248 BACTERIA IN RELATION TO PLANT DISEASES. 



Liquefaction begins irregularly in the upper layer mostly after 3 to 5 weeks, and slowly continues. 

 Consequently the variability is very great, just as is the browning. Entire liquefaction in 3 weeks 

 occurs very rarely even with weak gelatin, 4 weeks is rapid, and 2 and 2.5 months the rule, and under 

 paraffin oil more than 3.5 months are required. The melting-point of the gelatin exercises a great 

 influence. Always the gelatin with the highest melting-point liquefied most slowly (Non plus ultra 

 of Von Gehe & Co., Dresden), and this stained black, while other gelatins were not colored or only 

 became brown. These tests were made on 58 stab cultures from 15 isolations out of tobacco, 3 stab- 

 cultures from an Acalypha stem, and 2 cultures from an Ageralum stem. In Uyeda's gelatin-cultures 

 liquefaction with B. solanacearum was less rapid than with B. nicotianae, i. e., 5 to 6 weeks in place of 

 within less than 2 weeks. Because the rapidity of liquefaction is dependent on the melting-point and 

 on the alkalinity of the gelatin, I can not say that I have found any great difference between the 

 behavior of the bacteria investigated by Smith and by Uyeda. Moreover, not only are the different 

 Deli strains unlike in their behavior on gelatin, but there are also distinct differences between sister 

 cultures of the same strain. Because the time of liquefaction is three times as long for some of the 

 Deli cultures as for others, the liquefaction difference between B. nicotianae and B. solanacearum 

 pointed out by Uyeda is of importance only in case it is constant. 



A gar. In the beginning I obtained no blackening of agar-cultures. Later some cultures were 

 obtained which darkened agar. 



Potato. As for Smith's organism and Uyeda's, the Deli bacteria blacken slices of potato after 

 about a week. They become, for the most part, violet-black and not gray-black. Sometimes the 4 

 per cent glycerin solution, above which the potato rested on a short glass rod, was colored brown. 



Milk.- In Migula it is stated of B. solanacearum: "Milk becomes soapy, strongly alkaline, the 

 casein is not precipitated. " In his preliminary communication Uyeda says of B. nicotianae: "Sapo- 

 nifies milk," but in his complete work: "Milk first coagulated, but the coagulum is gradually dis- 

 solved and peptonized. " These two statements do not agree. 



The 20 isolations first investigated behaved for the most part alike, but different from the Ameri- 

 can and Japanese bacteria. Of 95 cultures from 14 isolations out of tobacco, 3 out of Acalypha, 2 out 

 of Ageralum, and i out of Physalis, all remained nearly unchanged, there was no precipitation of the 

 casein, but also not much alkali was produced [clearly, or the milk would have become translucent as 

 I have described, but perhaps the cultures were not held long enough. They should have been kept 

 under observation for at least 8 or 10 weeks]. To litmus the cultures remained feebly alkaline, and 

 were not distinctly acid to phenolphthalein. Only one isolation showed in 4 of the 6 cultures after 7 

 weeks, precipitation of the casein with weak acidity. 



Noticeable for the Deli isolations is the behavior in litmus milk. In 7 isolations [which are men- 

 tioned specifically] each one of the 20 cultures rendered the milk alkaline after 3 days, which reaction 

 progressed gradually. After 5 days each one was entirely alkaline. But two days later 16 of the 20 

 cultures had begun to form acid, first a small edge which became gradually broader, until finally the 

 whole column was colored red and the milk solid. [If this may be taken as the typical action of the 

 Deli tobacco organism and not the effect of undetected intruding saprophytes, or other non-compara- 

 ble circumstances, then we may conclude that it is not the same as Bact. solanacearum.] 



Neither in "Natura Milch, " from Bosch & Co., Waren, Mecklenburg, nor in boiled fresh Medan 

 milk was any chocolate-brown color observed, at room-temperature or at 36 C. [see pi. 41, fig. 3]. 



Cultures were also made in other milks, with similar results. From his table (p. 229) it would 

 appear that most were alkaline at first, and then acid with coagulation. A few remained alkaline, 

 and one of these was an isolation which soon lost its virulence. 



The results in milk may be summarized as follows: 



(1) The Deli isolations coagulate and peptonize as a rule the milk of the zebu (except 4 cultures of i isolation of the 

 95 from 20 sources) just as Uyeda's B. nicotianae. 



(2) With sterilized European milk all the isolations show in the beginning an alkaline reaction, just as B. sola- 

 nacearum Smith (concerning the strength of the reaction without figures nothing can be judged). The term "soapy" 

 I do not like. 



(3) Some (weakened?) isolations do not proceed any farther, the most begin afterward to ferment the sugar to 

 form acid and to throw down the casein. This is just the opposite of what Uyeda states for B. solanacearum [and Smith 

 also]. 



Conclusion. Because the Deli isolations have given three different results depending on the age 

 of the cultures and on the kind of milk used, the possibility is not excluded that Uyeda actually had 

 another bacterium, but it is just as likely that if he had worked with "Nutricia, " "Landbouw," 

 "Natura," or "Milkmaid" milk, he would have obtained the same results. Taken in connection 

 with the numerous other points of agreement this is very probable [see pp. 263, 264]. 



Need of Oxygen. This is plainly shown by the much slower growth of cultures under paraffin oil, 

 by the form of the gelatin-stab-cultures, by the slow clouding of the closed end of fermentation tubes 

 in comparison with the open end, and by its slow growth in an atmosphere of hydrogen in a Botkin's 

 apparatus. 



