370 YOUNGKEN— ON THE MYRICACEAE 



Methods 



Material from many plants of each species was macroscopically and 

 microscopically examined both in its fresh and preserved condition. All 

 of the preserved material was fixed in weak, medium and strong Flem- 

 ming's on the ground immediately after the position and nature of the 

 tubercles on the plants had been ascertained. Samples of each lot were 

 then dehydrated in gradually increasing strengths of alcohol, cleared in 

 cedar oil and xylol and imbedded in parafi&ne. Transverse, tangential, 

 longitudinal radial, and longitudinal tangential sections were then cut 

 6-10 microns thick and subsequently stained in several ways. The best 

 results were obtained with the Methylene Blue and Acid Fuchsin com- 

 bination, although satisfactory results were also obtained with a com- 

 bination of Safranin and Gentian Violet. 



The writer employed the following technique in isolating the endo- 

 phyte which produces the tubercles on M. cerijera, M. Carolinensis, M. 

 Macfarlanei, M. Gale, Comptonia asplenifoUa and probably most, if not 

 all, of these lesions on other plants of the Myricaceae. 



A tubercle cluster from a root of one of the seedHngs grown in the 

 University of Pennsylvania greenhouse was washed thoroughly with clean 

 water to remove all traces of adhering soil. It was then introduced into 

 a test tube containing 1:1000 corrosive sublimate solution for 20 seconds 

 in order to destroy any surface organisms. From this it was transferred 

 with sterile forceps to a test tube containing distilled water which had 

 previously been sterilized in the autoclave. Into this was introduced a 

 sterile scalpel and two of the tubercles were cut into small fragments. 

 These fragments were next transferred to 5 tubes of sterile slant agar by 

 means of a sterile platinum loop. The tubes containing the culture were 

 then stored in a dark closet at ordinary room temperature for several 

 weeks. All 5 cultures when examined revealed the presence of Acti- 

 nomyces rosettes, non septate thin filaments, and rods of different sizes 

 as well as coccus forms, all of which stained well by Gram's method. 

 The coccus forms are probably for the most part products of the degenera- 

 tion of the above filament.* Jordan supports this view in regard to 

 similar forms of Actinomyces found in cattle, sheep, hogs and man. 

 The Actinomyces rosettes were found to be present in the depth of the 

 agar. This shows the anaerobic nature of the organism. 



*From two of the above cultures, the writer has recently successfully grown pure 

 sub-cultures on coagulated horse serum in sealed tubes, kept at the temperature of 

 37.5'' C. 



