300 BOTANICAL GAZETTE [ocroBER 
much more rapidly by holding them under a faucet of running water, 
but this is not recommended to the beginner because of the danger 
of cooling the tubes too much. Number four of the tubes and add 
two with blank labels for reserve purposes in case of accident or 
special requirement. 
11. Preparations jor making the inoculations.—A lighted Bunsen 
burner and a platinum wire loop fastened into a glass rod are 
that is required. Wipe the table on which the work is to be done 
with a cloth moistened in some disinfecting solution. 
12. Inoculating tube no. 1; first attenuation.—Heat the platinum 
loop in the Bunsen flame, allow it to cool and dip into the root nodule 
crush. preparation, being careful not to include larger portions of 
tubercles, and introduce the loopful into test tube no. 1, shaking the 
platinum loop back and forth a few times within the medium. 
Replace the cotton plug in the tube, heat the loop in the Bunsen 
flame to a red glow, and place to one side. Now roll the test tube, 
kept in vertical position, between the hands to mix thoroughly the 
contents. Roll for five or six seconds. 
13. Inoculating tube no. 2; second attenuation.—Take two loop- 
fuls out of tube no. 1 and transfer them to tube no. 2; sterilize needle 
flames as before, and roll tube no. 2 as above. 
14. Inoculating tube no. 3; third attenuation—Take two oF 
loopfuls out of tube no. 2 and transfer to tube no. 3 and proceed . 
under section 12. 
15. Inoculating tube no. 4; fourth attenuation—Take three loop- 
fuls out of tube no. 3 and transfer to tube no. 4 and proceed as under 
section 12. 
Immediately after rolling, the tubes are t 
of warm water to prevent the media from coagulating. Clo 
cation to detail is very important in order to avoid confusion. 
well to rehearse mentally the different manipulations before beginning 
with the tube inoculations or attenuations. way 2 
16. Plating contents of inoculated tubes.—Tube n0- pas 
rejected, but it is preferable to set it aside for control one. aly 
Roughly estimated it contains several thousand rhizobia quite eve" 
distributed. Pour contents of tube no. 2 into Petri dish no. a os 
has been kept in a warm place (about 50° C.), spread the 
three 
o be replaced in the beaker 
se appl 
It 3s 
