1905] SCHN EIDER—CULTIVATION OF RHIZOBIA 299 
media. Microbes differ widely in this regard. For example, typhoid 
fever germs and others, survive in a 0.5 per cent. carbolic acid 
solution. 
8. Crushing the nodules.—Decant the last supply of water and 
transfer the tubercles to a thoroughly sterilized watch glass or similar 
small container (small salt cellar does well), and crush the tubercles 
with a sterile solid glass rod with rounded end. The crushed tubercles 
with the moisture that remained from the last rinsing form a pulpy 
mass. Cover with a sterile cover, such as a glass plate or a watch 
crystal, and set aside until ready to make the isolation cultures. If 
some time is required, the covered crush preparation should be 
placed upon a sterilized portion of the table and covered with a ster- 
ilized beaker or bell jar to present contamination by means of air 
microbes. 
9. Preparations jor isolating rhizobia.—It is ‘assumed that the 
desired culture media have been prepared in the usual way. I would — 
advise using the usual very slightly alkaline solid media of beef 
extract, salt, gelatin, and agar. Only sufficient agar should be used 
to give it solidity (about 1.5 per cent.). A number of test tubes 
about one-half to two-thirds full of the culture medium, sterilized by 
the fractional method, should be on hand ready for use. Six of these 
tubes should be placed in the steam sterilizer so that the medium may 
be thoroughty liquefied. This will require -half an hour or more. 
They should be placed in the sterilizer about the time that the work 
* cleaning the tubercles is begun. Three thoroughly sterilized 
Petri dishes should be on hand ready for use. Number these one, 
two, and three. 
- Preparing the test tubes jor inoculation.—When the culture 
medium in the test tubes is thoroughly liquefied, place the tubes into 
: beaker of moderately cool water. As soon as the water becomes 
ie hog heat of the tubes, replace it with fresh moderately i 
ay ile the tubes are still quite warm to the be place 
ae cos beaker of water having a temperature of about 50” C., the 
cag being to keep the medium liquefied. In this considerable 
ae S necessary. The medium must not be too hot, nor should 
ie Portion of it be allowed to coagulate. The tubes should feel 
arm. (not hot) to the touch when used. The tubes can be cooled 
