WILT-DISEASES OF TOBACCO. 249 



Reduction of Melhylene Blue. In 92 bouillon-cultures from 12 isolations, containing 0.002 per 

 cent methylene blue, 9 were bleached after i day, 31 after 2 days, 71 after 3 days, 85 after 5 days, and 

 87 after 14 days, except the uppermost 3 to 4 mm. The bleaching took place after i day in glucose 

 bouillon (3 cultures from each of 3 isolations out of tobacco, Mucuna, and Blumea). 



Reduction of Litmus in .\filk. Litmus is also wholly or partly reduced [see p. 264]. 



Reduction of Sodium Selenite. To obtain reductions the quantity of sodium selenite (NajSeOj) 

 used must be small. Growth and reduction was obtained with all strains in 2 days, using o.oi per 

 cent in peptone bouillon agar, but no growth where o. i per cent was used. 



Reduction of Nitrate. Nitrate is reduced when the Deli organism is grown in presence of glucose 

 or saccharose, but not when starch is used as the carbon food, as starch is not assimilable by this 

 organism. 



Diastase Production. As mentioned above, starch is not acted upon. In this respect the organ- 

 ism agrees with Smith's statements. 



Tyrosinase. Each one of 8 isolations grew using tyrosin as carbon-nitrogen food, but less 

 well than in peptone-water. None of the 34 cultures were actually blackened. "When one adds i to 

 5 per cent tyrosin solution to a culture of the bacillus, it takes on a red-black color quicker than with- 

 out this addition" (Uyeda). On repeating this experiment the result was again negative, but the 

 concentration of tyrosin was less. Without more ammonia or alkali Uyeda could not have made a 

 i to 5 per cent solution, since according to Erlenmeyer only one part of tyrosin dissolves at 2oin 2,454 

 parts of water. 



IndoL According to Uyeda, B. nicotianae produces a small amount of indol. The same is true 

 of the Deli cultures. With sulphuric acid and potassium nitrite the reaction of 22 bouillon-cultures 

 from 9 isolations was negative or nearly so. Repeating with 25 cultures from 10 isolations, 20 of the 

 cultures, after heating, gave a permanent red color of extremely variable intensity. It also happened 

 that a culture which gave no indol reaction like Uyeda 's B. solanacearum, after inoculation into 

 tobacco, gave a positive indol reaction like Uyeda's B. nicotianae. Yet is it doubtful whether indol is 

 really produced. According to Crossonini the indol reaction of Ehrlich is much sharper with para- 

 dimethylamidobenzaldehyd, and there are bacteria known as indol-formers on account of the old 

 reaction, which when tested by the new method show no trace of indol. Following this suggestion, 

 Honing tested 7 strains of the Deli tobacco-bacteria with the paradimethylamidobenzaldehyd in 

 alcohol with hydrochloric acid, but all with negative results. 



Hydrogen Sulphide. This substance is formed in very slight quantity, as Uyeda has also shown. 

 Strips of lead-acetate paper exposed for some days remained white or were only slightly blackened. 

 Of 1 8 cultures [kind not stated] from 9 sources only one gave a positive reaction. After the careful 

 boiling away of 32 cultures (10 isolations) the lead-acetate paper was black in only 10 tubes, and here 

 only a little around the edge, while 22 remained negative. From one and the same source sometimes 

 one culture was positive and another negative. 



Fat. Neither with osmic acid nor with Sudan III in alcohol or glycerine could the formation of 

 fat be shown. 



Glycogen. This substance is not formed by the bacteria (reagent, iodine-potassium iodide), nor 

 can it be used as a carbon food by the bacteria, as will be shown later. 



Disagreeable Odor. -This is noticeable from peptone-bouillon cultures, but varies greatly in cul- 

 tures from different sources. Isolation Ts resembled Uyeda's B. nicotianae, while others produced 

 scarcely any odor, like Uyeda's strain of B. solanacearum. 



Gas Production. Gas-formation does not occur in the closed end of fermentation tubes with 

 glucose, saccharose, or mannit. For the most part even in the presence of oxygen there is no trace of 

 gas-formation. Once very fine bubbles were detected in 24 glucose-bouillon cultures, 10 inoculated 

 out of tobacco, 7 out of Physalis, 2 out of Ageratum, 4 out of Mucuna, and i out of Blumea. This is 

 hardly a specific difference. 



Acid- and Alkali-formation. With exception of milk (in the second stage; i. e., after the alkaline 

 reaction), and with a nutrose-substratum, alkali is always formed. In normal growth the original 

 always feebly acid solution became plainly alkaline. In feeble growth the alkalinity was feebler or the 

 fluid had not yet reached the litmus neutral point. Acid-formation has not been detected with any 

 single sugar when the source of nitrogen was not casein. 



Temperature. Uyeda's statement that heating to 55 C. is fatal and Smith's that exposure to 

 52 C. for 10 minutes kills, is true of the Deli strains. The tropical climate (Medan lies in +3 north 

 latitude) appears not to have increased the maximum temperature. Bouillon-cultures exposed for 10 

 minutes at 55 C. appeared to be sterilized (5 isolations tested). We do not, with Uyeda, consider 

 3 C. difference in maximum temperature as specific, since 55 C. (for B. nicotianae) and 52 C. (for 

 B. solanacearum) are not the average of a great number of determinations with comparable material 

 from different sources (see under "Spores"). 



Resistance to Drying. This is slight, as already noted under "Spores. " 



