Tue INFLUENCE OF Low TEMPERATURE ON SOIL BACTERIA 1055 
asparaginate agar. The agar used in plating the Bacillus radicicola 
organism was the same as Wilson (1917) used in his studies. In the nu- 
trient solutions the saccharose was replaced by mannite, 1 gram to a liter. 
METHOD OF FREEZING, THAWING, AND PLATING THE BACTERIA 
In the case of the field soils, the samples were collected in the field 
under sterile conditions and 20-gram portions were placed in sterile liter 
flasks. The samples were then taken to the laboratory in a frozen con- 
dition and treated immediately, water at the different temperatures being 
used to thaw the samples. Plates were poured from the 20,000 and 
200,000 dilutions. Five plates were poured from each dilution in all 
eases. Ice and salt, liquid air, and outside temperatures, were used to 
freeze the laboratory samples. 
The Bacillus radicicola organism was grown in sterile sand, soil, and 
water cultures. In the case of the sand and soil cultures 1-gram portions 
were weighed out into small, sterile, test tubes under sterile conditions, 
and subjected to the various treatments. The samples subjected to the 
liquid-air treatment were lowered into the wide-mouth Dewar bulbs con- 
taining the liquid air. Care was necessary to prevent bursting of the tube 
in freezing. The plates were incubated at 30° C. and counts were made 
at the end of four and seven days. Five plates from each of three dilutions 
were poured from all samples. 
\ 
EFFECT OF RATE OF THAW ON BACTERIA COUNT 
SOILS FROZEN IN THE FIELD 
' It would seem that if the large number of bacteria in frozen soils as 
shown by the plate method is due to the breaking up of the clusters of 
organisms, the rate of thaw should have some effect. In order to test 
this point, samples of Dunkirk clay soil containing 33 per cent of moisture 
were taken from the field in a frozen condition and thoroly mixed, and 
20-gram portions were put into liter flasks. These samples were brought 
to the laboratory and plated by the dilution method. The only difference 
in the treatment was the temperature of the water blanks used in making 
the dilutions. The results are shown in table 1. 
From the results shown in table 1, the rate of thaw seemed to have 
a marked effect as shown by the counts on agar plates. The increased 
count due to the sudden thawing of the soil with water at 30° C. was 200 
