Sept. 15, 1925 
Plate Counts of Soil Microorganisms 
5Q3 
portions were withdrawn and plated. One gram of a clay loam field 
soil was put into the cup, and after a minute or two of vibration the 
siphon from the reservoir was started. The flow was adjusted so 
that the 1,900 c. c. would pass through in from 20 to 30 minutes. At 
the end, the suspension was mixed and further dilutions were made 
in sterile tap water in steps of 10 x, by putting 5 c. c. in 45 c. c. 
Four plates from the 1 : 200,000 dilution were poured on soil extract 
agar. 5 As a check on the effect of the vibrator, 10 gm. of the soil 
were added to 1,000 c. c. sterile tap water and shaken vigorously 
from 3 to 5 minutes. Dilutions were made in steps of 10 X by adding 
5 c. c. to 45 c. c., reaching the final dilution of 1 : 100,000 from which 
four plates were poured. (Data in Table I.) 
Table I .—Results of the first plating, together with series 2 which was made as a 
control to series 1 
Series 
Suspension by— 
Dilution 
Millions 
per gram 
soil 
Control 
from cup, 
per c. c. 
fHand___________ 
1 :100,000 
1 :200,000 
1 :100,000 
1 :200,000 
1 :200,000 
7.5 
14.6 
13.1 
22.6 
15.4 
1 . 
2 . 
(Vibrator_ 
•5,000 
fHand.. -___ 
■{Vibrator......... 
4,200 
6,500 
[Vibrator _ 
«Estimated. 
A glance at the last column of Table I is enough to condemn alcohol 
as the sterilizing agent of the vibrator cup. Although the effect of 
this contamination on the final dilution is small, it is a fruitful source 
of error. 
The next method of sterilizing consisted in soaking the parts of the 
vibrator cup in a solution of equal parts of formaldehyde and water 
for 15 minutes. The parts were then rinsed with sterile water, assem¬ 
bled aseptically, put in place over the vibrator, and filled with sterile 
water. At this point the gallic acid test for formaldehyde was made 
with 2 c. c. water from the cup. 6 If the test was positive, sterile 
water was run through until it became negative. One gram of soil 
was then added, and vibrated as in the preliminary experiment. 
The first trials with formaldehyde showed that the apparatus could 
be rendered practically sterile. It was the writers’ custom, however, 
to make four plates from the vibrator cup before every set of experi¬ 
ments, thus positively checking on the sterilization. In 13 different 
tests, representing a total of 52 plates, an average of only 2 colonies 
per plate was found, the highest number recorded being 7, while 
many plates were sterile. 
Whittles (14) used bleaching powder for the sterilization of the 
vibrator, and the writers tested this substance, too. Following his 
directions, the cup was taken apart, “ thoroughly washed in running 
water under the tap, rinsed with a solution of bleaching powder to 
which a drop of hydrochloric acid had been added; it was again 
washed thoroughly in running water, and then with a dilute solution 
* Field soil 500 gms., water 1,200 c. c., autoclave half hour at 15 pounds, or boil over a bare flame for 15 
minutes. Filter, make up to 1,000 c. c., add 0.5 gms. K2HPO4 and 20 gms. agar. Adjust to Ph 7 to 7.2. 
• To 2 c. c. sample add a few drops of a saturated alcoholic (absolute) solution of gallic acid and stratify 
over concentrated sulphuric acid. A blue ring indicates formaldehyde. . Sensitive to 1 part in 200,000. 
