DIAGNOSIS OF INFECTION WITH B. PULLORUM. 6 



birds were so incomplete concerning their histories, it was considered wise 

 to put all together and include them all in the tests. Birds Nos. 1, 2, 4, 

 5, 6, 7, 8, 10, 13, 18, 22, 48, 52 and 53 were all more than three years old, 

 and were sent to this laboratory through the kindness of Director H. J. 

 Patterson of the Maryland Agricultural Experiment Station, where the 

 author had started work to determine the possibility of artificial infection 

 of ovarian tissue by intravenous injections of the organisms, — work 

 which was interrupted before final results could be obtained.^ Jones,^ 

 however, was successful in his attempts to bring about ovarian infection 

 with Bad. pullorum by the injection of pure cultures of the organism into 

 the blood circulation of hens. 



All individuals retained for these tests were trap-nested and complete 

 egg records kept of each hen. 



Methods employed in the Examination of Eggs for Bacterium 



Pullorum. 



The object primarily in making the examination of all eggs laid by 

 these suspected hens was to determine if possible the presence of the 

 organism in the yolk, which would be of value in checking up the work 

 in connection with any of the serum reactions which later might prove 

 positive. The method used for these egg analyses was essentially that 

 used by Rettger.' Eggs were allowed to remain several minutes in car- 

 bolic acid (1-40) and dried with sterile absorbent cotton. The end of the 

 egg was sterilized by flaming, the flamed portion cut around with sterile 

 scissors. The albumin was carefully separated from the yolk and the 

 yolk inserted into a large test tube (Buchner type) containing about 

 30 c.c. sterile bouillon. In the first part of the egg-testing work fresh 

 eggs were studied, but later the eggs were incubated prior to the testing, 

 and in some instances sufficiently long for embryos to develop. In such 

 cases a sterile platinum loop or scissors were used to aid in freeing the 

 embryo from the shell and albumin. If embryo was very large it was 

 inserted into sterile bouiUon along with the rest of the yolk. The disin- 

 tegrated egg yolks in bouillon were placed in the incubator at 38° C. and 

 aUowed to remain there for varying lengths of time, the shortest period 

 being twenty-four hours and the longest two hundred and eighty hours. 

 After tubes were taken from bacteriological incubator the material was 

 thoroughly mixed and four samples streaked on four different tubes of 

 agar. These were placed in the bacteriological incubator and examined 

 macroscopically for the presence of the typical Bad. pullorum colonies 

 at the end of twenty-four, forty-eight and seventy-two hours. A tube 

 was not considered negative until it had been allowed to incubate for 



1 Work referred to by Dr. Rettger in Bulletin No. 74, Storrs Agricultural Experiment Station 

 Storrs, Conn., p. 162, line 12. 



2 Jones, F. S., " Further studies on bacillary white diarrhoea in young chickens." Report, 

 New York State Veterinary College, 1910-11, pp. 09-88. 



3 Rettger, L. F., and Stoneburn, F. H., Bulletin No. 68, 1911, Storrs Agricultural Experi- 

 ment Station, " Bacillarj' white diarrhcEa of young chicks" (second report). 



