The following points highlight the three methods for culture maintenance which seems to be generally used in the fermentation industries: 1. Preservation of Culture by Drying 2. Maintenance of Cultures by Storage with Limited Metabolic Activity 3. Preservation by Lyophilization.
Method # 1. Preservation of Culture by Drying:
These are different methods of drying of cultures given below:
(a) Dried on Silica Gel:
The higher survival rate was noticed at 4°C in comparison to storage at room temperature.
(b) Dried on Soil:
About 50% of the total cells remain viable after 20 years of storage. It is observed that 92-96% cells remain viable after 4 years. lijima and Sakene (1973) developed a method of drying bacterial cultures and bacteriophage under a vacuum at 2 to 5°C where a cotton plug acts as a dessicant to remove the water from the cells and the cells are more gently treated than in the lyophilization process. A some what similar method found useful with yeast after adding CaCO3 to the suspension and allow it to dry on the powder.
Method # 2. Maintenance of Cultures by Storage with Limited Metabolic Activity:
(a) Storage on Agar Slants:
Recent studies have suggested that storage under oil for 10 months did not change carbohydrate assimilation pattern for Mucor racemosus, Cunmghamella echinuata, Penicillium cyclopium and Aspergillus niger. Elliot (1975) found 95% viability when above microbes stored under oil for 1 year and when stored for 2 years, viability was 79%.
(b) Storage of Spores in Water:
Long term viability has been noted when spores of various fungi suspended in sterile distilled water and stored in a refrigerator. Similar success has been obtained with the bioassay organisms such as Saccharomyces cerevisiae and Sarcinia lutea suspended in weak buffer and stored in a refrigerator for more than a year.
(c) Storage at Frozen Temperature:
Yamasato (1973) studied the viability of 259 strains belonging to 32 genera suspended in 10% glycerol and stored at -53°C for 16 months. About 10% of the Gram positive bacteria and 3% of the Gram-negative bacteria lost viability quickly. Honey was suggested as a better adjuvant for frozen storage than glycerol.
Preservation by storage of cells or spore suspension in liquid nitrogen has been widely used since the initial advantages described by Sokolski (1984). Daily and Higgens (1973) reported the inclusion of 10% glycerol with 5% of either lactose, maltose, or raffinose in the suspending solutions increased the viability of spores, vegetative cells and Streptomyces mycelial fragments.
Moore (1975) observed the survival of plant pathogenic bacteria, was enhanced by suspending the cells in 10% skim milk prior to freezing and storage. The brewing yeasts have been difficult to maintain by lyophilization.
They were successful when the cell suspension was mixed with 10% glycerol and frozen at 1°C/min stored at -196°C. Similar success was noted in case of Thiobacillus ferrooxidans.
On the other hand, Lactobacillus bulgaricus concentrated cell suspensions lost viability after storage in liquid nitrogen and the addition of known cryoprotective agents to cell suspensions of the labile strains before freezing provided little or no protection. They also found that supplementing the growth medium with Tween 80 improved the storage stability of all strains. Fatty acid composition has also a direct relation with the survival of organisms.
Method # 3. Preservation by Lyophilization:
In addition to that the sterile glass ampoules are suspended in a carrier or protective agent such as sterile bovine serum or skim milk, rapidly frozen at low temperature, and dried in a high vacuum, the ampoules are then sealed and stored in a refrigerator.
If properly prepared and stored, most lyophilized cultures will remain viable for more than 10 years. In experiments with mycophages and bacteriophages, lyophilisation of suspensions in 5% sodium glutamate and 5% gelatin solution resulted in long-term survival.
E. coli phage T4 showed damaged head coats after lyophilization while the tail assembly did not damage and this may have the result of both osmotic shock and drying phase. In fact, the losses in titre of myco-bacteriophages during the lyophilisation can perhaps be related to the particle morphology, size, chloroform sensitivity, nucleic acid contents and osmotic sensitivity.
Ashwood Smith and Grant (1976) examined the incidence of mutants in E.coli and observed that single stranded DNA breaks either during or immediately after lyophilisation. It was concluded that addition of freeze-drying protective agents does not significantly affect the number of mutant cells, survival of E. coli and oxygen has no role in mutation induction.