452 DESTRUCTION OF TYPHOID AND COLON BACILLUS. 



is produced, in which case countiug" of the colonies is pisictically 

 iiiil>ossible. 



For this reason the followiuj;' method was used for each of the three 

 orgauisins, the staphylococcus pyo.uenes aureus, the bacillus coli coui- 

 munis, and the bacillus typhi abdominalis. 



To obtain an accurate measure of the effects produced by lights of 

 different intensity or of different colors, it is necessary to insure, as far 

 as possible, that the bacteria to be experimented on shall be uniforndy 

 distributed in the culture media. Tubes containing each 10 c. c. of 

 bouillon were inoculated with one drop of a bouillon culture and tJjen 

 placed in an incubator for twenty-four hours. A small quantity of 

 sterilized gravel was then added to the culture tube and it was thor- 

 oughly shaken, after which 10 c. c. of a one-half per cent salt solution 

 was added and the culture drawn into a Nuttall's dropping apparatus. 

 From this one-twentieth of 1 c. c. of the bouillon culture was dropped into 

 a tube of melted agar-agar, which was slowly and thoroughly agitated, 

 and the contents were then pouied into a l*etri dish, carefully leveled 

 on a leveling tripod over ice water. In the first method used the Petii 

 dishes were found to be so uneven on the bottom that the layer of 

 medium under the protective square was often very thick or very thin 

 as comi)ared with that about the circumference of the plate, and there- 

 fore c()ini>arisons made between the center and the circumference would 

 be in almost every case unreliable. To overcome this difticuUy Just 

 one-half of the plate was shaded with black paper or colored glass. 



The plates were then exposed to sunlight bottom upward, so as to 

 allow the sun to shine as directly as possible on the inoculated agar- 

 agar. At intervals of fifteen minutes a plate was removed and placed 

 in the incubator. The temperature of the plates during insolation was 

 always below ;U^ C, as shown by a thermometer with a blackened 

 bulb which was placed in the sun and the temperature noted every 

 fifteen minutes. Sunny, still days were utilized for insolation, begin- 

 ning at 10 a, m., during the months of October, November, and Decem- 

 ber. After insolation the plates, and also a noninsolated control plate, 

 were incubated for twenty-four hours. 



The colonies were counted in the following manner: A Xo. 1 eye- 

 piece was divided into fields (as done by ISTuttall in counting tubercle 

 bacilli) by introducing a disk of black cardboard which had a square 

 opening divided into four parts by two hairs placed at right angles. 

 This eyei)iece and an objective of low power were used in counting. 

 The percentage of germs destroyed by insolation was estimated from 

 the mean of four counts taken on both the insolated and the protected 

 halves of the plate. By this method an accurate statement can be 

 made regarding the difference in protective power given by the differ- 

 ent colors, not from simple observation, but by comparison of a definite 

 number of colonies counted. 



