242 



DIPHTHEniA IN THE THOPICS 



Bacillary rorm 

 predominates 

 on subculture 

 on agar 



Involution 

 forms 



reacted faintly to the Neisser stain (Plate XIV., fig. 1). A pure culture of these cocci 

 was obtained on serum and on agar, both in tube and on plates, and it was felt that 

 the case was worthy of extended investigation, especially as the man's throat continued 

 to yield these cocci for some time, although he became to all appearance perfectly well. 

 Owing to the pressure of other work it is unfortunate that the exact date on which his 

 throat was found free of these cocci was not noted, while it is a pity that he was not 

 given a single dose of antitoxin and its effect on the micro-organisms observed. 



However, it was soon found that on subculture on agar, i.e. within 48 hours, the 

 cocci assumed a bacillary form, so that at first there were coccal and bacillary forms 

 together, and then, in some cases, bacilli alone (Plate XIV., figs. 3 and 4), all the cocci 

 having assumed the rod form. Attempts to separate the coccal from the bacillary forms 

 by means of agar and serum plates entirely failed. They were evidently the same organism. 

 Every care was taken in the culture work. My assistant, Mr. Buchanan, worked with 

 one set of plates while I worked with another, and our results were identical. The 

 reaction of the agar used was always +10 on Eyre's scale, except on certain occasions 

 (vide infra). The bacilli, as they developed, proved to be both Gram-positive and Gram- 

 negative and to resemble B. diphtherise morphologically, especially when transferred to 

 broth. They showed metachromatic granules when stained with Neisser. Cultures were 

 incubated at 37° C. and then kept at winter room temperature (about 95° F.) exposed to 

 the light. On agar slope, examination with a magnifying lens showed the small, crowded, 

 white colonies to be discrete. 



It was found that original coccal cultures on blood serum slopes remained coccal. 

 The colonies were small and discrete, and the cocci, ou staining with Gram, proved 

 negative save for granules which, in a certain proportion of them, retained the stain. 

 Cultures on serum plates, kept at the room temperature, eventually showed both forms. 

 The agar cultures and subcultures showed the progressive change of cocci into bacilli. 

 The coccal form appeared to divide and produce a small diplo-bacillus and this gradually 

 became the apparently typical B. dijphtherim, showing polar bodies when stained by 

 Neisser's method, barred forms and forms like those shown in Plate IX., fig. 2, of 

 Nuttall's and Graham-Smith's standard work.' Involution forms exactly resembling those 

 of the Klebs-Loeffler rods eventually developed, and, in old agar cultures, the bacilli tended 

 to become Gram-negative. Natm-ally it was not sufiScient to base any conclusions on 

 morphology alone, and the following cultural reactions were determined. 



1. Ordinary alkaline broth. The growth exactly resembled that of B. diphtherim in 

 this medium, there being no turbidity but a formation of whitish granules at the 

 bottom and along the sides of the tubes. In older subcultures a slight film formed 

 on the surface which, on shaking, broke up and sank slowly to the bottom of the 

 broth. 



2. Glucose broth. In 1 per cent, glucose broth the organism, whether in a coccal or 

 a bacillary form, produced acid — slight in 24 hours, more marked in 48 hours. 



3. Milk. There was no clotting, but acid was produced, then alkali, and then acid 

 again, a curious alternating reaction being obtained. 



4. Litmus whey. The coccal form from serum cultures produced acid as did the bacillary 

 forms from agar cultures, but to a less extent. 



5. Mannite broth. In 1 per cent, mannite broth with Durham's tube, the cocco-bacillus 

 produced neither ^rn^i nov acid. 



' Nuttall, G. H. F., and Qraham-Smitb, Q. S. (1908), Tht Bacteriology of Diphtheria, Cambridge 



