52 REPORT OF THE BACrERIOLOGIST OF THE 
ing for distribution organisms which fix or gather atmospheric 
nitrogen, of which the following is a specification : 
This application is made under the act of March 3, 1883, chap- 
ter 148, and the invention herein described and claimed, if 
patented, may be used by the government of the United States 
or any of its officers or employees in prosecution of work for the 
government or by any other person in the United States without 
the payment to me of any royalty thereon. 
The invention relates to the process of growing these organisms 
and preparing them for distribution. 
The invention has for its object the production of more highly 
effective organisms and their distribution in a form preventing 
deterioration and easily applied in agriculture. All work that 
has heretofore been done in the cultivation of nitrogen-gathering 
root-tubercle organisms for use in agriculture has been done in . 
culture media containing either decoctions of the leguminous 
plants, from which these specific organisms in each case were ob- 
tained or in media containing some other available form of com- 
bined nitrogen not free or atmospheric. When there is available 
combined nitrogen in the medium, the organisms instead of de- 
pending solely upon the atmospheric nitrogen for their nitrogen- 
supply draw upon the nitrogenous materials of the culture me- 
dium—such, for example, as proteids, nitrates, ammonium com- 
pounds, ete.-—for which reason they do not develop their full nitro- 
gen-gathering power and rapidly deteriorate. 
By my process the organisms are first obtained from the tuber- 
cles or swellings on the roots of the leguminous plants—such as 
clovers, cow-peas, beans, etc. After the tubercles are thoroughly 
washed and surface sterilized in the ordinary ways the interior 
of the tubercle is cut out under sterile conditions and mixed in 
a medium consisting of water containing about one per ct. com- 
mercial agar-agar, about one per cent. maltose sugar or cane- 
sugar, (the former being the better,) about .02 to .05 per ct. 
magnesium sulfate, and about 0.1 per ct. monobasic potassium 
phosphate. This solution is made up in the ordinary way and 
sterilized according to ordinary bacteriological processes. It 
differs from ordinary culture media for bacteria only in the 
absence of a source of combined nitrogen. The agar may be 
‘varied above or below the amount suggested. The maltose or 
