Dec. 15, 1920 Effect of Temperature and Humidity on Citrus-Canker 449 



The writer has always noticed this zone on potato plugs, especially in 

 young cultures. 



Preliminary tests on inoculated potato plugs with iodin solution 

 showed that the narrow white zone was completely free from starch, 

 while it was surrounded by a small light band of red and blue, indicating 

 that the decomposition of the starch was slowly taking place. In old 

 cultures the cell walls were separated, showing that the middle lamella 

 had been attacked and dissolved. Wolf (77) and Doidge (z) have re- 

 ported similar observations. Thus, by the use of soluble starch agar 

 and potato cylinders, the growth of the organism as well as the rate of 

 enzym action at different temperatures could be measured directly. 

 The soluble starch agar was made up as follows : x 

 12.0 gm. shredded agar. 

 5.0 gm. soluble starch (Merck), according to Lintner. 

 .5 gm. potassium phosphate (dibasic). 

 .5 gm. magnesium sulphate. 

 .5 gm. sodium chlorid. 

 i.ogm. ammonium sulphate, 

 i.ogm. calcium carbonate. 

 1,000 cc. distilled water. 



Two methods of measuring the growth of the organism presented 

 themselves: first, the pouring of dilution plates and measuring the growth 

 formed from a single bacterium by means of an enlarged projection 

 through a fixed camera, and second, the placing of a definite amount of 

 inoculum on the agar and measuring the increased diameter of the colony. 



The most serious objection to the first method was that the plates 

 could not be poured at the temperatures to which they were subsequently 

 exposed. The minimum temperature for the growth of the citrus-canker 

 organism is approximately 2 to 4 C. lower when this method is used. 

 Also the initial growth at temperatures between 5 and 15 is greater. 

 This is due to the fact that all materials are at room temperature when 

 the inoculations of the plates are made and, furthermore, there is a 

 definite time limit required to bring the plates or tubes to the tempera- 

 ture of that of the case. 



In the second method a 2-mm. loop was pressed gently on the hardened 

 agar at three or four points on the plate, so that the inoculum remained 

 on the spot made. The increase in the diameter of the colonies was then 

 measured from day to day. This method is not so accurate from the 

 standpoint of measurement as the first, but it gives much more compara- 

 ble results, when the temperature and time factors are considered. 



All the plates were poured at the same time, care being taken to get 

 the agar in the plates of the same thickness. They were then placed in 

 the various temperature cases overnight, so that at the time of inocula- 



1 A modification of the starch agar used by McBeth and Scales. (McBeth, I. G., and Scales, F. M. the 



DESTRUCTION OF CELLULOSE BY BACTERIA AND FILAMENTOUS FUNGI, U. S. Dept. Agr. Bur. Plant Indus. 

 Bui. 266, p. 26-28, 1913.) 



16917°— 20 4 



