DIPHTHEROID ORGANISMS 39 



Experiments with Mellon Strain Cultivated on Blood Medium. This culture, 

 obtained directly from Dr. Mellon, was morphologically like a typical diph- 

 theroid containing barred, granular, split and clubbed forms, and was relatively 

 free from very minute ovoid bacteria, so far as could be revealed by a routine 

 smear examination. The strain had been cultivated on blood agar for about 

 1 year and showed well-developed adaptability to this medium. 



STUDY WITH PURE LIXES OF C. ENZYMICUS BACILLARY TYPE 



Sheep blood dextrose agar plates were smeared with a platinum loop lightly 

 inoculated with the original culture. After 24 hours' incubation at 37 C. single 

 colonies were taken for seeding a second series of plates. This process was 

 repeated for 10 generations, using in each instance but 1 colony for the 

 subculture. A single colony, when examined in a smear, always snowed the 

 typical bacillary appearance, yet was not "pure" insofar as type was con- 

 cerned. From 40 to SO separate colonies selected from each generation were 

 studied microscopically and invariably ovoid and very small coccoid forms 

 could be found in goodly numbers. By carefully examining numerous fields 

 the interesting fact is revealed that a small diplococcus or coccus type is 

 present. Painstaking study of very many microscopic fields is necessary before 

 passing judgment on the presence or absence of these types, but they can be 

 found none the less. Whether or not these diplococci are appearances caused 

 by unusually short ovoid or coccoid forms, I cannot say, since the line of 

 demarkation between these types is not marked, but, as seen, they were dis- 

 tinctly spherical and appeared in pairs. Although the contrast was sufficiently 

 striking with a magnification of about 1,500 diameters, a high power Jens with 

 a magnification of 2,500 dispelled any doubt. Where two such organisms were 

 united, one could distinguish the point of union from that which is observed 

 in coccoid and small rod-forms. With these observations to go by, Mellon's 

 contention regarding his bacillary culture becomes untenable, and it is perhaps 

 inaccurate to say that "a culture of the C. enzymicus was prepared from a 

 single colony whose antecedents had shown no diplococci." The9retically as 

 well as practically the culture is absolutely pure but we are not justified, under 

 any circumstances, in assuming that a single biotype can be so obtained. It is 

 not that it is difficult to obtain the bacillary form from the coccus, but 

 that it is difficult, if not impossible, to get the bacillus without the coccus. 

 Agar" slants, inoculated with single colonies from various generations plated out 

 according to the technic described, always contained cocci. 



"Transition" of Bacillus to Coccus. The technic of Mellon was repeated 

 carefully in all details, Meat infusion veal glucose broth 1.6 to phenolphthalein 

 was enriched with sterile rabbit serum to the amount of about 5 c.c. per liter. 

 Flasks and tubes were prepared containing 30 c.c. and 10 c.c. of the medium, 

 respectively, and tested for sterility before use. A single colony from the 

 8th generation of plates made with the bacillary types, previously described, 

 was planted in a tube of this medium, incubated at 37 C. for 24 hours and 

 then at 28 C. for 3 days. From this tube the sediment was transferred to a 

 flask of the same broth and incubated at 37 C. After different intervals 

 smears were made from supernatant fluid and sediment and examined. At 

 no time was any change to a coccus observed, and the picture did not differ 

 in any degree from that seen under ordinary conditions on agar slants. 

 Before we can affirm that the morphologic appearance represents the actual 

 mechanism of transformation, it is essential that a culture of cocci be obtained 

 and propagated for several generations from material showing this "transi- 



