Proceedings: Preservation of marine and fresh water algae by means of freezing and freeze-drying

Several species of marine and fresh water algae have been isolated from various habitats. Recently they were examined for their viability after freezing and freeze-drying procedures.The addition of suspending agents to algal cultures has resulted in greater viability for most of the green algae, but has shown little effect on the blue-green algae.It is considered that the preservation of algae by means of freezing and freeze-drying procedures are of great benefit as they offer the possibility of long-term preservation of viable collections, especially for patent depository for industrial applications.     

Proceedings: Preservation of Mycoplasmatales and L-phase variants in the American type culture collection by freezing and freeze-drying

The ATCC collection of Mycoplasmatales (with the exception of Thermoplasma) have been freeze-dried using a final concentration of 12% sucrose in the recommended growth media. Longevity studies show that storage at ?30 °C results in acceptable viability. Improved methods for the preservation of strains of Neissera L-phase variants are being investigated.     

Proceedings: The preservation of dermatophytes in parasitic form by freezing and freeze-drying

Preservation of M. canis infecting hair at room temperature, in the frozen state and as freeze-dried material was examined. The isolation of the fungus was equally successful from those hairs preserved at room temperature for 6 months as compared with fresh hair samples, but beyond 6 months the isolation became less successful. No successful isolations were made from hairs preserved at room temperature beyond $\text{2}\text{1}\text{2}$ years.Good recovery of M. canis infecting hair was also obtained after freezing and freeze-drying without any detectable change in morphological and functional characteristics.     

Proceedings: Preservation of bacteria by freezing at moderately low temperatures

In order to get some basic information for the development of a long-term preservation method by freezing at moderately low temperatures, the viability of 259 strains belonging to 32 genera and 135 species was measured. Cells were suspended in 10% glycerol and stored at ?53 °C for 16 months. About 93%, 88%, and 74% of aerobic bacteria gave viable cell counts higher than 10⁵/ml, 10⁶/ml, and 10⁷/ml, respectively. About 10% of gram-positives and 3% of gram-negatives gave viable cell counts lower than 10⁵/ml. There seemed to be some species—and genus—specificity with respect to viability after frozen storage and liquid paraffin-seal storage. Strains of coryneform bacteria, genera of the family Enterobacteriaceae, and the genus Pseudomonas were generally resistant. Pseudomonas putrefaciens proved to be specifically sensitive. Lactic acid bacteria were subject to sublethal injury, requiring special recovery media. Psychrophilic bacteria were very susceptible to frozen storage. All the tested strains of acetic acid bacteria survived frozen storage well both in 10% glycerol and in 10% honey at ?28 °C for 4.5 years. Honey proved to be a better adjuvant for frozen storage than glycerol. It was suggested from the results that for many kinds of bacteria, long-term preservation by freezing at moderately low temperatures might be possible when appropriate procedures are applied.     

Preservation of tissue mucins by freeze-drying and vapour fixation

Summary Histochemical studies previously undertaken showed that tissue mucins (glycoproteins) of the rat submaxillary salivary gland and other organs are preserved very satisfactorily by formaldehyde vapour treatment applied after freeze-drying of the tissue.It was thought desirable to confirm these histochemical findings by quantitative chemical data. This was performed by studying the effect of formaldehyde vapour treatment on the solubility of proteins in the freeze-dried rat submaxillary gland.Large quantities of protein (about 30 to 60 per cent of the dry weight) could be removed by aqueous extraction from the freeze-dried control samples, which had not received any formaldehyde vapour treatment, but very little protein (about 0.5 to 4 per cent of the dry weight) could be extracted from those samples which had been exposed to this vapour at 50°C for 3 hours.Each of the experiments performed confirmed this overall picture, but there were differences in the amount of protein extracted among the control samples, as well as among the formaldehyde vapour treated ones; it has been suggested that these differences were due to variations in the proportion and/or type of protein present, most probably caused by fluctuations in the content of secretory mucins.In part fulfilment for the Doctorate of Philosophy, University of London.     

Proceedings: Freezing and freeze-drying of Spirulina platensis

A species of blue-green alga, Spirulina platensis, is extremely susceptible to freezing and drying. However, young cells grown autotrophically with a high intensity of light were resistant to freeze-thawing if the rate of temperature change in the operation was in the range of 20–50 °C/min. Several amino acids, gum arabic, and gelatin were effective in protecting cells from injury caused by freezing.In the case of drying only gum arabic and gelatin could protect cells from injury. The gum arabic-plug method of freeze-drying was shown to be the most suitable method for the maintenance of viability in this alga.The freeze-thawed or freeze-dried cells grew to form abnormally long cells after a period of long lag in the first stage of transfer.     

Proceedings: A method for preservation of bacteria and bacteriophages by drying in vacuo

An efficient and practical method was established to preserve bacterial strains and bacteriophages. The method is characterized by drying without freezing and by use of a cotton wool plug (nonabsorbent) to prevent contamination. Drying conditions were examined by measuring temperature, vacuum, and residual moisture of the samples. From the measurement, it was found that the cotton wool plug acts as a buffer and a desiccant. Thus, the specimens reached optimal conditions during storage. Another point of advantage is that the temperature of the specimen during the drying procedure was 2–5 °C; therefore, the evaporation of the water is rapid and the time of completion is shorter than that during lyophilization.     

