﻿ETIOLOGY OF DENGUE FEVER. 115 



tion, which was then ejected, a very little being allowed, to remain in 

 the needle; the syringe was then filled with blood by plunging the needle 

 into a prominent vein of the forearm and withdrawing the blood very 

 slowly until the barrel of the syringe was full ; the blood was then ejected 

 into small sterilized glass tubes and kept at room temperature in the 

 incubator or in the lower compartment of an ice box, the latter in 

 order to give any organism undergoing a part of its life cycle in a 

 cold-blooded animal, surroundings congenial to its development. 



In making blood cultures in bouillon, 10 cubic centimeters of blood 

 obtained from the median basilic vein were added to 250 cubic centi- 

 meters of bouillon contained in 500 cubic centimeter flasks, and in- 

 cubated at temperatures of from 26° to 36.05° C. 



In preparing our culture's the utmost care was taken to avoid infec- 

 tion, everything being rendered sterile that could be, but despite all of 

 our precautions the majority of our cultures sooner or later became con- 

 taminated with various forms of bacteria or yeasts. 



(b) Citrated blood cultures. — In eight cases we have endeavored to 

 secure cultures of the organism causing dengue by citrating blood ob- 

 tained from dengue patients at various periods of the disease. In none of 

 these have we been able to demonstrate any organism which we can 

 consider to be of any etiologic significance; in none of the tubes of citrated 

 blood did we encounter any organism resembling, in the least, a protozoon, 

 and all of the bacteria observed were evidently contaminations. A small 

 diplococcus occurred in two cases, but in the light of our later work on 

 filtered blood this obviously is of no significance. 



(c) Bouillon blood, cultures. — In twelve cases we used bouillon blood 

 cultures, allowing them to incubate for as long as eight weeks. The 

 majority of the flasks became infected, but in four cases the blood cultures 

 did not show any growth at the end of eight weeks, when they were 

 destroyed. A staphylococcus grew in one in forty-eight hours, a diplo- 

 coccus in three in seventy-two hours, accompanied by a large spore- 

 bearing bacillus in two of the cases ; a- short, thick, motile bacillus, 

 together with a staphylococcus in one, in four clays and various spore-bear- 

 ing baccilli in the remainder. These organisms we believed to be conta- 

 minations and therefore we did not perform any experimental work with 

 them. Our conclusions regarding them were confirmed by the result of 

 our experiments with filtered blood 



As the result of our culture experiments we were forced to conclude 

 that no organism was found constantly enough in the cultures to warrant 

 us in regarding it as having an etiological relationship to the disease, 

 especially as a number of them remained sterile, although kept for as long 

 as eight weeks. 



