SPECIAL FOKMS OF CONJUNCTIVAS INFECTION 193 



When kept cool and in the dark, or hermetically sealed in an incubator, diph- 

 theria bacilli retain their vitality for many months ; they have considerable powers 

 of resistance against dryness and cold. Moist heat, on the contrary, kills them at 

 60 C. very rapidly. 



The development of acidity in bouillon is best demonstrated by titration with 

 1 in 40 normal caustic soda (phenolphthalein as indicator) ; it especially occurs 

 in sugar bouillon. For cultures in 1 per cent, sugar bouillon twenty hours old 

 Lehmann and Neumann, used 3 c.cm. caustic soda ; forty hours old, about 6 c.cm. ; 

 and for cultures in bouillon without sugar about half these amounts. 



The passage of toxin out of the bacilli into the bouillon only begins with the 

 cessation of acid formation. Old cultures, therefore, after filtration show typical 

 toxic activity ; fresh twenty-four-hour cultures do not, unless containing the bacilli. 



Staining Reactions. The diphtheria bacilli stain very intensely by Gram's 

 method. It has been stated that old degenerate bacilli lose their colour. This 

 has no practical importance in diagnosis, and when examining fresh cultures we 

 can, with absolute certainty, rely on obtaining an intense colour with Gram's stain. 

 I must again emphasize the fact that an even staining with this method can only 

 be guaranteed when the material has been evenly spread out. In places where 

 clusters of bacilli lie in dense masses of secretion the deeply-placed bacilli are often 

 unstained (the same is true for xerose bacilli). 



Intensely stained with Gram, the young small bacilli appear of an even colour ; 

 in the older and longer forms a more intense staining occurs at the poles. Seg- 

 mentation, or granule formation, occurs in the large club-shaped masses, and narrow 

 stained sections alternate with clear intervals (zebra staining). This segmentation 

 is much more obvious in cultures and with Loffler's methylene blue or Eoux's 

 mixed stain (see p. 17). The Babes-Ernest bodies, segments, or polar bodies then 

 appear blue or violet ; when Loftier' s method is used the intervals are either light 

 blue or unstained, and when Koux's is used they are light green. The long rods or 

 threads, especially obvious in involution forms, may be taken on superficial examina- 

 tion to resemble Streptococci. 



The granules are first formed at the poles, and are most clearly seen in that 

 situation ; they are best displayed by M. Neisser's stain (acetic methylene blue and 

 Bismarck brown). A preparation is to be considered as positive and absolutely 

 diagnostic when the bacilli have the typical slender form, are arranged in the charac- 

 teristic manner, and at the ends of their yeUowish-brown bodies occasionally, too, 

 in their intermediate parts show dark blue granules ; not merely here and there, 

 but in every example. 



In secretion preparations this method may give very good results, and in such 

 cases a probable diagnosis of diphtheria is justifiable. On the other hand, a negative 

 result with Neisser's stain in a secretion preparation is of no value in the opposite 

 sense. 



According to M. Neisser, the following conditions are necessary for the diagnostic 

 value of this method : the cultures must be fresh, of the first generation, and grown 

 on good LofHer's serum at a temperature of 34 to 36 C. Cultures which in nine to 

 twenty hours show the typical staining and typical form were considered by him to 

 be true diphtheria. Older generations, after longer propagation, do not conform to 

 this rule. It is important to have a good Loffler's serum, and to make certain that 

 this is the case every new batch of tubes should be tested with an inoculation of 

 true diphtheria. 



Pseudo- Diphtheria Bacilli. 



Besides the toxic diphtheria bacilli, we find on various mucous membranes, both 

 in a state of health and of disease, rod-shaped organisms, which are more or less 



13 



