478 MEMOIKS OF THE NATIONAL ACADEMY OF SCIENCES. 



1. Prepuration of ciiUnres nucd for inocidation of af/ar-iif/ar. — TiibfS of bouillon, each contain- 

 ing 10 cc, are inoculated with one drop of a bouillon culture and placed in the incubator for 

 twenty-four hours. A small quantity of sterilized gravel is then added to the culture tube and 

 it is shaken thoroughly, so as to separate the germs and disperse them evenly throughout the 

 bouillon. Ten cubic centimeters of a one-half ])er cent salt solution are then added, and the 

 culture is drawn into a dropping apparatus like the oue used by Nuttall for counting tubercle 

 bacilli, which he describes as follows (Bulletin of the Johus Hopkins Hosjiital, May-June, 18'Jlj : 

 "It consists of a finely graduated burette, to which a finely drawn-out dropper is attached by a 

 rubber coupling. From the upper end of the burette proceeds a piece of rubber tubing which 

 leads — a glass stopcock intervening — to a mouthpiece, which can be placed at the highest point 

 of the apparatus out of danger of any possible contamination." By simply filing a snuill groove 

 with a triangular tile on one side of the aperture in the stopcock, the groove gradually fading 

 as it proceeds away from the opening, a most effective and simple means of controlling the rate of 

 dropping was found. When ready to use the stopcock is turned enough to allow about twenty 

 drops to a cubic (centimeter. 



i'. Inoculation of agar-agar. — Oue drop of the bouillon culture was dropped iuto a tube of 

 melted agar-agar, which was slowly and thoroughly agitated, aud then jioured iuto a Petri plate 

 placed upon a leveling tripod over ice water. 



3. Isolation of cultures. — The plates, when solidified, were placed in an east window, bottom 

 upward, aud half of each i)late covered with black paper or glass of different colors. A ther- 

 mometer with a blackened bulb was placed in the sun to note the temperature. At intervals of 

 fifteen minutes a plate was removed and placed in the incubator. A control plate made at the 

 same time as the others was placed immediately in the incubator. 



1. Counting the colonies on plates. — An eyepiece was divided iuto fields, as done by Nuttall, 

 by introducing a disk of black cardboard, which has a square opening divided into four parts by 

 two hairs placed at right angles. This eyepiece and an objective of low power were used in 

 counting. The colonies in each field were counted iu exactly the same portion of each half of the 

 jjlate and from the sum of these numbers the per cent was estimated. Thus the ])er cent 

 destroyed by insolation was ascertained, aud also an estimate of the difference of protection 

 afforded by the different colors used. The i^lates were insolated on clear, sunny, still days. If 

 the sun became obscured during the period of insolation the plates were counted to note the 

 effect, but the per cent was not used in the final estimation. The temperature of the plates 

 during the insolation was kept below 34° G. in every instance. Each jjlate used for counting was 

 insolated for either fifteen, thirty, forty-five, sixty, ninety, one hundred and five, or one hundred 

 and twenty minutes, and then placed in the incubator. Plates uniform in size and as level as 

 could be obtained were used, so that 10 cc. of the medium would be evenly distributed by using 

 the leveling apparatus. 



Cultures were also insolated for varying periods of time. A culture of the staphylococcus 

 pyogenes aureus exposed to the rays of the sun for nine hours gave six colonies on the insolated 

 portion. The number of colonies on the other half, protected by red glass, was also lessened. 

 Plates of the bacillus coli communis were iusolated for six hours, one-half of each plate being 

 covered with either red, yellow, or blue glass. Five colonies were Ibuiul on the iusolated portion 

 of the plate protected by red glass and a few colonies at one edge of the plate covered with 

 yellow glass. Under the glass the number of colonies had decreased somewhat. On the plate 

 with a blue-glass shade eight colonies were found on the iusolated half and the same number 

 on the protected part, the result being that at the end of six hours the colonies had been equally 

 destroyed on both sides of the plate. Four plates of the bacillus typhi abdominalis shaded with 

 black, red, orange, or blue glass were insolated three hours. From these,.six colonies were found 

 on the insolated half of the i)lates, protected by black, red, and orange. Not a colony was found 

 on the entire plate, which was half shaded with blue. 



Diffused //r//;/.— Plates were made and exposed in the same manner as used for suulight. 

 Clear, sunny days were taken for the cxjieriments, the plates being placed in a light part of a 

 room and exposed for periods varying from fifteen minutes to two days, aud then placed in the 

 incubator for twenty-four hours and counted as before. Many plates were made, using the 



