The following points highlight the top six methods that are used for preservation of organisms. The methods are: 1. Agar Slant Culture 2. Agar Slant Culture Covered with Oil 3. Saline Suspension 4. Preservation at Very Low Temperature 5. Preservation by Drying in Vacuum 6. Lyophilization or Freeze Drying.
Method # 1. Agar Slant Culture:
Agar slants are prepared in vitro. After inoculation slants are incubated for a period of 24h and then stored in a refrigerator. These cultures require periodic transfer after six months.
Method # 2. Agar Slant Culture Covered with Oil:
The agar slants are incubated after inoculation until profuse growth appears. These are then covered with sterile mineral oil to a depth of 1 cm above the tip of the slanted surface. Transfers are made by removing a loopful of growth; touching the loop to the glass surface to drain off excess oil in the medium and then preserving the initial block culture.
Method # 3. Saline Suspension:
High concentration of sodium chloride is used as inhibitor of bacterial growth. Bacteria are suspended in 1 % salt solution in screw cap tubes to prevent evaporation. The tubes are stored at room temperature and transfers are made on agar slants.
Method # 4. Preservation at Very Low Temperature:
The organisms are suspended in a nutrient broth containing 15% glycerol, or in skimmed milk containing 7.5% glucose. The suspensions are frozen and stored at -15°C to -30°C.
The ready availability of liquid nitrogen (-196°C) has provided another means of preservation of stock cultures. In this procedure, the cultures are frozen with a protective agent (glycerol or dimethyl sulfoxide) in sealed ampules. The frozen cultures are kept in liquid nitrogen flask.
Method # 5. Preservation by Drying in Vacuum:
The organisms are dried over CaCl2 in a vacuum, then stored in the refrigerator. The organism survives longer than when air dried.
Method # 6. Lyophilization or Freeze Drying:
The microbial suspension is placed in small vials. A thin film is frozen over the inside surface of the vial by rotating it in a mixture of dry ice or alcohol or acetone at a temperature of -78°C. The vials are connected to a high vacuum line. This dries the organism while still frozen. Finally, the ampoules are sealed off in a vacuum with a small flame.
These cultures can then be stored for several years at 4°C. To revive microbial cultures, it is merely necessary to break up the vial aseptically to which suitable sterile medium is added. After incubation, growth appears which allows them for further transfer. The process permits the maintenance of a large number of cultures without variations in the characteristics of the cultures which generally reduce the danger of contamination.