COBB S DISEASE OF SUGAR-CANE. 61 



ing is much slower (4 to 7 days) . The fluid is never what might be called turbid, but pseudo- 

 zoogloeae are sometimes present in small numbers, and there is a small amount of pale yellow 

 precipitate (Ridgway's primrose yellow or a little deeper, to his maize color or Naples yellow). 

 A 3 mm. loop from a feebly clouded bouillon culture 24 hours old clouded feebly 10 c.c. of 

 + 15 standard beef-bouillon on the third day at 25 C. ; a 2 mm. loop of well-clouded bouillon 

 6 days old did the same at the end of about 24 hours. After 7 days' growth in peptonized 

 bouillon one could usually see a pencil through 70 mm. of the cloudy fluid (4 tubes, one 

 behind another). 



Notes of December 5, 1902, on six tube-cultures of Bacterium vascularum, made November 12, 

 1902, in standard peptonized bouillon (stock 712), as checks for thermal death-point experiments: 

 Fluid moderately cloudy in each one; rolling clouds on shaking; no pellicle, and in most no trace of 

 rim ; in others the merest outline of a whitish rim. Can not detect pseudozoogloeae even with the 

 hand-lens. Precipitate distinctly yellow and 5 mm. broad. Pencil easily visible through 2 of the 

 tubes (in series), less plainly so through 3, barely visible through 4, not visible through 5. These 5 

 tubes make a diameter of 9 cm. Organism not a copious grower in this bouillon. After 2 to 3 months 

 the fluid becomes clear, but is not browned (once slightly). There is no rim, or only a faint whitish 

 one which may be 2 to 3 mm., or more, in width. In only one set of transfers was a pellicle noted; 

 this was thin and easily shaken down. Prismatic crystals are often present. After 70 days about 7 

 to 14 mm. breadth of the pale yellow, fine-grained precipitate may be present on the bottom of the 

 test tube. 



Experiments with bouillon of different degrees of acidity and alkalinity, i. e., titrating 

 + 25, +15, o, and 20 with phenolphthalein, have given the following results: The +15 

 bouillons clouded first and in the end gave most precipitate. At the end of 9 days the 

 + 15 showed the best growth, i. e., 8 or 10 times as much precipitate as in neutral bouillon; 

 the neutral bouillon the next best, then + 25 ; the 20 bouillon was very feebly clouded with 

 almost no precipitate. Six days later the order was : Neutral, densely clouded, i. e. , more so 

 than +15, but less precipitate; + 15, well clouded, heaviest clouding in the uppermost centi- 

 meter; +25, as well clouded as lower part of +15 and uniformly, but much less precipitate 

 than in +15; 20, feebly clouded. In a later experiment, using 6, 4, neutral, +26, 

 + 28, and +30 bouillons (Mr. Johnston's notes), the best growth was in the acid bouillon; 

 + 26, +28, and +30 were very heavily clouded, the neutral bouillon well clouded (probably 

 corresponding to E. F. Smith's "moderately clouded"), and one of the 6 tubes lightly 

 clouded. The other 6 tube and both 4 tubes were clear. All of the cultures were 

 transfers from the same tube a beef-bouillon culture 8 days old. The acid was that of 

 beef-juice. 



Greig Smith obtained no growth in acid fluids such as wort and cane juice, and scanty 

 growth in neutral fluids, but does not state what fluids nor what indicator was used. 



The organism grew in beef-bouillon with phenolphthalein, with or without the addition 

 of potassium formate, but in neither case did the fluid become red. 



There is a moderate clouding of nitrate bouillon, but no reduction of nitrates to nitrites 

 (RGS., EPS.). At the end of a week in this medium, inoculating from cultures on slant 

 agar, all of 6 tubes were uniformily clouded, free from pellicle and rim, no pseudozoogloeae 

 were visible to the naked eye in the unshaken tubes, and the penholder was distinctly visible 

 through three tubes, and barely so through four, placed one behind another. Similar 

 results were obtained by inoculating from six potato cultures. 



Litmus-milk-cultures (pi. 11, figs. 10, n, 12) gradually become a deep indigo-blue (30 

 days, 54 days, 74 days, 6 months). Some tubes 38 days old are recorded as deep blue, near 

 Saccardo's atrocyaneus (sapphire of Standard Dictionary, nearly). The bluing begins to 

 be noticeable about the fourth or fifth day. No acid is produced. The milk was lavender 

 or lilac when inoculated. There is no reduction of the litmus or precipitation of the casein, 

 the milk remaining perfectly opaque and fluid. The milk does not become viscid (45 days, 

 75 days). A distinct yellow precipitate was formed, but in most tubes there was very little 



