LABORATORY WORK 345 
The study of the nitrifiers is too difficult to be undertaken by elementary 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 14 c.c. and five more with {9 c.c. of the water 
to be tested. Place in the incubating oven for twenty-fourhours. If gas appears in 
the closed arm sufficient to fill it from 1/9 to 34 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 1/9 c.c. tubes it will 
indicate that there are probably more than 10 of the gas-producing organisms per 
c.c. of the water. If it appears in the 14 c.c. tubes but not in the {0 it indicates 
that there are less than ro perc.c. This presumptive test is only of value in suggest- 
ing suspicion, but insufficient to state the presence of sewage contamination. 
No. 28. Bacteria from the Root Tubercles of Legumes.—The bacteria that 
cause root tubercles may be obtained as follows: 
Prepare the following mixture: 1 gm. potassium phosphate (monobasic); 0.5 gm. 
magnesium sulphate; ro gm.saccharose; 1o gm.agar. Dissolve in rcooc.c. tap water 
by boiling and filter through cotton. Place in test-tubes and sterilize by steaming on 
three successive days, or in an autoclave for 15 minutes at 15 pounds pressure. 
Select some vigorously growing legume, not too large, and with a spade dig up 
its mass of roots still embedded in soil. Wash away the soil from the roots and 
probably plenty of tubercles will be found attached. Remove a small tubercle with 
forceps, wash in tap water and then immerse in a solution containing Soo c.c. water, 
I gram corrosive sublimate, and 2.5 c.c. HCl. It should be immersed in this for two 
to three minutes to sterilize the surface. Then the tubercle is held between folds of 
filter-paper that has been moistened in the sublimate solution and a gash is cut in it 
with a hot knife. A platinum needle is then sterilized and some of the central mass 
of the tubercle removed and placed in a drop of sterile water on a slide. Place a 
drop of sterile water in each of several Petri dishes and transfer a small loopful of the 
drop on the slide containing the tubercle contents, into the water drop in each of the 
Petri dishes. Pour into each a tube of the agar, allow to harden and incubate 
at 70°. Slimy, transparent colonies will appear in three to four days and may 
be isolated and studied in the usual manner. 
No. 29. Bacteriological Analysis of Market Milk.—Collect in sterile bottles a 
number of samples of market milk from different milkmen. Keep cool with ice 
until they are brought to the laboratory and then plate at once. Dilute 1,000, 
place 1 c.c. in each of two or three Petri dishes and add a tube of ordinary agar. 
Incubate at 98° for twenty-four hours, count the bacteria in each plate and determine 
the average. This is the procedure commonly used in making bacteriological analy- 
ses of miscellaneous samples of market milk. If there is any reason for thinking the 
milk is very old a higher dilution is necessary. If, on the other hand, the examina- 
tion is to be made of milk known to be good and in cool weather, a dilution of 100 is 
better. 
No. 30. Bacteria in Sour Milk.—Allow some milk to stand till sour, but not 
