132 HISTORY AND CAUSE OF THE COCONUT BUD-EOT. 



set out after their arrival from a locality in Florida where the bud-rot 

 does not occur. They were just beginning to make good growth. 

 The following notes are from Miss McCulloch: 



Seedlings 6 to 12 inches out of nut: At base of stalk a spot was washed with 1:1000 

 HgClo, rinsed in sterile water, then with sterile needle a puncture made to heart or 

 center of stalk. The bacterial growth from agar slant was washed off in sterile water 

 and this water (cloudy with bacteria) was injected with a hypodermic needle into the 

 center of the plant. Inoculations Nos. 1 to 4 from agar slants 1 to 4 Hitchings strain of 

 Bacillus coli; inoculations Nos. 5, 6, and 7 from agar slants 1, 2, and 3 of VI-ll-V-09 

 strain of Bacillus coli. After inoculation the agar from the tubes was taken out on the 

 cotton plug and bound over the point of inoculation. Checks Nos. 8 and 9 were 

 punctvu-ed with sterile needle and the binding of agar and cotton put on as with 

 inoculated plants. 



Inoculation No. 4, collected June 8: The path of the inoculating needle is brown. 

 In the youngest inner leaf there is a brown, water-soaked area about the inoculation 

 point; extends 1.5 centimeters above and 1 centimeter below the inoculation point. 

 Brownish tissue 1.5 centimeters above used for plating. Tissue was washed in 

 alcohol, merciu-ic chlorid, and water, and then crushed in the test tubes. 



June 10: No colonies on these plates. They have been at 37° C. for 24 hours, and at 

 room temperature for 24 hours. A new set of plates was poured from same tubes. 



June 13: A few white colonies only, on original plate; discarded. Same with 



second set. 



Inoculation No. 5: Inoculating needle missed the center of the growth. There is 

 no discoloration about the path of the needle except in the leaf base last punctured, 

 where there is considerable water-soaked, reddish tissue, some of it 3 centimeters 

 from inoculation point. Some of this diseased tissue farthest from inoculation was 

 washed in alcohol, mercuric chlorid, and water, and crushed in test tubes. No organ- 

 isms responding to Bacillus coli tests were isolated. 



Inoculation No. 7: The central leafstalk seems unaffected by the inoculation. 

 The base of the leaf just outside this shows discoloration and is slightly water-soaked 

 around the opening made by the needle. All the dark part was cut off from the 

 remainder of the leaf. The hole was laid open — the loose soft part in the opening 

 removed — then the whole discolored part was immersed in 95 per cent alcohol 15 

 seconds, then in HgClo for 2 minutes, and washed in several changes of distilled 

 water for half an hour. The material was then crushed finely in beef bouillon and 

 allowed to stand with frequent shaking for 3.5 hours before plates were poured. After 

 2 days at 37° C. only a few white colonies appeared on the plate. Plates were again 

 poured on the Dolt's synthetic medium, but as before only white colonies appeared. 

 Plates discarded. 



The remaining inoculations of May 7 were examined by the writer, 

 using the customary precautions of rinsing in alcohol and soaking in 

 mercuric chlorid. The results were as follows: 



Inoculation No. 1, collected on Jimc 8: The tissues about 2.5 

 centimeters above inoculation point showed a browning; a rather dry 

 rot. Plates were made in the usual way on June 9 in Dolt's syn- 

 thetic medium and incubated at 37° C. 



June 10: Plate lb thickly sown with irregular, luxuriant pink colonies. 

 Plate 1 c^, no colonies. 

 Plate Ic, numerous pink colonies. 

 Plate lb, Five round pink colonies; one irregular mass. 

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