THE INFECTIOUS DISEASES. 287 



one minute. The proportions are 2 c.c. of a ten-per-cent solution of formalin to 100 c.c. 

 of strong ethyl alcohol. The organisms are most easily found and studied in prepara- 

 tions colored with thionin. The formula for the stain is 20 c.c. of a saturated solu- 

 tion of thionin in fifty-per-cent alcohol, added to 100 c.c. of two-per-cent aqueous 

 solution of carbolic acid. The stain requires several days to ripen and then keeps indefi- 

 nitely. Its action is rapid, requiring only about fifteen seconds to color the malarial 

 organisms deeply. 



In order to demonstrate the nuclear chromatin of the malarial parasites it is neces- 

 sary to use special stains, the most useful being a modification of that originally devised 

 by Romanowski for this purpose and improved by Giemsa. 1 Two substances are neces- 

 sary; first, an aqueous solution of "methlyene azure II., Grubler," 8 dgm. to the 

 litre, and second, an aqueous solution of eosin, " extra water-soluble, HOchst. " These 

 two stock solutions are permanent if kept in dark-colored bottles. The smears to 

 be stained are fixed in methyl alcohol for about ten minutes. The staining mixture 

 is prepared by adding 1 c.c. of the methylene azure II. to 10 c.c. of the eosin solution. 

 The staining mixture is poured into a Petri dish and the slide immersed in the fluid 

 with the blood side down. The process is complete in from fifteen to thirty minutes 

 The slide is washed off for about ten seconds with a strong stream of distilled water, 

 dried in the air without heat, and embedded in dammar dissolved in xylol. A simpler 

 method has recently been published, 2 which is better adapted for clinical work. The 

 smear is fixed for two minutes or more in methyl alcohol, then stained for ten seconds 

 with a 1:1,000 aqueous eosin solution, the latter allowed to run off the slide, and the 

 smear again covered with a few drops of a one-fourth-per-cent solution of methylene 

 azure II. In about from fifteen to thirty seconds the staining is complete. The slide 

 should be washed in distilled water, dried, and examined directly with an oil-immersion 

 lens, no cover-glass being necessary. 



INFECTIOUS DISEASES OF UNKNOWN ORIGIN. 



Notwithstanding the great increase in our knowledge of the infectious diseases made 

 possible within the past few years by the new methods of cultivation of micro-organisms 

 and by animal experimentation, there are still several diseases obviously of this class 

 whose inciting factors are wholly unknown to us. We are now beginning to realize 

 that we must not confine our search in this class of diseases to the bacteria alone ; but 

 that among the protozoa may be found most important infective agents. In this latter 

 group of organisms the culture methods applicable to the bacteria do not furnish the 

 necessary data for the establishment of etiological relationships. While the significance 

 of the malarial protozoan has been determined without cultures, the desirability of arti- 

 ficial culture methods in the study of protozoa is daily becoming clearer. 



In the search for the inciting agents of infectious diseases which have as yet baffled 

 investigation, it should be borne in mind that it is quite possible that bacterial and 

 other micro-organisms may exist which are ultra-microscopic ; that is, so small as to be 

 invisible with such microscopic resources as we at present possess. In one instance, the 

 infectious peripneumonia of cattle, Nocard and Roux by a special technique have 

 been able to isolate an organism so small that its morphological characters could not be 

 learned even with the highest available magnification. Experiments with the filtration 

 of infectious material, especially from certain communicable diseases of animals, 

 through porcelain filters whose pores are so fine as to retain ordinary bacteria, have 

 shown that the infective agent may pass these filters and though revealing no morpho- 

 logical elements is still virulent. The possibility of the existence. of ultramicroscopic 

 organisms must then be held in mind in our summaries of infectious diseases whose in- 

 citing factors, though persistently sought, are still unknown. 3 



Cent. f. Bakt., vol. xxxii., 1902, p. 307. 



* Wood, Medical News, August 8th, 1903, p. 248. 



3 For a most interesting and suggestive summary of recent investigations on infec- 

 tious diseases of unknown origin, see Ilektoen, Jour. Am. Med. Assn., August 15th and 

 22d, 1903; see also Roux, " ' Invisible ' Microbes," Bull, de 1'Inst. Pasteur, tome i., 1903, 

 pp. 7 and 49, bibl. 



