22 



naturp:, formation, and activities 



stration that this substance can also be 

 produced in liquid media. This is of prime 

 importance, since antibiotics must be ob- 

 tained in liquid media for large-scale produc- 

 tion. 



Since the production in liquid media is the 

 chief concern of an industrial microbiologist, 

 many large-scale screening programs bypass 

 completely the cross-streak test and inocu- 

 late their isolates directly into liquid media, 

 which are usually incubated on shaking 

 machines to furnish the aeration necessary 

 for the growth of most actinomycetes. 



The following scheme outlines the basic 

 screening procedures discussed above : 



spraying the test organism (Stansly, 1947) 

 on the surface of the plate or by flooding this 

 surface with a water-agar suspension of the 

 test organism. 



One of the disadvantages of these methods 

 was that they did not permit the testing of 

 activity against more than a single test 

 organism. Another difftculty encountered in 

 the screening of actinomycetes is that media 

 selective for the growth of these organisms 

 are often not good for antibiotic production. 



An adaptation of Lederberg and Leder- 

 berg's replica plating method was described 

 in IQoS by Lechevalier and Corke. The 

 substrate was plated out with a dilute sus- 



I. Selection of natural substrates as a potential sovirce of actinomycetes 

 II. Isolation from substrate of actinomycetes in pure cultures — 



i 



Selection ot test organisms ttj r-i j- + <-• <• i- ^ i 



. u- u 4^-1 ■ <-• ill- Confrontation ot actinomycetes and 



against which antibiotics —* ^ ^ ■ i- i ■ t- 



are desired 



test organisms on solid media 



I 

 IV. Growth of actinomycetes in li(iuid media *— 



i_ " 

 V. Determination of aiitil)iotic activity in liquid media 



Various modifications of this general 

 scheme ha^^e been made, and a number of 

 special methods have been devised. 



Special Screening Techniques 



The crowded-plate technique, used in the 

 early studies on the isolation of antibiotic- 

 producing organisms, consisted in plating 

 out hea\'y suspensions of natural substrates. 

 Organisms growing on such plates, sur- 

 rounded by a zone of inhibition, were 

 selected for further work. This method did 

 not permit the development of slow-growing 

 actinomycetes which were submerged by 

 bacterial growth before they had a chance to 

 elaborate any antiliiotic. 



Attempts have been made to difterentiate 

 at an early stage between antibiotic-produc- 

 ing organisms and nonproducers bv inoculat- 

 ing a test organism directly on plates in 

 which the substrate had been plated out. 

 This inoculation was accomplished either In' 



pension of the natural stil)strate to obtain 

 well-isolated colonies. These were trans- 

 ferred with a sterile velveteen stamp to a 

 series of plates containing media thought to 

 be favorable for the production of anti- 

 biotics. After incubation, the plates were un- 

 molded and turned upside down. The back 

 of each plate was inoculated with a test 

 organism. Colonies ha\'ing the desired 

 spectrvmi of activity could then be isolated 

 from the original plate from \A'hi('h all 

 transfers had been made. 



New techniciues have been used to detect 

 types of biological activities undetectable by 

 pre\'ious procedures (Gause, 19.58). Such 

 was the method of Alurat cf al. (1959) which 

 was designed to detect antil)iotics active 

 against intracellular bacteria. The method 

 consists in allowing brucellae to be phago- 

 cytized by guinea pig monocytes. The re- 

 maining extracellular brucellae cells are 

 killed with streptomycin. The living brucella- 



