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. a.s.suming 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.

4. Place a square wire basket of about 50 tubes capacity close to the left of the test-tube rack, for the reception of the inoculated tubes.

5. Take a sterile 1 c.c. pipette from the case, pick up a sterile rubber washer with forceps and push the point of the pipette into the central hole.

6. Put down the forceps on the bench with the sterile points projecting over the edge. Without taking the tube from the rack remove the cotton-wool plug from tube A, and lower the pipette, with the rubber washer affixed, on to the open mouth of the tube; with the help of the forceps to steady the washer, push the pipette on through the hole until the point of the pipette has reached to within a few millimetres of the bottom of the tube (see fig. 219).

7. Adjust in the same way a pipette and a washer in the mouth of each of the other tubes, B, C, D and E.

8. Set the electric signal clock to ring for the commencement of the experiment and at subsequent intervals of 2-1/2, 5, 25 and 30 minutes.

9. Take up 0.5 c.c. of B. coli emulsion in sterile pipette graduated in tenths of a cubic centimetre and stand by.

10. As soon as the bell rings lift the pipette from tube A with the left hand and from the charged pipette held in the right hand deliver 0.1 c.c. of B. coli emulsion into the 1:100 solution. Then replace the pipette and washer.

[Ill.u.s.tration: FIG. 219.--Test-tube rack.]

11. Raise the tube with the left hand and shake it to mix germ and germicide, whilst returning the delivery pipette in the right hand.

12. Repeat the process with tubes B, C, D and E; then drop the infected delivery pipette in the lysol jar. The inoculation of the five tubes can be carried out very expeditiously, but a period of 10 seconds must be allowed for each tube.

13. When the bell rings at 2-1/2 minutes blow through the pipette in tube A (this agitates the germ + germicide mixture and ensures the collection of a fair sample); allow the mixture to enter the pipette, and as the column of fluid extends well above the terminal graduation, the right forefinger adjusted over the b.u.t.t-end of the pipette before it is lifted will retain more than 0.1 c.c. of the mixture within the bore when the point of the pipette is clear of the fluid in the tube. Touch the point of the pipette on the inner wall of the tube, and allow any excess of fluid to escape, only retaining 0.1 c.c. in the pipette.

14. At the same time, with the left hand remove Bile Salt Tube No. 1 from the upper tier of the rack, take out the cotton-wool plug with the hand already holding the pipette (the relative positions of pipette, plug and culture tubes being practically the same as those of platinum loop, plug and culture tube shown in Fig. 68, page 74).

15. Insert the point of the pipette into the subculture tube, and blow out the mixture into the medium--replug the tube and drop it into the wire basket. Replace the washer-pipette in tube A.

As soon as the point of the pipette has entered the mouth of tube A it may be released, since it has already been so adjusted that it just clears the bottom of the test-tube, and the elastic washer will prevent any damage to the tube.

Steps 13, 14 and 15 occupy on an average 10 seconds.

16. Repeat steps 13, 14 and 15 with each of the other tubes B, C, D and E.

17. Repeat these various steps 13-16 when the bell rings at 5, 25 and 30 minutes.

18. Place all the inoculated tubes in the incubator at 37 C.

19. Examine the tubes at intervals of 24 hours, and record the results in tabular form as shown in Table page 491 (the figures in the squares indicate the number of hours at which the changes in the medium due to the growth of B. coli first appeared).

20. If a consideration of the tabulated results indicates strengths of Germicide-x lethal at 2-1/2 and 30 minutes the final test can be arranged, but if this result has not been attained, sufficient evidence will probably be available to enable a second trial test to be planned which will give the required information.

~Final Test.~--

c. _Determination of Phenol Coefficient._--

_X-Disinfectant._--This comprises two distinct tests, one of the Germicide-x, the other of the standard phenol.

1. Arrange five test-tubes clearly marked in the lower tier of the rack.

2. Pipette into each 5 c.c. respectively of the five percentage solutions of x-disinfectant which the trial run has already shown will include those affording lethal values at 2-1/2 and 30 minutes.

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.

4. Arrange further 20 tubes of bile salt broth numbered 21-40 in two rows in a second smaller rack which can be stood on the upper tier of the rack as soon as the first 20 tubes have been inoculated.

5. Place a square wire basket of about 50 tube capacity close to the left of the test-tube rack, for the reception of the inoculated tubes.

6. Adjust a sterile 1 c.c. pipette in the mouth of each of the tubes, A, B, C, D and E, by means of a washer, as previously described.

7. Set the electric signal clock to ring for the commencement of the experiment and subsequently at 2-1/2, 5, 10, 15, 20, 25, 30 and 35 minutes.