the glass transition curve at Tg' indicates the lowest concentration of glycerol that, in theory, will vitrify. In practice, the lower temperatures on the melting curve are unlikely to be reached owing to the high viscosity preventing the...
References
1. Polge, C., Smith, A. U., and Parkes, S. 1949 Revival of spermatozoa after dehydration at low temperatures. Nature London 164, 166. 2. Sakai, A. 1966 Survival of plant tissues at super-low temperatures. IV Cell survival with rapid cooling and rewarming. PJant Physiol. 41, 1050-1054. 3. Mutetwa, S. M. and James, E. R. 1984 Cryopreservation of plasmodium chabaudi. II. Cooling and warming rates. Cryobiology 21, 552-558. 4. Stacey, G. N., Byrne, E., and Hawkins, J. R. in press DNA fingerprinting...
Experimental Determination of Glass TransitionCollapse Temperature
3.3.1. Determination of Glass Transition Temperature by Modulated Differential Scanning Calorimetry The principle of modulated differential scanning calorimetry and its application to developing freeze-drying conditions is described elsewhere 8 . 1. Aliquots of the sample in the intended lyophilization buffer or a range of buffers are prepared and an 80-pL aliquot is dispensed per pan and the pans crimped with an appropriate crimper. 2. The sample pan and an empty crimped reference pan are...
Encapsulation and Dehydration
Fabre and Dereuddre 19 developed this cryoprotective approach and applied it for the first time to Solanum phureja. The following method has been adapted for Ribes spp. 20-22 and as such, has been used to assist in cryop-reservation technology transfer and training programs that also incorporate PVS2 and controlled-rate freezing methodologies. It may be applied with appropriate modification and optimization to other shoot-tip systems. 1. Transfer shoot-tip meristems to 3 w v alginate solution...
Chemical Additive Vitrification
These methods are largely based on those originally developed by Uragami 9 and Sakai 11 and has since been adapted extensively by Kim et al. 17 and Thin et al. 18 to produce different derivative methodologies. These use different combinations of cryoprotectants applied at higher concentrations than would be the case for standard cryoprotection. They also include protocol permutations that incorporate pregrowth and dehydration treatments that assist the recovery of more sensitive genotypes 11 ....
Cryoprotective Dehydration
This method is adapted from those of Niino et al. 8 , Uragami 9 , Bagniol and Engelmann 15 , and Sherlock et al. 16 . 1. Remove nodal segments or shoot tips 2- 5 mm from plantlets and detach expanded leaves using a scalpel see Note 20 . 2. Culture the prepared explants in a series of media containing 0.5- 0.75 M of osmotic agent selected from sucrose, mannitol, or sorbitol prepared on standard solid medium for 1-5 days see Notes 13 and 17 . 3. Transfer plant tissues to standard medium in order...
References 1
1. Adams, G. D. J. 1995 The preservation of inocula. In Microbiological Quality Assurance A Guide Towards Relevance and Reproducibility of Inocula, Brown, M.R.W and Gilbert, P., eds. , CRC Press, London, UK, pp. 89-119. 2. Fanget, B. and Francon, A. 1996 A Varicella vaccine stable at 5 degrees C. Dev. Biol. Stand. 87, 167-171. 3. Adams, G. D. J. 1996 Lyophilization of vaccines. In Vaccine Protocols, Robinson, A., Farrar, G. H., and Wiblin, C. N., eds. , Humana Press, Totowa, NJ, pp. 167-185. 4....
Preparation Suspension in Lyoprotectant
All culture transfers should be carried out using aseptic techniques in an appropriate microbiology safety cabinet for the organisms to be preserved. It is important that all local safety requirements are adhered to. 1. Make a spore or conidia suspension in 12 w v Tyndalized skimmed milk see Note 1 . 2. Add 50-200 pL spore suspension, depending on the concentration to a borosili-cate freeze-drying ampoule see Note 2 . 3. Close the ampoule with a cotton plug. 4. Cool the propagule suspension to...
The Effect of Rate of Change of Temperature
That degree of understanding provided a starting point for the development of practical freeze-preservation techniques for a range of cells, but it soon became clear that reality was considerably more complex. First cooling rate, and then warming rate, was found to be important determinants of survival and Lovelock's theories did not account for such kinetic effects. In 1963, Mazur discovered that the rate of change of temperature was important because it controlled the transport of water...
Introduction Uvn
Yeast cultures are held in long-term storage in the National Collection of Yeast Cultures NCYC http www.ncyc.co.uk by two methods 1 freeze-dried in glass ampoules, and 2 under liquid nitrogen using glycerol as a cryoprotectant. Freeze-drying is a generally accepted method for yeast storage, having the advantages of conferring longevity and genetic stability, as well as being suitable for easy worldwide postal distribution of the cultures in glass ampoules. However, preservation by freeze-drying...
Notes
1. Lyophilization is a process that is widely used in biotechnology in a range of different situations. It may be performed on a small benchtop dryer with little process control or in a multimillion pound industrial lyophilizer whose operation is carefully monitored and controlled by computer systems and described in detailed written operating protocols. Laboratory-scale lyophilization differs from the production process commonly applied to the industrial preparation of biological biotechnology...
The Drying Cycle
For clarity it is usual to separate the drying cycle into primary drying the sublimation stage and secondary drying or desorption. The first step in the drying cycle is defined as primary drying and represents the stage where ice, which constitutes between 70 and 90 of the sample moisture, is converted into water vapor. Sublimation is a relatively efficient process although the precise length of primary drying will vary depending on the sample formulation, cake depth, and so on. During primary...
Sublimation and Drying 34
Under atmospheric conditions, liquid water is converted into vapor by warming, a process defined as evaporation. However the three states of water ice, liquid, and vapor coexist at the triple point and illustrates that at subatmospheric pressures ice can convert directly to vapor by sublimation. Ice sublimation from a frozen sample results in an open, porous, dry structure where solutes are spatially arranged as in the original solution or suspension. In contrast to evaporation, where...
Other Issues for LongTerm Storage
Over time liquid nitrogen freezers become clogged with a build up of ice sludge, which can accumulate microbial contamination from environmental sources 36 . Thus, long-term storage vessels will benefit from periodic cleaning to remove the ice sludge and it is also helpful to carefully disinfect recovered ampoules. The use of double-sealing methods for ampoules or storage boxes will also help to provide protection against microbial contamination. Serious and lethal viral cross-infection of...