ISOLATION. ll)i:N'ni"l('\llo\, ('ri,ll\ ATloN, AM) I'lM ;Si:in' A'IMoN 



(Mil ill tilt' suhslratc niul tlic loriii;il ion of 

 sponilnl iiin' Ixxlics. This iiKikcs il possible 

 not only io dtMnoiisti'iitc the acliinl ijrcsciicc 

 of sucli oi'gaiiisnis, hut also Io (lilTcrciit iatc 

 and rrcotiiiizc ('(>rtaiii tu'oad iiioi|)lio|o<;i('al 

 fj;r()ups. It is also possit)lt' to use this met hod 

 for llic stud\' of the criccts of \arioiis tical- 

 nuMits, siK'h as the additions of plant and 

 animal residues and hnic to the soil, response 

 of actinoniycetes to diffei-ent methods of 

 fertilization and croppiii.ti', and the relation 

 between saprophytic and plant paiasitic 

 forms to the root systems of i)laiits. 



Cholodny observed that the direct micro- 

 scopic method does not si\e so accurate a 

 picture of the abundance of actinoniycetes 

 in the soil as does the contact slide method. 

 The latter, however, has one distinct disad- 

 vantage, since it does not permit estimation 

 of the relative abundance of actinomj'cetes 

 in the soil or in other natural material, such 

 as foodstuffs, manure, or water. 



Conn obser\ed that an increase in the 

 moisture content of the soil favored a change 

 in the microbial population from that of 

 actinoniycetes and fungi to bacteria. Waks- 

 man, Umbreit, and Cordon were able to 

 record the change in the actinomycete popu- 

 lation of composts by the use of the contact 

 slide. Numerous forms were detected that 

 could not be found readily by other methods. 



For the microscopic examination of colo- 

 nies of actinomycetes, Xishimura and Ta- 

 wara used the agar-cylinder method. This 

 consists in pouring 25 ml of nutrient agar 

 into a sterile Petri dish and allowing it to 

 harden. A pre\'ioiisly sterilized cork borer, 

 about 8 mm in diameter, is plunged through 

 the agar at several sites on the plate. Each 

 agar cylinder is dug out carefully with a 

 sterile hooked wire needle. A loopful of spore 

 suspension of the culture is placed on the 

 surface of each agar cylinder. With the aid 

 of a flame-sterilized pincette, a previously 

 sterilized clean coverglass is pressed down 



oxer each cylinder so that the corners of the 

 coN'erglass ;ire e(|iiidisl ant from the center 

 ot the agar cylinder. The |)etri dish is co\-- 

 eicd and inciiliated .il "JS ( '. When the cul- 

 ture has icached the proper stage of devcl- 

 opmeiil, one of the co\'ergIasses is remo\'ed 

 from the agar cylinder with the >terili/:ed 

 pincette. The growth of the niyceliuni clings 

 to the iintlersurface of the co\-erglass, form- 

 ing a ring. The microscopic examination of 

 the periphery of this circle makes it possible 

 to observe both aerial and vegetative myce- 

 lium, in an undisturbed condition, and also 

 to follow the growth of the organisms at 

 every stage of their development. 



Permanent slides are stained and pre- 

 pared as follows. The ring of the growing 

 mycelium on the undersui'face of cox-erglass 

 is fixed by means of 2 oi- 'A drops of abs(jlute 

 methanol for 10 minutes. The preparation is 

 then washed with tap water and dried before 

 it is stained. Among the stains tested, the 

 following gave the best result: Giemsa solu- 

 tion, crystal violet, eosin, methyl violet, 

 hematoxylin, methylene blue, and carbol 

 fuchsin, the first two stains allowing a clear 

 picture of the sporulation process. Numerous 

 modifications of the staining of actinomycete 

 colonies and their previous cultivations on 

 specially prepared media or under special 

 conditions have been described. 



Among the various modifications of the 

 methods of examination of colonies of ac- 

 tinoniycetes, the cellophane procedure de- 

 serves further consideration. Erikson (1940) 

 grew cultures of actinomycetes on sterile 

 cellophane placed on agar of different com- 

 positions, l^ortions of the growth were re- 

 moved at different inter\'als. Both the sub- 

 strate and aerial mycelium could thus be 

 stained and examined. The cellophane bear- 

 ing the growth is stained for 30 minutes in 

 a 70 per cent butanol solution of Sudan IV, 

 dipped in 70 per cent ethanol, washed in wa- 

 ter, and mounted on slides. This method was 