Proceedings: The phospholipid degradation and cellular death caused by freeze-thawing or freeze-drying of yeast

Freeze-thawing or freeeze-drying of yeast cells increased the amount of total lipid and phospholipid extractable to the level of the cell's total lipid contents. However, the amount of total lipid and phospholipid extractable from intact cells was usually less than half of these values.Phospholipase activity was apparent after freeze-thawing or freeze-drying of the cells, and phospholipids in the cells were decomposed to diglyceride and phosphoryl groups. Lipase activity was higher at pH 3–4.5, but at pH 6, practically no activity was noted.The cells incubated in medium at pH 6 after freeze-thawing or freeze-drying showed higher survivals than the cells incubated at pH 4.4 after the same treatments.     

Preservation by freezing of potentially probiotic strains ofLactobacillus rhamnosus

The aim of this work was to detect the best conditions to preserve by freezing potentially probiotic strains ofLactobacillus rhamnosus isolated from food. Four strains isolated from Parmigiano Reggiano cheese, the commercial strainLactobacillus GG and the type strain ATCC 7469T were used in the present study. Two different pre-incubation times (5 and 24 h), three protective media (Skim milk, Skim milk plus glucose and MRS plus glycerol) and two storage temperatures (?20 and ?80 °C) were used for a preservation period of 90 days. A sensible loss of survival of the strains was detected and the acidifying activity decreased depending on the different factors analysed. Moreover, plate counts performed in MRS plus bile salts evidenced that a considerable percentage of cells suffers damages deriving from cold. This study showed that the growth phase of the cells plays an important role for the resistance to the storage by freezing. Finally, Skim milk had the best protective action, showing the highest activity at ?80 °C.     

Proceedings: Protectants in the freeze-drying and the preservation of vaccinia virus

Purified and unpurified vaccinia virus suspension were prepared from calf dermal pulp or chorioallantoic membrane of developing hen eggs, infected with vaccinia virus strain Ikeda. For the preparation of more stable dried product, single and combined protectants composed of a mixture of sodium glutamate with soluble starch, polyvinylpyrrolidone (PVP) or sodium carboxymethyl cellulose (SCMC), were compared in the course of the freeze-drying and preservation process.Single protectants sodium glutamate or peptone were effective in the freeze-drying and the preservation of both purified and unpurificd vaccinia virus products. There was an optimal concentration of sodium glutamate to be added to the suspension for the preservation of the dried products, especially the purified products.Combined protectants were effective in the purified products when the concentration of sodium glutamate exceeded the optimum necessary for the preservation.     

Long-term preservation of mouse spermatozoa after freeze-drying and freezing without cryoprotection

The widespread production of mice with transgenes, disrupted genes and mutant genes, has strained the resources available for maintaining these mouse lines as live populations, and dependable methods for gamete and embryo preservation in these lines are needed. Here we report the results of intracytoplasmic sperm injection (ICSI) with spermatozoa freeze-dried or frozen without a cryoprotectant after storage for periods up to 1.5 years. Freeze-dried samples were stored at 4 degrees C. Samples frozen without cryoprotection were maintained at -196 degrees C. After storage, spermatozoa were injected into the oocytes by ICSI. Zygotic chromosomes and fetal development at Day 15 of gestation were examined after 0, 1, 3, 6, 9, and 12 mo of sperm storage. When fresh spermatozoa were used for ICSI, 96% of resultant zygotes contained normal chromosomes, and 58% of two-cell embryos transferred developed to normal viable fetuses. Similar results were obtained when spermatozoa were frozen without cryoprotection and then used for ICSI (87% and 45%, respectively; P > 0.05) and after 12 mo of sperm storage (mean of six endpoints examined: 87% and 52%, respectively; P > 0.05). Freeze-drying decreased the proportion of zygotes with normal karyoplates (75% vs. 96%; P < 0.001) and the proportion of embryos that developed into fetuses (35% vs. 58%; P < 0.001), but similar to freezing, there was no further deterioration during 12 mo of storage (mean of six endpoints examined: 68% and 34%, respectively; P > 0.05). Live offspring were obtained from both freeze-dried and frozen spermatozoa after storage for 1.5 yr. The results indicate that 1) the freeze-drying procedure itself causes some abnormalities in spermatozoa but freezing without cryoprotection does not and 2) long-term storage of both frozen and freeze-dried spermatozoa is not deleterious to their genetic integrity. Freezing without cryoprotection is highly successful, simple, and efficient but, like all routine sperm storage methods, requires liquid nitrogen. Liquid nitrogen is also required for freeze-drying, but sperm can then be stored at 4 degrees C and shipped at ambient temperatures. Both preservation methods are successful, but rapid freezing without cryoprotection is the preferred method for preservation of spermatozoa from mouse strains carrying unique genes and mutations.