166 ROYAL SOCIETY OF CANADA 



rotated and a sensible quantity of the cruslied bacteroidal tissue adlieres 

 to its point. The needle is then touched into a drop of sterile water 

 in a sterile Petri dish until the water is turbid. The needle is thrust 

 again into the nodule and touched into a small drop of water on a clean 

 cover glass. In this way three cover glasses are prepared and spread, 

 to be stained later. Each of several sterilized Petri dishes receives 

 first a drop of sterile water and then the drop is inoculated with a 

 loopful, or more, of the turbid suspension from the first Petri dish. 

 The agar media, previously melted and near the temperature of solidifi- 

 cation, are poured into the several Petri dishes in such a manner that 

 the suspension and the medium are mixed throughout and yet some- 

 what unequally. Then the colonies which develop may be crowded in 

 parts of the plate but will be well isolated in other parts. If desired, 

 suitable gelatin media may be poured into some of the plates. These 

 are interesting for study, but cultures for propagation and inoculation 

 are taken from the nitrogen-poor agar plates only. AYhen the media 

 have solidified, the plates are marked and incubated at 20° C. 



The films on the cover glasses are now fixed lighth' in the flame 

 and stained (see Staining reactions). 



5. Colony Formation. — The colonies of the Ps. radlcicola in ash- 

 sugar-agar media vary with the plant from which the cultures are made 

 and with the composition of the media, yet they are characteristic. 

 Colonies may appear in some plates in three or four days at 20° C, 

 hut the plates made from the same nodules at the same time, but in 

 different media. 



The deep colonies are circular, elliptical, or triangular with rounded 

 corners. Elliptical colonies often present two opposite lateral-rounded 

 outgrowths. The outline is sharply defined, but deep colonies some- 

 times appear as irregular patches. The colonies are granular, white 

 by reflected light, and brownish by transmitted light. Deep colonies 

 do not grow so large as surface colonies, bu.t deep colonies may grow 

 1.5 mm. to 2 mm. long and half as wide. Submerged colonies may 

 rise to the surface and then they take on the form and appearance of 

 surface colonies, except that they show at the centre the form of the 

 colony from which they arose. 



The surface colonies are raised, round, wet, entire, shining and 

 white. They appear like drops of melted paraffin, at first gleaming 

 and transparent, then translucent, then gradually more turbid and 

 opaque. At first they are watery, then more mucilaginous, and then 

 may become so highly viscid as to draw out in long threads when 

 touched with the needle. When the surface colonies become viscid the 

 deep colonies do also. After surface colonies appear they grow steadily 



