1905] SCHNEIDER—CULTIVATION OF RHIZOBIA 297 
cautions in order that satisfactory results may be obtained. After 
numerous trials I have finally found the following methods to give 
the most satisfactory and most uniform results. 
1. Securing tubercle-bearing roots.—Taking it for granted that it 
is desired to isolate and cultivate the rhizobium of some herbaceous 
leguminous plant, it is of considerable importance that the roots 
should be taken from plants growing in soil free from contamination 
with surface sewage, away from thickly populated areas, as vacant 
lots in large cities, etc. The object is to obtain tubercles which are 
comparatively free from foreign bacteria. Experiments with roots 
and nodules from plants growing in vacant lots in Chicago showed 
that they were covered externally with multitudes of microbes, which 
interfered very considerably with the rhizobia experiments. 
Select healthy looking plants growing in loose soil, dig up the 
Toots carefully; shake off the dirt very carefully, as the nodules are 
quite easily removed. By means of a sharp knife cut off root por- 
tions having well-developed single nodules or small groups (two or 
three) of nodules, and place them in a clean sterile container in which 
‘0 carry them to the laboratory. Do not take roots with nodules 
that appear quite dark (bluish hue) at the base, as these are old 
tubercles and are quite badly contaminated with foreign soil microbes. 
Do not take roots with large tufts of nodules for similar reasons, and 
for the further reason that these nodule clusters are difficult to clean. 
_ 2. Cleaning and garbling roots.—At the laboratory look the mate- 
nal over carefully. Remove all foreign vegetable substances. 
Reject all roots or rootlets showing decay or discoloration. By means 
of the pocket knife cut away root portions which are undesirable. 
Now place this carefully garbled material under a faucet and let the 
Water run over it to wash away dirt and sand. Care is necessary, 
aS a strong flow or careless handling may remove the most desirable 
nodules, Remove all dirt, using a small brush, if necessary, to remove 
§ing sand and soil particles. 
o = seis nodules.—After this cleansing, look the material over 
a y: By means of a pair of small, blunt-pointed, clean, sterile 
id - pick off young, clean-looking, but well-developed nodules 
tho Pp them into a small, clean, sterile beaker about half full of 
Toughly boiled water (boil this water yourself for half an hour). 
