ON TECHNIQUE 53 



of this zone as outside of it; and where relatively large numbers 

 of measurements are made, this error tends to cancel itself out. 

 While on theoretical grounds none of these methods of measurement 

 are as accurate as could have been obtained, for instance, by the use 

 of a filar micrometer the large number of measurements made serves 

 to give at least a reasonably close approximation to the true measure- 

 ment, and because of the much greater length of time required, it 

 would have been practically impossible to make all these measure- 

 ments by use of a micrometer. 



In the case of the colon bacillus (Chapter V) and the cholera 

 vibro (Chapter VIII) I have attempted to illustrate the average size 

 and form of the cells by means of composite photographs. These 

 were prepared from the tracings of the cells on cards as described 

 in the paragraph above. The proper exposure for one card was de- 

 termined, and then all of the cards were photographed on one plate, 

 each receiving a proportional share of the entire exposure. The 

 cards were all so placed that the center of each cell came at the same 

 point on the plate, and that the long axes of the cells coincided sis 

 nearly as possible. If the cells were curved, the cards were so placed 

 that the major curvature always came on the same side. 



Nearly all of the measurements have been made from slides pre- 

 pared as above described, using the negative staining method of Beni- 

 ans, where the organisms are surrounded by a field of dark blue 

 Congo red. This procedure was chosen because it seems to give 

 the least amount of shrinkage of any of the methods used for making 

 permanent microscopic preparations of bacteria. The image is not 

 that of the bacterium itself as it appears after it has been dried and 

 fixed, but of the space which was occupied by the cell before it 

 underwent the shrinkage which occurs during drying and fixing. This 

 fact, previously stated by Eisenberg, can be easily demonstrated, for 

 with the large organisms, at least, one can see the shrunken cell 

 within the space which is not occupied by the dye, there being a 

 considerable zone between the cell and the edge of the dye. In a 

 preliminary series of measurements of a preparation of Bacillus mega- 

 therium, measuring first the living cells suspended in water, then the 

 clear spaces in the Congo red preparation, and finally a dried fixed 

 slide stained with methylene blue, it was found that the measure- 



