WILT-DISEASES OF TOBACCO. 247 



The greatest length observed after staining with earbol fuchsin and measuring without a cover 

 glass was 1.8/1. In old cultures they are shorter, i. e., 0.5/j, and then look more like cocci than rods. 



In nearly all the preparations the bacteria are single or united in pairs. Chain-form colonies, 

 reported by Uyeda in 3-months-old cultures, were seen only in glycocoll-glucose solutions, where 

 arose twisted threads of 20 or more cells, among which were swollen single cells which no longer 

 stained like the others. 



Motility. In 1 or 2 day old bouillon-cultures the bacteria often show self-motility which gradu- 

 ally ceases. Agar-cultures appear less favorable for its demonstration. Also, when freshly isolated 

 out of tobacco, the examination is mostly negative. 



Flagella-staining by Van Ermengem. More than 3 fiagella I have not seen on a free-lying bac- 

 terium, but whenever a pair lie close together there are more than 3. 



Capsules. Contrary to Uyeda, I have not been able to find any capsules, not even in agar-cultures 

 2 to 3 months old stained with earbol fuchsin or by Friedlander's method. 



Spores. According to Uyeda, B. nicotianae forms spores in cultures poor in nutrient substances, 

 but B. solanaecarum does not. Since Uyeda neglects to say how he demonstrated the spores, speak- 

 ing neither of germination nor of resistance to dry air or high temperature, and not even of staining, 

 he must still show proof (see under Polar staining). For the Deli strain, spore-staining with earbol 

 fuchsin and methylene blue yielded no positive result, not even after mordanting with chromate of 

 potash (Chroomzure Kali). 



The slight resistance of these bacteria to heat makes the existence of spores very improbable. 

 Tests were made of 5 strains grown for 16 days in glycocoll-glucose medium by exposing them for 2 

 minutes at 8o C. and then during the next 3 minutes cooling the water down to 40 C. From each 

 of the 5 cultures so treated 3 transfers were made to peptone bouillon, but all remained sterile. The 

 experiment was repeated using 4 strains grown in bouillon 66 days. These cultures were plunged 

 into water at 92 C, which in 2 minutes cooled to 83 C. and showed after 3.5 minutes a temperature 

 of 40 C. Three transfers were then made to fresh media from each of the 4 strains, but the 1 2 remained 

 sterile. The check tubes clouded in 24 hours, showing that each one of these strains was still living. 

 Finally, 15 sterile strips of filter paper (3 from each culture) were wet with fluid from 5 cultures 26 

 days old, representing 5 different strains, placed in sterile Petri-dishes, and incubated in a thermostat 

 at 36 C. for 45 hours, after which they were transferred with sterile forceps to sterile bouillon. In 

 none of the 15 transfers was there any growth. Moreover, Uyeda's contention that spores develop 

 best in exhausted media is in contradiction to the statements of Schreiber, and also those of Lehmann 

 and Osborne. These authors find bacterial sporulation proceeds better in nutrient- rich material. 



Polar Staining. Polar staining is very plain, even on material taken directly from the diseased 

 tobacco. In order to avoid confusing this with occurrence of spores, slides were fixed in the flame and 

 also with alcohol and colored with Loeffler's methylene blue, with Ziehl-Nielsen's earbol fuchsin undi- 

 luted for some seconds, and diluted (1 : 10) during a minute, and with 0.01 per cent watery methylene 

 blue solution during 5 to 30 minutes. 



The question now arises whether Uyeda mistook polar staining for endospores. 



Gram's Slain. The Deli-tobacco bacillus is Gram-negative. There were tested at the same 

 time Bacillus coli communis (negative) and Diplococcus enteritis (positive). 



PHYSIOLOGY. 



Generally 10 strains of the tobacco bacteria from different localities were investigated at the 

 same time. After a couple of weeks they were again inoculated into tobacco to keep the grade of 

 virulence and always afford comparative material. 



Bouillon (1 per cent Liebig's meat extract, 1 per cent Witte's peptone, 0.5 per cent salt, acid or 

 neutral). This is generally clouded throughout in 12 to 24 hours. Often the growth is plainly 

 strongest above, which appearance disappears after two days. Some strains then develop a pellicle 

 like Uyeda's B. nicotianae, but others behave like .Smith's B. solanaecarum, forming a pellicle first 

 after 1 or 2 weeks. 



The Deli strains also stain the fluid brown, but not all of them to the same degree. A series of 36 

 cultures (3 from each strain) showed typical differences. The 3 cultures from one strain were colored 

 brown after about 3 weeks, and those from 3 other strains became dark-brown during the same time, 

 while cultures of the 8 other strains were only a little stained. On far more than 200 bouillon-cul- 

 tures a black ring around the pellicle was never observed, but in all a gray- white precipitate. 



Gelatin. The behavior of the Deli strains on gelatin is somewhat different from that Uyeda 

 described for B. nicotianae and recalls rather that of B. solanaecarum; that is, the liquefaction proceeds 

 more slowly. Stab-cultures are funnel-form above and thread-form below, and sometimes a pellicle 

 spreads slowly over the surface. Later short projections develop along the track of the stab, and 

 liquefaction may occur in the clefts of these outgrowths. 



