PRESERVATION OF CULTURES 51 



A gar-Slant Method 



The method most generally employed for maintaining mold cultures, 

 and the one which has been successfully used by the writers for many years, 

 may be termed the agar-slant method. This involves the periodic transfer 

 of spores from old agar slants, or plate cultures, to new agar tubes. The 

 composition of the substratum is varied to suit particular requirements and 

 groups of organisms. In our work we regularly employ the Czapek's 

 solution agar. Few, if any, strains make their maximum growth on this 

 medium, but it has been our experience that they maintain exceptionally 

 well any characteristic morphological or physiological features which may 

 characterize them. It is necessary to know the expected viability of all 

 cultures to be maintained, and to gauge the intervals of transfer accordingly. 

 With the Aspergilli, transfer every 8 to 9 months is sufficiently frequent for 

 all species, with the possible exceptions of A. citrisporns and A. itaconicus, 

 and a period of one year is not too long for most forms. In practice it is 

 advisable to handle separately the few very short-lived species, and to set 

 the regular period of transfer well within the known viability period of the 

 remaining forms. Transfer of the general collection at least once each year 

 insures a complete survey, within the yearly period, of all strains main- 

 tained. New tubes are inoculated at least in duplicate, while triplicate 

 preparations afford a desirable margin of safety. The new cultures are 

 incubated for 2 to 3 weeks, or until a good crop of spores has developed. 

 Incubation at room temperature is suitable for most of the Aspergilli, 

 although a few forms such as the large-spored members of the A. glaucus 

 group sporulate more abundantly at 20° C. The correctness of the cultures 

 is then checked with a wide-field binocular or a 10 X pocket magnifier, the 

 plugs are poisoned to preclude any possibility of subsequent contamination, 

 and selected tubes are placed in storage. Cultures can be stored for 

 reasonable periods at room temperature; certain species will remain viable 

 for many years at 24° to 26° C. The viability of most species is materially 

 lengthened and the possibility of progressive variation reduced by storage 

 at 2° to 4° C. (i.e., above any danger of actual freezing but sufficiently low r 

 to prohibit further growth and possible dissociation). Tubes of any de- 

 sired size may be employed. We have found lipless tubes 15 by 125 mm. 

 to be quite satisfactory since they provide adequate culture surface and at 

 the same time require much less storage space than the larger tubes com- 

 monly in use. Each culture should be maintained at least in duplicate, 

 with the different tubes of each pair stored in separate refrigerators. With 

 the accidents and failures of refrigeration, the possible escape of toxic gases, 

 or the possible ingress of contaminations that escape the usual inspection, 

 the maintenance of not less than two complete series of strains is a necessary 

 precaution. If natural conditions, such as temperature and relative hu- 



