PRESENT STATUS OF [NVE8TIG ATIOH OF BEE DI8EA8E8. 29 



the drop of Quid, while tin- specimens can be K » - 1 ► t permanently and compare 1 

 w'nii one another. 



in order to study the growth of the spores, i used n cultivation on Hie agar 

 agar cultivating material which bad been kepi al the temperature of the body 

 for fourteen days, and which consisted almosl entirely of spores, though a few 

 bacilli were present. As the result «•!' several experiments, I have gol n • 

 of preparations which have been taken .-n various times i r> minutes, 30 inin 

 utes, l" minutes, 1 bour, l ' hours, 1 hour 50 minutes, 2 bours, 2 bourn 20 

 minutes, 2 bours 50 minutes, 2 hours 55 minutes, .". hours 20 minutes, I hours 

 L'(» minutes, 5 hours, 5 hours 35 minutes, 5 bours 10 minutes, and 7 hour- 50 

 minutes), and the course <>f events is shown in Plate X.° The bacilli 

 stain with various anilin dyes best, l think, with methyl-violet; bul the 

 spores resemble the spores of other bacteria in not taking on the stain. The 

 cover glasses on which the organisms are dried .-ire passed three times through 

 the gas flame and floated on the surface of a fairly strong waterj solution of 

 methyl-violet for one or two hours. They are then washed in water, and 

 afterwards laid in weak acetic acid I I per cenl i till no more stain comes out. 

 'They are again washed iii water, allowed to dry at the ordinary temperattTre, 

 and mounted in Canada balsam. A spore-bearing cultivation shows the bacilli 

 stained violet and the spores unstained, with the exception of their outline, 

 which is of a faint violet color. In most cases no trace of the rod in which 

 the spore was formed can be seen. The first change which is observed on 

 cultivation is that in many cases the outline of the rod in which the spore was 

 formed becomes faintly visible. This can be seen in 15 minutes, and is. I 

 think, simply due to swelling by the fluid, as it is also evident to sonic extenl 

 in the case of spores soaked in water for the same length of time. In from 

 halt* an hour to an hour it is evident that the bacilli which were present in the 

 original material are beginning to multiply, and a considerable number of rods 

 are now seen containing spores. It is evident that these spores are newly 

 formed, as extremely few bacilli containing spores were seen in the original 

 material, whereas in the preparations taken from a half hour to an bour a 

 considerable number are present. That some of the rods, instead of growing 

 by lissimi. at once proceed to form spores is probably to he explained in this 

 way. When the cultivation was removed from the incubator, some bacilli were 

 growing by fission, some were forming spores, and some had passed into ;i state 

 ready to form spores. The first go on growing by fission, the last complete 

 their spore-formation, which was arrested by removal from the warm tempera- 

 ture. That actively growing rods would not have formed spores so early 

 is evidenced by the facts observed in the second series of observations on the 

 formation of spores. The next thing that is observed is that several of ihe 

 spores take on the stain, and are as intensely violet as the adult bacilli. The 

 number of the spores which take en the stain in this way goes on increasing 

 as time passes, till in about \'>>uv hours almosl all the spores slain violet. In 

 three hours the first indication of sprouting of these spores becomes evident. 

 The stained part ol the spore loses its oval shape, becomes elongated, and i> 

 soon seen to burst through tin- spore-capsule at one part, it then presents the 

 appearance of a short rod. with a pale envelope embracing one end. This rod 

 gradually leaves the spore-capsule and then goes on multiplying as n full 

 grown bacillus. In specimens taken from four to five hours all stages of 

 growth can he seen, and the remains of ihe ruptured spore-capsules are evident. 



The bacilli appear to grow mainly by fission, hut I have seen appears 



•' Not reproduced here. 



