316 MUTATION AND PLANT BREEDING 



isolates of an antibiotic-producing culture are grown using a suitable 

 solidified medium, and their antibiotic production measured by 

 streaking a sensitive indicator organism on the uninoculated portion 

 of the plate. Various refinements of this method have been devised, 

 the most elaborate is a "top-layering" method in which the well 

 developed colonies of the producing organism are covered with an 

 inoculum of a sensitive organism. The size of the zone of inhibition 

 of the sensitive organism around a colony of the antibiotic producer, 

 or the ratio of zone to colony size, is used as a measure of production. 

 A positive growth response may be substituted for inhibition by using 

 an antibiotic-requiring organism, such as streptomycin-dependent 

 Escherichia coli (36) or macrolide-dependent Micrococcus (64). 



While logical in theory, these rapid test methods are difficult in 

 practice. The antibiotic-producing organism must be plated in con- 

 venient number, develop at a constant rate to the same extent, posi- 

 tion of colonies on the test plates must not influence results, a medium 

 adequate for growth of both the antibiotic-producing and sensitive 

 organisms chosen, and the producing organism must be isolated free 

 of the test organism. This method has been disappointing with the 

 exception of hyphal tip isolations of Penicillia (44). Nonproducing 

 isolates can be recovered. There is usually little correlation between 

 increased inhibition zone diameter and the yield in submerged fer- 

 mentation (14). Again, as with correlations of yield and morphology, 

 one can go backwards but not forwards. Possible explanations for an 

 absence of correlation are that the test organism is inhibited by 

 factors other than the antibiotic, and that antibiotic yields obtained 

 by colonial growth on the surface of media are not responsive to the 

 same factors controlling yield in submerged fermentation. 



A popular approach to isolates with increased yield has been 

 the selection of antibiotic-resistant variants (113). An isolate produc- 

 ing a specific level of antibiotic is assumed to be resistant to this 

 level, regardless of whether the antibiotic is being produced. Isolates 

 of greater resistance are assumed to be immune by virtue of increased 

 yield. As an initial screening method for new cultures or for "degen- 

 erated" cultures, this technique is effective but fails for variants 

 resistant to greatly increased amounts of antibiotic. These highly 

 resistant isolates produce no antibiotic and are often asporogenous. 



A method based on a different rationale consists in selecting vari- 



