EXPERIMENTS WITH THE PEACH ORGANISM. 61 



tissues for puncturing. The slime was rubbed over an internode, 

 pricks were made through it, and the wounds were rubbed again with 

 the platinum loop. Check punctures were made on the opposite side 

 of each one of these shoots, and a little higher up, or else upon twin 

 branches. The first 12 plants were inoculated from as many colo- 

 nies. The remainder were inoculated from 4 slant agar cultures 

 made December 2 from as many colonies on the same poured plate, 

 the tubes having remained in the thermostat at 30° C. for two days, 

 and the surface being covered with a copious growth. There was 

 also an abundance of cloudy fluid in the bottom of the tubes and this 

 fluid was pricked in very thoroughly. There were always a greater 

 number of check pricks than of punctures with the infected needle. 

 Usually 20 punctures were made with a sterile needle on each plant. 

 All of the punctures were near the tops of the shoots in tender tissues, 

 i. e., the ones most certain to give results. 



Result.— J smuary 14, 1908: All but 3 of these 36 plants (92 per 

 cent) yielded distinct tumors in the inoculated part. None of the 

 more than 600 check punctures on the same plants showed any ten- 

 dency to form tumors. 



May 15, 1908: Photographs were made (PL XIII, fig. 1). 



November 16, 1908: About one-third of the daisies inoculated 

 December 4 of last year are still living; the rest have died this sum- 

 mer. The largest tumors are 2 inches in diameter." 



In tliis connection the following notes on the origin and appear- 

 ance of the bacteria used for these inoculations will be of interest. 

 The crown-gall of the peach was scraped, washed, and the denuded 

 surface further deadened by plunging into alcohol and then for five 

 minutes in 1:1,000 mercuric chloride water. The interior of the 

 knot was then entered b}" means of a sterile scalpel and scraped into 

 sterile bouillon, from which the poured plates were then made. The 

 scrapings fi*om this tumor were thrown in considerable quantity into 

 three different tubes of bouillon. Plates were poured from each 

 one of these and also from bouillon dilutions of the same. The 

 tubes which received the scrapings were marked Aj, Bj, Cj, and the 

 dilutions were marked Aj, Bj, Cj. Apparently, not a great many 

 living organisms were in the knot, and the dilutions did not yield 

 satisfactory plates. The number of bacteria in the plates appeared 

 to bear a constant relation to the amount of infectious material put 

 in, i. e., those plates which were sown thickest gave the most colonies. 

 The plates were incubated at room temperatures varj'ing from 20° 

 to 23° C. The plates were poured b}^ Miss Florence Hedges, using 

 + 15 peptonized beef bouillon containing 1 per cent of agar. The 



a The largest natural tumor observed on the daisy was on a root and measured 4J by G by 3 inches. 

 Tourney figures much larger ones from the almond. (See also poplar, PI. XXIII.) 

 213 



