SEARCH FOR ANTIBIOTICS: SCREENING PROGRAMS 



21 



which inhibits the growth of all other micro- 

 organisms. Porter et al. (19(i0) advocate the 

 use of an arginine-glycerol agar. 



The addition of selective inhibitors per- 

 mits reduction of the number of fungi or 

 true bacteria and helps in the isolation of 

 actinomycetes in pure cultures. Corke and 

 Chase (19o6) have used with success the 

 antifungal antibiotic cycloheximide to elim- 

 inate fungal growth. Lawrence (1956) re- 

 duced the number of contaminating bacteria 

 and fungi by pretreating the samples to be 

 plated out for 10 minutes with a 1:140 

 dilution of phenol. The successful use of 

 centrifugation of suspensions of substrates 

 to be plated out, as a means of separating 

 actinomycetes from other microorganisms, 

 was reported l)y Rehacek in 19")G. The 

 suspensions were centrifuged for 20 minutes 

 in tubes which were subjected to 904 X g 

 on the surface and 1609 X g on the bottom. 

 Under such conditions most actinomycetes 

 were not sedimented, whereas most other 

 microorganisms were. 



Another method consists in increasing the 

 number of actinomycetes present in a soil 

 sample before plating out. Tsao ct al. (1960) 

 dried soil samples and then incubated them, 

 buffered with calcium carbonate, in a moist 

 atmosphere. This resulted in an increase in 

 the percentage of viable actinonwcetes in 

 these soil samples. 



For the isolation of actinomycetes in pure 

 culture, there is, of course, no universal 

 medium. The authors consider yeast extract- 

 glucose agar the best general medium for 

 this purpose. In certain countries where 

 standard preparations of yeast extract are 

 not available, a bakers' yeast-salt medium 

 is a good substitute (Lechevalier and 

 Tikhonienko, 1960). 



It is always desirable to rememljer that 

 the use of various other media may permit 

 the growth of organisms which might not 

 grow, or which might grow poorly, on yeast- 

 glucose agar. 



The media used for the actual cross-streak 



Table 1 

 Cross-sireak test of 269 actinomvcetes freshly 



isolated from soil and from manure 

 Comparison between results obtained on nu- 

 trient agar and on yeast-glucose agar. 



Mycobacterium smeg- 



matis 607 



.1/. smegmatis 105. . . 

 -1/. smegmatis 7992. . 

 .1/. phlei 23 



Percentage of total number tested 



On nutrient agar, 

 width of inhibi- 

 tion zone 



1-10 

 mm 



11-20 

 mm 



10 



11 



10 



27 



>20 

 mm 



On yeast-glucose 

 agar, width of 

 inhibition zone 



1-10 

 mm 



11-20 

 mm 



>20 

 mm 



3 



2 



19 



test must l)e favorable for growth of both 

 the actinomycete and the test organisms. 

 The ideal medium for such tests should also 

 be free from chemicals that might inhibit 

 the action of the antibiotics produced by the 

 actinomycete. The selection of such a me- 

 dium must, of course, be highly empirical. 



The influence of the medium used in the 

 cross-streak test and the results obtained are 

 illustrated in Table 1. A group of 269 freshly 

 isolated strains of actinomycetes were tested 

 for activity against four strains of fast 

 growing, nonpathogenic mycobacteria on 

 two different media, nutrient agar and yeast- 

 glucose agar. The same amount of agar (15 

 ml) was used in all plates to permit com- 

 parison of the width of inhibition zones. 

 One will note that approximately the same 

 percentage of strains formed narrow and 

 broad zones of inhibition on both media, but 

 that the percentages of medium width zones 

 were higher on nutrient agar than on yeast- 

 glucose agar. This suggests that certain 

 types of antibiotics responsible for the 

 medium width zones were not formed or 

 were inacti\'ated by yeast-glucose agar. 



Demonstration that an antagonist can 

 produce a diffusible substance efYective upon 

 the test organisms chosen in a given screen- 

 ing program must be followed bj^ demon- 



