344 LABORATORY WORK 
platinum loop and examine (stained) under microscope. Note the immense number 
of bacteria. 
No. 23. Manure and Sewage.—Place a small drop of sewage and a small bit of 
manure in separate water blanks. After thorough mixing remove a loopful in each 
case and transfer to a second water blank for further dilution. After again mixing 
transfer a loopful of this second dilution to melted agar or melted gelatin, gently 
agitate and then pour into Petri dishes. Allow to grow for two or three days and 
note the number of colonies, indicating the great numbers of bacteria in the original 
materials. A quantitative determination can be made if desired by using 1 c.c. of 
the sewage or r gram of manure, and diluting 100,000 times. 
No. 24. Soil Bacteria——For general study the agar or gelatin media already 
described may be used. A rather more satisfactory medium, however, is plain 
gelatin dissolved in tap water without the addition of other ingredients, but with its 
acidity adjusted and with clarification by means of white of egg as described above. 
Plates made with almost any agar or gelatin medium will show numerous soil or- 
ganisms. An incubation of several days at as low a temperature as possible (70° or 
under) is advisable, because at higher temperatures the most rapidly growing bac- 
teria crowd out the numerous organisms that grow slowly on ordinary culture media. 
The relative number of bacteria in different soils may be determined as follows: 
Select two soils for study, preferably one rather sandy and the other filled with 
humus. Obtain a sample by mixing the soil well with a spade and take to the labo- 
ratory about 100 grams. Mix thoroughly the sample and weight 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. Transfer 1 c.c. of this to a second 99 c.c. water blank 
and mix well. Transfer zr c.c. to a 9 c.c. water blank and mix again. From this 
transfer 1 c.c. to a Petri dish and then pour upon it the contents of a melted agar or 
gelatin tube. Mix in the vsual way, harden, and after four to seven days count the 
number of colonies in the two soils. 
No. 25. Denitrifying Bacteria.—Make a broth containing 1,000 c.c. water, 1 
gm. peptone and 2 gm. potassium nitrate. Fill a few fermentation tubes and sterilize 
by steam. Inoculate several with a little soil from different 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 logs 
to the soil. 
No. 26. Nitrogen Fixers.—Their action may be shown as follows: Make a 
solution containing: MgSO, 2 gm., KsHPO, 2 gm., CaCl 0.2 gm., dextrose 2 gm., 
citric acid 5 gm., FeCl, traces, water (distilled), 1,000 c.c. Make the reaction 
neutral, taking great care not to pass beyond the neutral 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 
aa nave been assimilated from the air. The isolation of these bacteria is very 
ifficult, 
