36 FEEDERICK EBERSON 



bility of bacterial type comes up and it is essential that the cardinal 

 points be investigated. Two methods of attack were followed in my 

 studies with C. enzymicus : cultural and serological. 



CULTURAL STUDY OF MUTATION 



Two strains of C. enzymicus were studied. One was obtained from the 

 Museum of Natural History and had been cultivated on plain agar for several 

 months. Smears stained with Loeffler's methylene blue showed typical diph- 

 theroid bacillary forms with the usual coccoidal or short rod and extremely 

 small forms (Fig. 14). The second culture was sent by Dr. Mellon. This strain 

 had been cultivated on blood agar for about 1 year and in smear preparations 

 appeared to be practically free from coccoidal forms, showing large unevenly 

 staining bacillary types, varying in size and morphology. 



Museum Strain of C. Enzynricus. A subculture was made on plain agar 

 slants and the 24-hour growth examined. Stains were made from different 

 areas and examined with a ^ immersion, using a magnification of approxi- 

 mately 1,400. In all instances diplococci and coccilike organisms were seen 

 grouped like staphylococci and at times singly. Careful examination showed 

 clusters of bacillary forms in small numbers (Fig. 15). 



The next step was to obtain a pure culture of the coccus type. Agar plates 

 (meat infusion agar 1.0 to phenolphthalein) were streaked with a platinum 

 loop touched to oeiginal culture and several discrete colonies fished. In all 

 cases a single colony was used to prepare a second plate culture and from this 

 generation of single colonies, other plates streaked successively until the 10th 

 generation. Each generation was studied for morphology before seeding. 

 Pure coccus forms were obtained in every case (Fig. 16). No difficulty what- 

 soever was experienced in procuring a coccus form from the original culture. 

 The real hardship lay in trying to obtain the bacillary type in relatively pure 

 culture, free from cocci. 



It is a well known fact that blood and serum mediums exert a favorable 

 influence on the diphtheroid bacteria, causing the development of large clubbed 

 and barred forms. Taking advantage of this fact, and the fortunate "trans- 

 formation" effected on plain agar, it becomes possible to demonstrate the 

 mechanism of variability in diphtheroids. 



Again using the original culture of C. enzymicus I transplanted it to sheep 

 blood agar and examined the culture after 24 hours' incubation. Smears 

 showed cocci and numerous bacillary forms. When subcultured on the same 

 medium the bacillary forms had almost completely outgrown the cocci and a 

 typical diphtheroid picture was seen. As generation after generation on this 

 medium was studied, it was found that cocci disappeared, although not entirely. 

 At times it required careful examination of the microscopic field to discern 

 them among the larger barred and granular forms. A most interesting point 

 was next observed. When subculturing on sheep blood, transfers were made to 

 plain agar slants from the first generation on sheep blood medium. After 

 24 hours' incubation the agar tubes were found to contain cocci only. As 

 progressive generations on blood medium were transferred to plain agar it 

 became more and more difficult to suppress the bacillary types and to demon- 

 strate the coccus. By the use of various mediums and numerous cross sub- 

 inoculations it appeared quite clear that the selection of biotypes was dependent 

 on the environment. These details are shown in Chart 1. Certain cardinal 

 points may be observed by studying the diagram in which evidence is adduced 



