In order to determine whether or no the test germs have been destroyed, observations must always be continued--when growth appears to be absent--up to the end of seven days before recording "no growth." _h. Identification of the Organisms Developing in the Subcultivations after Contact in the Germ + Germicide Solution._-- This is based on the naked eye characters of the growth in the bile salt broth, supplemented where necessary by plating methods, further subcultivations upon carbohydrate media and agglutination experiments. The sign (+) is used to indicate that growth of the test organism occurred in the subcultivations, and the sign (-) to indicate that the test germs have been destroyed and no subsequent growth has taken place. METHOD.-- _Apparatus Required_: Sterile test-tubes (narrow, not exceeding 1.3 cm. diameter). Test-tube rack (Fig. 219). Sterile graduated pipettes in case, 1 c.c. (in tenths). Sterile graduated pipettes in case, 5 c.c. (in c.c.). Circular rubber washers, 2.5 cm. diameter with central hole, sterilised by boiling immediately before use, then transferred to sterilised glass double dish. Electric signal clock or stop watch. Sterile forceps. Sterilised glass beads. Shaking machine. Grease pencil. _Material Required_: Percentage solutions of germicide-x (_vide_ page 481). Percentage solutions of pure phenol (_vide_ page 482). Aqueous emulsion of B. coli (_vide_ page 481). Tubes of bile salt broth. ~Preliminary Tests.~-- _a. Inhibition Coefficient._-- Determine the lowest percentage of germicide-x which inhibits growth of B. coli in the bile salt broth, and the highest percentage which fails to inhibit (page 311). On the result of this experiment determine the bulk of medium required in the subculture tubes and the percentage solutions to be employed in the trial trip. Assuming the inhibition coefficient to be 1:1000, it will be quite safe to employ the ordinary culture tubes containing 10 c.c. medium in the subsequent experiments. _b. Trial Trip._-- Determine the lethal effect of a series of five solutions of germicide-x (say 1:100, 1:250, 1:300, 1:500, 1:600) at contact times of 2-1/2, 5, 25 and 30 minutes in the following manner: 1. Arrange five test-tubes marked A to E in the lower tier of the test-tube rack. 2. Into tube A pipette 5 c.c. germicide-x 1:100 solution. Into tube B pipette 5 c.c. germicide-x 1:200 solution. Into tube C pipette 5 c.c. germicide-x 1:300 solution. Into tube D pipette 5 c.c. germicide-x 1:500 solution. Into tube E pipette 5 c.c. germicide-x 1:600 solution. 3. Arrange 20 tubes of bile salt broth in the upper tier of the test-tube rack in two rows, those in the front row numbered consecutively from left to right 1-10, those in the back row 11-20.