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aO, organism not present; +, organism present.

From 5% stock phenol solution (1:20) dilute further to make 1:80 and 1:90 dilutions. 5 min, 10 min, and 15 min correspond to a set of transfers; for example, after 30 s, transfer loop from second subculture tube and continue process for each successive dilution; 5 min after makeing first transfer, repeat for 10 and 15 min intervals.

g. Remove stopper just enough to quickly add 5 mL 20% KI solution, taking care that no Br2 vapors escape, and immediately stopper the flask.

h. Shake thoroughly, remove stopper, and rinse it and neck of flask with a little H2O so that washings flow into flask.

i. Titrate with 0.1 N Na2S2O3, using starch indicator. (Mix approx 2 g finely powdered potato starch with cold H2O to a thin paste, add approx 200 mL boiling H2O, stirring constantly, and immediately discontinue heating.) Add approx 1 mL Hg, shake, and let stand over the Hg (1 mL 0.1 N KBr-KbrO3 = 0.001569 g phenol).

% phenol in stock solution = (30 mL 0.1 N Na2S2O3 solution from tritation) x 0.001569 x 1333 x 100/1000

where: 30 mL 0.1 N KBr-KbrO3 solution added.

0.001569 = g phenol equivalent to 1 mL 0.1 N KBr-KbrO3 solution.

1333 = dilution factor.

1000 = original volume phenol stock solution. j. If necessary, adjust solution to 5.00 ± 0.05% phenol by adding H2O or phenol. k. Keep in well-stoppered amber bottles in cool place, protected from light.

3. Potassium bromide-bromate solution 0.1 N: Standardization: Transfer 30 mL to I2 flask, and add 25 mL H2O, 5 mL 20% KI solution and 5 mL HCl. Shake thoroughly and titrate with 0.1 N Na2S2O3, using starch indicator.

4. Transfer loop: Make 4-mm id single loop at the end of a 50-75-mm or 4-mm loop fused on a 75-mm shaft. Then bend the loop at 30° angle with stem.

5. Water bath: Maintain a constant temperature of maintaining 20 ± 0.2°C.

7. Glassware: Sterilize all glassware for 2 h in hot air at 180°C. (Place pipets in closed metal containers before sterilizing.)

3. Methods 3.1. Technique

1. Make a 1% stock solution of the disinfectant to be tested. (You can use other concentrations; the convenient dilution depends on the anticipated dilution.) Keep in a glass-stoppered cylinder.

2. Make final dilutions, from 1% stock dilution, into test tubes and remove all in excess of >5 mL. The range of dilutions should cover killing limits of disinfectant in 5-15 min and should at the same time be close enough for accuracy.

3. From 5% stock phenol solution (1:20), dilute further to make 1:90 and 1:100 dilutions, and place in medication tubes.

4. Place these tubes (containing 5 mL each of final dilutions of disinfectants and phenol) and tubes containing test culture in an H2O bath at 20°C and leave 5 min.

5. Add 0.5 mL of the test culture to each of the dilutions at time intervals corresponding to intervals at which transfers are to be made. Thus, by time 10 tubes have been seeded at 30-s intervals, 4.5 min have elapsed, and a 30-s interval intervenes before transfer to the subculture begins.

6. Add culture from graduated pipet large enough to see all tubes in any one set. In inoculating test tubes, hold them in a slanted position after removal from bath, insert the pipet to just above the surface of the disinfectant, and run in the culture without letting the tip touch the disinfectant.

7. After adding culture, agitate gently but thoroughly to ensure even distribution of bacteria, and replace in the bath 5 min after seeding. For the first test tube, transfer one loopful of the mixture of culture and diluted disinfectant from the test tube to the corresponding subculture tube. To facilitate transfer of uniform drops of antibacterial mixture, hold the tube at a 60° angle, and withdraw the loop so that the plane of the loop is parallel with the surface of the liquid.

8. After 30 s, transfer one loopful from the second test tube to the second subculture tube, and continue the process for each successive dilution; 5 min after making the first transfer, begin the second set of transfers for a 10-min period, and finally repeat for a 15-min period.

9. Gently agitate the test tubes before taking each interval loop subsample. Before each transfer, heat the loop to redness in a flame, and flame the mouth of every tube. Sterilize the loop immediately after each transfer (before replugging tubes) to allow time for cooling. Use care in transferring and seeding to prevent the pipet or needle from touching the sides or mouth of the test tube, and see that no cotton threads adhere to the inner sides or mouth of the tubes.

10. Incubate the subculture for 48 h at 37°C and read the results.

Example of Methods for Calculating Phenol Coefficient Number3

Disinfectant (X) Dilution

S min6

l0 min6

lS min6

l:300

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