320 LABORATORY WORK. 
soil for the study of various soil problems requires special methods and spe- 
cial care too difficult for elementary work. The relative number of bac- 
teria in different soils may be determined as follows: Select two soils for 
study, preferably one rather sandy and the other filled with humus. Ob- 
tain a sample by mixing the soil well with a spade and take to the labo- 
ratory about 100 grams. Mix thoroughly the sample and weigh out one 
gram. It is best to do this after passing the soil through a sieve. Place 
in a 99 c.c. water blank and shake vigorously for two minutes. Transfer 
i c.c. of this to a second 99 c.c. water blank and mix well. Transfer i c.c. 
to a 9 c.c. water blank and mix again. From this transfer i c.c. to a Petri 
dish and then pour upon it the contents of a melted agar or gelatin tube. 
Mix in the usual way, harden, and after two to four days count the number 
of colonies in the two soils. This will give the relative numbers approxi- 
mately only. To obtain them exactly allowance must be made for the 
water in the two soils. 
No. 25. Denitrifying Bacteria. Make a broth containing 1000 c.c. 
water, i gm. peptone and 2 gm. potassium nitrate. Fill a few fermentation 
tubes and sterilize by steam. Inoculate several with a little soil from differ- 
ent localities. Incubate at ordinary room temperature for several days. 
Gas will appear in the closed arm if denitrifiers are present. This gas is 
mostly nitrogen and represents so much loss to the soil. The bacteria can 
be isolated by the plate method if desired. 
No. 26. Nitrogen Fixers. Their action may be shown as follows: 
Make a solution containing: MgSO 4 2 gm., K 2 HPO 4 2 gm., CaCl 0.2 gm., 
dextrose 2 gm., citric acid 5 gm., FeCl 3 traces, water, (distilled), 1000 c.c. 
Make the reaction neutral, taking great care not to pass beyond the neu- 
. tral point. Place some of this in a flask, sterilize by steam, and inoculate 
with a little soil. Incubate at ordinary room temperature. After two or 
three weeks, growth will be evident and usually a membrane appears on 
the surface. This membrane contains nitrogenous matter and since there 
is no nitrogen in the culture medium as above made, the nitrogen must 
have been assimilated from the air. The isolation of these bacteria is 
very difficult. 
The study of the nitrifiers is too difficult to be undertaken by elemen- 
tary students. 
No. 27. The Presumptive Test for Bacillus coli. This test is frequently 
made to determine whether water is suspicious. Fill fermentation tubes 
with lactose bouillon and inoculate five tubes with 1/2 c.c. and five more 
with- i /io c.c. of the water to be tested. Place in the incubating oven for 
twenty-four hours. If gas appears in the closed arm sufficient to fill it 
from i/io to 1/4 full, the test is positive and the water probably contains 
B. coli. To determine this absolutely requires further tests that cannot 
be given here. The purpose of using the different amounts of water is to 
give a rough idea of numbers. If gas appears in all of the i /io c.c. tubes 
it will indicate that there are probably more than io of the gas-producing 
organisms per c.c. of the water. If it appears in the i /2 c.c. tubes but not 
in the i/io it indicates that there are less than io per c.c. This pre- 
