4 THOMPSON YATES LABORATORIES REPORT 
In the method which I finally adopted, nutrient agar, carbolized in the proportion 
of i in i ,000, was used as a medium for the isolation of the B. coli. I first undertook 
a series of experiments to determine how great a proportion of phenol might be used 
without inhibiting too much the growth of the B. coli itself. Control plates of ordinary 
uncarbolized agar were employed for comparison, and the inoculations were made from 
pure cultures of B. coli commune. I found that the presence of 2 parts of phenol in 
1,000 parts of the medium rendered it quite sterile and 1.75 parts rendered it nearly 
so ; 1.5 parts of phenol exercised great inhibition (Plate I, Fig. 4), and 1.33 parts 
caused the colonies to be distinctly smaller and fewer in number (Fig. 3). It appeared 
that 1 part of phenol in 1,000 parts of the medium was the limiting quantity which 
could safely be employed (Fig. 2). 
The medium is prepared as follows : — Nutrient agar is prepared in the usual 
way, and filtered. While still melted one gram of pure phenol is added to each 
900 c.c. of the liquid, which is at once distributed, 9 c.c. being introduced by means 
of a Lode's distributer into each test tube. These should have been previously plugged 
and sterilized. The tubes containing the carbolized agar are sterilized for twenty 
minutes, in a Koch's sterilizer, at ioo°, and this has always been found to render tubes 
and medium quite sterile. 
That there is no loss of carbolic acid during this sterilization has been proved 
in the following way : — Tubes of agar, containing phenol in the proportion of 5 in 
1,000, were placed in a Koch's sterilizer ; the end of the tube was closed by an 
india-rubber stopper and delivery tube, the other end of which dipped beneath the 
surface of water in another test tube. After 20 minutes the water in the receiving 
test tube, and washings from the delivery tube, were tested with ferric chloride and 
found to give no trace of colouration, this test having previously been proved delicate 
enough to demonstrate the presence of phenol in water in a solution containing only 
.5 parts per 1,000. A 4 per cent, solution of phenol in water, similarly treated, gave 
the same result ; and, even when boiled, gave no trace of phenol in the distillate. 
Hence one may assume that no phenol is lost during the sterilization, nor during 
subsequent melting of the tubes before plating. 
When a sample is to be analyzed tubes of carbolized agar, prepared as above 
stated, are melted by being plunged into boiling water, and are then placed in a large 
water bath, the temperature of which remains constant at 45°, to 47 0 C. One cubic 
centimetre of the sample, or of the diluted sample, is added to the melted tube, which 
is then poured. By this addition the concentration of the phenol in the tube is 
lowered from 1 in 900 to 1 in 1,000. The plates are incubated at 42 0 C. for 48 hours, 
and then are examined, experience having shown that practically no more colonies 
develop after that time. 
It was at first feared that the numbers of B. coli on this medium might be 
diminished, and experiments were made to see if this were so. Pure cultures of 
