1917] VETERINARY MEDICINE. 783 



before death. If the increase in leucocytes just before death is occasioned pri- 

 marily by the hog-cholera virus or is due to a secondary infection with Bac- 

 terium suipestifcr or suisepticus can not be determined." Two pigs showing a 

 high ante-mortem leucocyte count were secondarily infected with B. suipestifer. 



The staining method used for blood smears is briefly as follows : The air- 

 dried smears were stained in a methyl alcohol solution of eosin, methylene blue, 

 and toluidin blue for three or four minutes, immersed for a few minutes in 96 

 per cent alcohol, washed in water, superficially dried, and then floated for 16 

 hours on a diluted Gierasa solution (1: 10) alkalized with two drops of 1 per 

 cent sodium carbonate or borax solution to 10 cc. The smears were then thor- 

 oughly washed in running water, air-dried, and mounted in cedar oil or in 

 paraffin oil. This method is considered to be superior to that previously de- 

 scribed. 



Some observed histological changes in hog cholera which will be reported 

 later in detail, together with the macroscopic changes, are noted. 



For the cultivation of the virus both the blood and organs of pigs which suc- 

 cumbed to hog cholera were used. The blood was collected aseptically, defibri- 

 nated, centrifugalized, and the serum passed through a Berkefeld filter. The 

 filtrate was tested aerobically and anaerobically for common bacterial contami- 

 nation. 



(;)nly perfectly sterile serum was used for culture purposes. Unfiltered sterile 

 carcinomatous ascites and sterile unfiltered horse serum were used as culture 

 media. Either was placed in sterile test tubes to which a piece of fresh kidney 

 or liver tissue from a guinea pig or rabbit was added, and covered with sterile 

 paraffin oil. The tubes were then incubated for a week at 37° C. Tests were 

 made to insure complete sterility. 



To the sterile culture tubes filtered hog-cholera serum equal in amount to the 

 cxilture medium was added by means of a sterile pipette. In one case blood 

 taken directly from the heart which proved to be sterile was added directly to 

 the culture medium. Cultures were made from the organs by taking pieces 

 aseptically removed from the dead animal and immersing them in unfiltered 

 sterile carcinomatous ascites or unfiltered sterile horse serum and then cov- 

 ering with sterile paraffin oil. The tubes which showed a high secondary in- 

 fection were discarded. Those which were but slightly contaminated after in- 

 cubation for a week were filtered through filter paper and then through a Berke- 

 feld filter. The filtrates so obtained were used for subcultures, as previously 

 described. 



After two or three weeks the culture medium showed a slight opalescence 

 which gradually disseminated through the liquid. In cultures made from 

 filtered virus with the addition of a piece of fresh tissue a groAvth was ob- 

 served in four weeks. In others, however, where the presence of the cocci 

 was demonstrated microscopically, hardly any change in the culture medium 

 could be noted. Several hundred cultures so prepared were examined micro- 

 scopically. The data reported confirm the previous microscopic findings. 



With the staining method described it was possible to demonstrate large 

 masses of microorganisms attached to the red cells. These findings corrob- 

 orate those of Meyer (E. S. R., 32, p. 475), who showed that hog-cholera virus 

 adhered tenaciously to the red blood cells and that it was impossible to re- 

 move the virus by repeated washings with normal saline solution followed by 

 centrifugalization. 



It is indicated that " as soon as sufficiently distant subcultures are obtained, 

 such that the transmission of the original virus is absolutely excluded, animal 

 experiments will be made to furnish conclusive proof that this organism is the 

 causative agent of hog cholera." 



