26 MISCELLANEOUS PUBLICATION 9 55, U.S. DEPT. OF AGRICULTURE 



At times degeneration involves such diagnostic features of cultures 

 as the production of aerial mycelia and of spores by actinomycetes; 

 the production of dextran by leuconostocs, streptococci, and lacto- 

 bacilli; and the formation of sexual spores by molds andyeasts. The 

 pathogenicity and antigenicity of many plant and animal disease- 

 producing micro-organisms are often either lost or modified when 

 maintenance entails serial transfer. 



With certain bacteria, yeasts, and molds, oiled cultures sometimes 

 are practical for prolonging the life of stock cultures. An ordinary 

 slant culture is converted to an oiled culture by covering it with sterile 

 mineral oil. If the medium is carefully and completely immersed in 

 oil, the culture may remain intact for several months or even years. 

 Certain disadvantages and dangers of this method, however, have 

 restricted its use in many laboratories. First, oil clings to the loop, 

 hook, or needle used to remove a part of the growth and spatters when 

 the wire is flamed. Also, there is considerable danger in transferring 

 pathogenic micro-organisms, which may be disseminated by the spat- 

 tering oil. Even if these disadvantages were to be overcome, it is 

 obvious that this method is but a modification of the serial-transfer 

 refrigerator-storage method and therefore has its disadvantages also. 



The use of soil cultures is advantageous in maintaining certain 

 molds, actinomycetes, and bacteria. For this purpose fertile garden 

 soil is sterilized in test tubes by autoclaving at 121° C for an hour or 

 more. The sterile soil is then moistened with about one-third of its 

 volume of a broth culture or with a suspension of spores of actino- 

 mycetes or molds. The broth culture should contain a high propor- 

 tion of endospores in the case of aerobic or anaerobic sporeforming 

 bacteria. The resulting soil culture is then allowed to dry at room 

 temperature in the case of molds, actinomycetes, and nonsporef orming 

 bacteria. 



When the culture is composed of sporeforming bacteria, it may be 

 dried in a vacuum oven at about 45° C. This procedure is preferred 

 for Clostridia intended for use in the production of solvents, because 

 it has been found that yields are maintained best by means of spore 

 inocula. The combination of drying and heat largely destroys vege- 

 tative growth. Soil cultures may be refrigerated as are other types 

 of stock cultures. An active culture may be obtained from a soil 

 culture by transferring a loopful of soil to a tube of appropriate 

 medium and incubating it under suitable conditions. 



Soil cultures are not satisfactory for preserving either yeasts or 

 most nonsporeforming bacteria, but where appropriate they have 

 certain advantages over the other methods described. Because of the 

 dryness of soil cultures, the environment is essentially nonnutritive and 

 the cells are practically dormant. Under these circumstances there is 

 less likelihood of variation. Also, soil cultures seem to have a lon- 

 gevity that varies with the type of micro-organism, but is appreciably 

 longer than that for cultures kept as agar or broth cultures. Aerobic 

 sporeformers seem to remain viable and unchanged for several years, 

 but some anaerobic sporeformers die in less than 2 years. 



The best method for maintaining cultures of most bacteria, yeasts, 

 and molds is lyophilization or freeze drying (13) . It has the follow- 

 ing advantages. First, a culture preserved in lyophil is safe from 



