182 ROYAL SOCIETY OF CANADA 



well, or add the ashes to the water when it is boiling hot and boil 

 for a minute; filter within an hour through absorbent cotton by means 

 of a vacuum pump. To fill 100 parts of the filtrate add 1 part of 

 agar and 1 part of maltose, heat in steam, boil a minute over the flame 

 and heat again in steam to dissolve the agar. Filter the hot solution 

 through absorbent cotton by means of a vacuum pump. In filtering 

 it is well to use a small plug of cotton thrust tight into the neck of 

 the funnel, and to renew the plug each time after a litre or so has 

 been filtered. When the medium is all filtered its reaction may be 

 tested, using phenolphthalein as an indicator. The ash water may have 

 been alkaline — 9° to — 12°, but the agar medium will be neutral 

 or only faintly alkaline, not more than — 1°. Siphon off the filtered 

 medium into two-ounce French square, narrow-mouthed bottles of flint 

 glass. Each bottle should receive about 35 c.c, which will nearly half 

 fill it. Thrust a plug of cotton in the mouth of eacli bottle and 

 sterilize in the autoclave at 10 pounds steam pressure for one-half to 

 one hour, or in flowing steam for one hour on each of three successive 

 days. Eemove the bottles from the autoclave, incline them and allow 

 the medium to solidify so as to present a large surface for inoculation. 

 The medium will not usually be found favourable for growth when 

 first made, but will be fit for use after two or three weeks and will 

 improve with age. 



Inoculation and Growth of Nitro-cuUures in Bottles. — A mother 

 culture for each species of legume is isolated the previous year or early 

 the same spring, and from this several tubes or bottles of ash-maltose- 

 agar are inoculated to be used from time to time for inoculating the 

 bottles intended for distribution. These last may be inoculated by 

 means of a sterile pipette containing the culture mixed with sterilized 

 water, or it may be done by transferring a bit of the surface of the 

 agar in the bottle by means of a platinum spatula. In two or four 

 days at 25° C, the growth accumulates at the point of inoculation and 

 gravitates down the inclined surface of the agar as a raised, transparent, 

 wet-shining band which increases in width. The wedge of agar may 

 now be shaken over so that the inoculated surface comes against the 

 glass. By shaking back and forth a few times, all five faces become 

 inoculated and in four to twelve days on favourable media there is a 

 copious, wet-shining, mucilaginous or slimy growth over the whole sur- 

 face of the agar and the culture may be used, or it may be kept for 

 a month if desired, and there will be further increase in growth. Just 

 before mailing a culture, the cotton plug is replaced by a cork which 

 is well flamed before inserting. The printed label is attached, the bottle 



