3o8 DISINFECTANTS AND ANTISEPTICS 



testing the efficiency of substances for use as food preservatives, or for the preserva- 

 tion of protein and carbohydrate materials, methods may be employed which prove 

 or disprove the capacity of such substances to prevent the growth of bacteria. Anti- 

 septics for use in treating or counteracting infections or for ridding the skin and mu- 

 cous membranes of pathogenic micro-organisms should be tested in a manner which 

 simulates as far as practicable the conditions under which these reagents are used in 

 practice. When antiseptics, such as the ordinary licjuid preparations, are used in such 

 a manner that short-time contact only is assured, the germicidal test such as described 

 above for disinfectants is used. However, a temperature of 37° is employed instead of 

 20°. In this case M. aureus' is the test organism, and the ability to kill it in 5 minutes 

 is the criterion of the efficiency of such liquid antiseptics. 



The following types of preparations are examined by what may be called the "fil- 

 ter-paper method." The method, briefly, is as follows: 



Number 2 Whatman Filter paper is cut into pieces about 0.5 cm. square, placed in a 

 test tube, plugged with cotton, and sterilized in the hot air oven at not over 170° C. The 

 desired number of these sterile squares are then immersed in a 24-hour broth culture of the 

 test organism, M. aureus. These paper squares, impregnated with the culture of M. aureus, 

 are then fished out with a sterile culture wire (bent at the end) and transferred to the anti- 

 septic. They are kept immersed in this preparation for 5 and 15 minutes, and they are 

 then transferred to a tube of broth (10 cc.) of the composition given above. By shaking 

 thoroughly at intervals over a period of 5 to 10 minutes, the excess of the antiseptic is washed 

 off from the paper squares. The pieces of paper are then fished out and transferred to another 

 tube of broth (10 cc.) and incubated at 37° C. for 48 hours. The tubes are then observed for 

 growth. 



Preparations that may be tested by this method are antiseptic oils, antiseptic 

 powders, antiseptic toothpaste, antiseptic dyes, shaving cream, and antiseptic soaps. 

 In the case of soaps, a thick lather must be made before the filter-paper squares are 

 used. Lozenges can also be tested in the same way after a concentrated solution has 

 been made. 



Antiseptic salves and ointments, however, must be examined by a special pro- 

 cedure which is, briefly, as follows: 



M. aureus of normal resistance is grown at 37° C. in the broth described above and trans- 

 ferred in this medium for three consecutive days. One-tenth of a cc. of a i-ico dilution of 

 this culture is added to 15 cc. melted nutrient agar contauiing 2 cc. normal sterile blood 

 serv.m at 45° C. (1.5 per cent agar in the broth), the culture thoroughly mixed in the agar 

 and poured into a sterile p^tri dish and allowed to cool at room temperature. As soon as 

 this inoculated agar has hardened, the salves and ointments, previously melted at 37° C, are 

 streaked over a small surface of the inoculated agar and the plate, inverted, incubated at 37° 

 C. for 48 hours. A vaseline control streaked in the same way is also included in this test. 

 After being incubated it will be noted that colonies of M. aureus grow immediately adjacent 

 to the vaseline control and even under it. There is no active ingredient in pure vaseline 

 which will prevent the growth of M. aureus. However, in effective antiseptic salves and 

 ointments a part of the active ingredients contained in them is absorbed into the agar 

 and prevents the growth of the organisms.^ Therefore, the plate will show a clear zone around 



' The M. aureus used must exhibit resistance to phenol as indicated above. 

 ' The organisms in the clear zone surrounding the antiseptic preparations are not only inhibited, 

 but usually actually killed except near the edge, as may be proved by subculture into broth. 



