CULTIVA'T'ION AND PRESERVATION OF PENICILLIA 79 



method." This involves the periodic transfer of spores from old slant or 

 plate cultures to new agar tubes. For most species, Czapek's solution agar 

 can be used satisfactorily. For perithecial forms it is better to use malt 

 or corn meal agar for reasons already cited (see p. G8). For a few species, 

 such as Penicillium albicans Bainier, some medium such as steep agar 

 containing abundant amino nitrogen is desirable. Transfers should be 

 made frequently enough to maintain the viability of the most short-lived 

 species. We have found from our experience that 8 to 9 months repre- 

 sents a safe, and not too tedious, interval. New tubes are inoculated in 

 triplicate. After a proper incubation period the least characteristic cul- 

 ture is discarded. The remaining two cultures are retained for pi-eserva- 

 tion, one being added to our stock collection and the other being held in a 

 reserve collection. Both collections are stored in separate refrigerators at 

 a temperature of 2° to 4°C. (fig. 20). Storage at this temperature mate- 

 rially lengthens the viability of many species. Stock cultures maintained 

 in this way should be periodically recultivated in petri dish cultures and 

 carefully examined to confirm the authenticity and typical character of 

 the individual strains. 



Lyophil Preservation: The preservation of molds in lyophil form has been 

 practiced at this Laboratory since 1941 and results obtained up to this 

 time lead us to consider it the most desirable method for preserving cul- 

 tures of the Penicillia. The techniques employed have been described 

 in detail elsewhere, by Raper and Alexander (1945a) and by Thom and 

 Raper (1945), hence need not be repeated here. It is sufficient to say that 

 the process involves the suspension of conidia in sterile blood serum or 

 some other protein-rich medium. The resulting suspension is distributed 

 into small glass tubes which are attached to a manifold and are then im- 

 mersed in a freezing bath at a temperature of —40 to — 50°C. The sus- 

 pensions are frozen instantly. Evacuation of the system by means of a 

 vacuum pump protected by a conventional water-trap is initiated and the 

 cultures are completely desiccated from the frozen state. When drj^, 

 the tubes are sealed off with a gas-oxygen torch so that each of the indi- 

 vidual preparations retains within itself the degree of vacuum present in 

 the system when the seal was made. An apparatus of the type employed 

 is shown in figure 21. Lyophil preparations are stored in a refrigerator, 

 although it is not known that this precaution is necessary. Cultures of 

 many Penicillia preserved in this way (Raper and Alexander, 1945) have 

 been tested for viability at the end of a five year period and, without ex- 

 ception, have been found to retain their cultiu-al and morphological char- 

 acteristics. Strains preserved in lyophil form appear to retain their 

 physiological and biochemical characteristics. The capacity to produce 

 penicillin was found to remain unaltered in penicillin-producing strains 



