TOP-ROT OF SUGAR-CANE IN JAVA. 83 



In any case, the top-rot has microscopically not the least resemblance to the yellow dis- 

 ease of the hyacinths. The disease shows as little characteristic symptoms in other respects. 



The same is true of both the other forms. 



In the cracks and holes of Pokkah-bong one may also meet with bacteria and even 

 moulds, and the same is true of the inside of young plants attacked by the Heart-disease. 



In order to learn what bacteria were to be found in the decayed tops, Wakker states 

 that he took two courses: (1) direct transfers to tubes of agar; (2) poured plates. 



Went, in his preliminary investigation, had met with but one bacterium; Wakker, on 

 the contrary, found several. For his experiments Wakker used test-tubes with a slanting 

 surface of nutrient agar-agar containing 975 gm. of water, 3 gm. pepton, 6 gm. cane-sugar, 

 1 gm. extract of meat, 15 gm. agar-agar. He obtained the folio wing results from his transfers: 



Test I. On March 7 by means of a sterilized needle I derived from a rotten top just cut through, 

 infectious matter which was transferred to agar-agar tubes. There developed : 



1. Thick, whitish drops, which soon flowed down. On examination they appeared to be short, 

 thick rods which without exception were surrounded by a jelly covering. As appears from the de- 

 scription and picture byCobb they are identical with his Bacillus vascularum, and likewise with the one 

 found by Went. 



2. Flat, almost invisible coverings which appeared to consist of a micrococcus. 



Both kinds are without movement of their own. The appearance of the two bacteria naturally 

 made new tests necessary. The micrococcus was preliminarily designated No. 1. 



Test II. On March 31, with as much antiseptic precaution as possible, from a fresh cut top-rot 

 stalk I soaked a small quantity of glassy tissue from the inside of the red line, in sterilized water, and 

 of this I transferred some drops to melted agar-agar. This was poured out into four glass boxes and 

 left to stiffen. After 15 hours a number of colonies had developed, the bacteria of which were also 

 numbered. 



No. 2. Small yellowish colonies which consisted of a non-motile micrococcus in very large 

 numbers. 



No. 3. Many larger, opalizing drops, which consisted of short, thick bars, without movement of 

 their own and with a jelly covering. 



No. 4. Spread everywhere in the agar-agar, long, thin bars, without movement of their own, but 

 sometimes with spores, which formed no real colonies. 



After heating the boxes to ioo C. in the steam sterilizer the latter alone were left and now formed 

 also clear colonies in the form of a white layer. 



Lastly, also thin, short bars, with an active movement of their own, which occurred in colonies 

 resembling externally those of No. 3. 



Test III. The foregoing test was repeated with the white rotting tissue of the top on April 4. 

 This gave the following results : 



No. 5. Long, thin, crooked non-motile bars. 



No. 6. Short, thin bars with active movement of their own. 



No. 7. Long non-motile bars containing spores. 

 In all three tests only those forms are numbered of which pure cultures were made in test-tubes. 

 They were for a time all nurtured separately until it appeared that they were : 



Nos. 1, 2, a Micrococcus. 



No. 3, Bacillus vascularum Cobb. 



Nos. 4, 5, 7, Bacillus subtilis. 



No. 6, the last one named under test II. 



Thus four are left which will be further distinguished with the numbers 2, 3, 6, and 7. 



The question now was which of these might be considered the cause. To this end served the 

 following : 



Test IV. On April 16 I introduced pure 2-day old, liquid cultures of the bacteria Nos. 1 to 7 into 

 the closed leaf-sheaths (Kokers) of seven sprouts which had developed in small pots. In each pot 

 there was still another sprout which was not infected. The pots were marked with the same numbers 

 as the bacteria. That same day the bacteria were microscopically examined and of each a new cul- 

 ture was made on agar-agar. On examination these appeared later to be pure cultures. 



In view of the approach of the dry monsoon the pots were placed under a gangway in the shadow. 



