Lyophilization
In this process culture is dried rapidly in vacuum from the frozen state. The material is frozen by a suitable method and then dried by sublimation of ice. The sublimation is effected by exposure to an atmosphere of very low pressure (e.g.0.01 mm Hg or less) which is dried by a chemical desiccant or refrigerated condenser. The dried material is preserved in vacuo in hermatically sealed ampoules which are stored / in the dark, either at room temperature or preferably, in a refrigerator at 4-5°C. This is a suitable means of preserving stock strains of bacteria, complement and antiserum required for reference or standardization purpose. For lyophilization the following steps should be used :-
-Grow the microorganisms on culture media. A satisfactory bacterial culture medium must contain available source of the constituents as the survival of the bacteria on freeze-drying is greatly influenced by the nature of the medium in which these are suspended. Nutrient broth containing 1% peptone and meat extract is satisfactory medium for the most resistant organisms, e. g Streptococcus pyogenes and Staphylococcus aureus and the moderately resistant e.g enterobacteriaeceae and brucellae. Broth culture of these may be dried directly.
-Check the colonial morphology and purity of the cultures. It should be identified by biochemical tests if necessary to ascertain the purity of cultures.
– Single colony of the culture should be plated on culture media in order to have confluent growth of the organism in pure form.
-Harvest growth from a confluent area of growth in a suitable quantity of any of the following cryoprotective stabilizers, which are necessary for the poorly resistant organisma such as Neisseria gonorrhoeae, Vibrio cholerae and Haemophillus influenzae.
3(i) 5% sterile skimmed milk ori
1i) 30% sucrose broth (1 part) and sterile inactivated horse parts serum (4 part )
(iii) Sucrose Glutamate-Dextron stabilizer, whose composition is as follows;
Sunrise. 5.0 gm
Sodium glutamate 1.0 gm
Dextran. 5.0 gm
0.15 MNSS 100 ml
Sterilize the solution by filtration. Suspend the bacterial growth in above solution.
-Add presterized sucrose powder @ 30% w/v and dissolve.
-To 1 part of the bacterial suspension, add 4 parts of sterile inactivated horse serum.
-Dispense 0.5 ml into sterilised, pre-necked ampoules and freeze dry. Freezing must be very rapid, with the temperature lowered to well below 0°C 9e.g 0 to -20° C), since slow freezing would prolong exposure to the denaturing influence of the suspending salt solution as it was concentrated to its eutectic level by the formation of pure ice crystal.
-Store at temperature +1 to -20°C.
