ISOl.AlloN, IDIAril'lCAI'IoN, ( TI/I'IN' A'l'loN , AM) I'K I :SI ;K\ ATlON 



\H 



(M'li l\('u;i()ii;il Ixcscaich Lalior.-ilorics of the 

 r. S. Department of Aij;ii('iiltiire. Lyopliili- 

 zatioii has been iclied upon e\clnsi\ cly, toi- 

 so\'(M"al years, tor the preseix at ion of tlie 

 ha('t(M'ia, includinii the act inoniycetes. \o 

 aetinoni>'cele was encouiUered w hicli did not 

 siif\"i\'e lyophih/.at ion. With few exceptions, 

 tlie lyophiHzed cultures lia\e icniained \iahle 

 for as l()n<i as tlie>' ha\'e l)een under ohser\'a- 

 tion, some l)eins 14 years old. 



Pridham vi al. (19o()) made a detailed 

 stuily of the different media fa\-oral)le to 

 the mainteuanee of the desirable properties 

 of actinomycete.s. A eomparison was made 

 of 500 [^trains grown on 19 different media. 

 Veast-glucose and tomato-paste oatmeal 

 agar allowed the growth of 85 to 8(3 per cent 

 of the cultures with th(^ formation of abun- 

 dant aerial mycelium. I'otato-glucose agar, 

 however, gave only 12 per cent viabilit}'. 

 Glucose-asparagine agar and synthetic 

 (known as Czapek's) agar were among the 

 worst . 



Soil has lieen found very effective in 

 maintaining actinomycete cultures. Several 

 methods are used in the preparation of the 

 soil cultures. A good, fertile soil is selected 

 and freed from roots and pebbles. One-hun- 

 dred gm portions of such soil are placed in 

 250-ml Erleiyneyer flasks. If the soil is acid, 

 1 gm of ground CaCO.3 is added. If the soil 

 is poor in organic matter, 0.25 gm of dried 

 blood or casein is added. 



Pridham ct al. (1956) described the follow- 

 ing method. About 2 gm of finely ground, 

 loamy soil is placed in each of a muiiber of 

 10- by 100-mm tubes. These are plugged 

 with cotton and sterilized four times for 30 

 minutes at 121° C, on alternate days. At the 

 end of the fourth sterilization period, tubes 

 (selected at random) are tested for sterility 

 by the addition of broth and incubation at 

 room temperature (25 to 80° C) for 1 week. 

 In no instance did growth of microorganisms 

 occur in the soil-broth suspensions. The de- 



tails of the method were lurlher descriix-d 

 ;is follow s: 



"Soil cullui-es wei-e prepared by inoculat- 

 ing 10 ml of biol h in a 25- by 150-mni culture 

 lube with the ^lowlli from a slant culture 

 of a |)arl iculai' si ivpt oni>-ces. The inoculated 

 tubes were |)lace(l on a lotary shaker and 

 incubated with shaking foi' 2 days al 28 

 to 80° C Two ml of the broth culture were 

 pipetted into a lube of sterilized soil and the 

 suspension thoroughly mixed with the tip of 

 the pijK'tte. The tubes were plugged and 

 allowed to air-dry al I'oom lemperattu'e for 

 2 to :\ weeks. During this period, growth 

 usually occurred in the soil-broth mixture, 

 appearing on the surface as a mat that was 

 often covered with abundant aerial myce- 

 lium." 



Other (experiences wer(> reported by P'rom- 

 mer (195G). 



Additional iNIethods and Media 



Numerous additional reports are found in 

 the literature on the methods for the culti- 

 vation of actinomycetes, and for the study 

 of their morphological and physiological 

 properties. Some of these methods are spe- 

 cialized in nature, as, for example, the meth- 

 ods for the isolation of antibiotic-producing 

 strains of Streptomyces (Waksman ct al., 

 1940, 1947), and the principles of the screen- 

 ing program for antibiotic-producing organ- 

 isms (Waksman and Lechevalier, 1951). 

 Additional stu(li(\s have been made by ^^dyi- 

 Xagi and Szabo (1957). The standard media 

 (Waksman, 1950) used in the growth of 

 actinomycetes, both for descriptive pur- 

 poses, and for morphological and physiologi- 

 cal investigations, will be gi\-en in \'ol. II, 

 Appendix 2. Details of staining procedures 

 of cultures of actinomycetes will be given 

 in ^'ol. ir. Appendix '■]. 



Tresner and J^ackus proposed another sim- 

 ple method for the preservation of cultures 

 of actinon\ycetes. Spores of the organisms 

 are streaked on appropriate agar media in 



