THE CUCURBIT WILT: TECHNIC OF ISOLATION 137 



at the time the plates are poured inoculate a tube of bouillon 

 or of peptone water by squeezing into it a drop of the viscid 

 fluid, let it stand 18 to 24 hours, and then pour from it a second 

 set of plates, inoculating them very sparingly from the original 

 tube, if it is well clouded, and rather copiously if it is clear. 

 Inoculate also from dilutions of the same. Save all tubes, or 

 at least the dilutions until results are known. The diseased 

 stems must also be retained until results are known, unless 

 others are easily procurable. They may be kept in the ice box. 



For inoculation purposes use cucumber, muskmelon, squash. 

 These should be planted in a warm place 2 months before they 

 will be needed, should be potted off early, and shifted to larger 

 pots frequently to keep them growing rapidly. Watermelons 

 may be inoculated for contrast. These cucurbits require houses 

 having a day temperature varying from 65° to 75°F., and a night 

 temperature about 15°F. lower. Avoid chilling the plants as 

 then they are very subject to mildew. If any mildewed plants 

 appear they should be removed from the house immediately. 



Inoculate by needle-pricks (10 to 20 to make sure, but on 

 some a lesser number for comparison) , using young (2- to 6-day) 

 agar-streak, potato, peptone, or bouillon cultures. Prick the 

 blades of well-developed vigorous leaves. Make check pricks 

 on other leaves of the same plant, or upon the same leaf on the 

 opposite side. In both cases group the pricks. Examine the in- 

 oculated leaves frequently from the second day on. Do not 

 over-water so as to complicate by root-rot. Do not tear the 

 leaves in making inoculations. 



Determine 



For the organism. Morphology. — In various media: size 

 in microns, form, aggregation of elements if any, i.e., chains, 

 filaments, pseudozoogloeae, etc., motility on margin of hanging 

 drop (using a high-power dry lens or an oil immersion lens, with 

 a long working distance) ; absence of spores (heat, spore stains) ; 

 presence and distribution of flagella (van Ermengem's stain); 

 stain one of the viscid, cobwebby threads on a clean slide 

 using carbol fuchsin and study under a high magnification. 

 Try capsule stains, Gram's stain. Do involution forms occur? 



