48 MORPHOLOGIC VARIATION 



lated, but aeration was accomplished by thoroughly shaking the flasks 

 every half hour during growth. Samples were removed from the 

 flasks with sterile pipettes. 



The difficulties met with in broth cultures can be obviated by 

 using solid media, agar slants, the cells being sufficiently concen- 

 trated and obtained practically free of products of the medium itself 

 by simply scraping them off with a wire loop. The use of agar, 

 however, introduced certain other difficulties. In an ordinary agar 

 slant the medium varies in depth from the bottom to the top, and the 

 conditions of growth are therefore not uniform throughout the tube. 

 As a matter of fact, growth is heavier and probably also more rapid 

 in the lower part of the tube. In order to obtain a representative 

 sample from agar slant cultures, then, it is necessary to suspend 

 the entire growth in water and remove a sample from this suspension 

 for examination. This necessitates using at least one agar slant cul- 

 ture for each observation, and therefore the preparation of a large 

 series of agar slant cultures as uniform as possible in size of seeding 

 and conditions of growth for the entire period of observation. This 

 was approximately accomplished by using a large series of measured 

 test tubes of practically uniform diameter. These were obtained by 

 first boring two holes into a piece of sheet brass, differing in diameter 

 by about one-half millimeter. Test tubes as they came in the com- 

 mercial packages were then selected by means of this piece of brass, 

 only those tubes being chosen which would pass through the larger 

 opening but not through the smaller one, the variation in diameter 

 of the tubes thus being practically negligible. These tubes were 

 then filled, each with 5 c.c. of agar carefully measured, and were 

 also sloped, as far as possible, at the same angle. The tubes, there- 

 fore, were probably sufficiently uniform for the purposes, the diffi- 

 culty being in obtaining uniform inoculation. 



Seeding was accomplished by preparing a suspension in sterile 

 water, or by using a peptone or broth culture, and one loopful of 

 such a suspension was spread as uniformly as possible over the sur- 

 face of each slant. Undoubtedly the loopfuls varied somewhat in 

 size, and more particularly it was difficult to maintain uniformity 

 of distribution of the seeding over the surface of the agar slant. 

 At any rate there was a certain amount of unavoidable variation in 



