14 MASS. EXPERIMENT STATION BULLETIN 148. 



tlie desired dilution and thoroughly shaken each tube to afford a com- 

 plete mixture of the agglutinative sera and Bacterium, pullorum all was 

 placed in the bacteriological incubator at 38° and readings made of the 

 macroscopic agglutinative picture at the end of twenty-four, forty-eight 

 and seventy-two hours. All tests were controlled, i.e., test fluid and 

 agglutinative sera. 



A positive macroscopic agglutination reaction is evident when the 

 formation of fine, flake-like masses settle to the bottom of the tube into 

 uneven heaped-up masses at the bottom and sides, leaving the super- 

 natant fluid clear. This reaction is usually very prompt, and with sera 

 of marked potency it is very clear and definitely defined. Controls should 

 always be kept for check of test fluid, and check of diluted serum in car- 

 bolated salt solution. 



The test fluid used for our work at first was composed of the 6 tested 

 strains of Bacterium pullorum preserved by 0.5 per cent, carbolic acid 

 and kept on ice. The serum was used continuously until no positive re- 

 actions would result in a serum known to be positive, and from this it was 

 possible to determine about how long a serum could be retained under 

 proper conditions and be in an active state for use in making the test. 



In tables 7 and 8 which follow can be seen the results of the 

 macroscopic tests on the birds carried out by three different technicians 

 working independently. The technician is denoted in the column of that 

 legend as 1, 2 and 3. It is indeed interesting to note that the work of 

 the three technicians checks very well, and from the summary of the work 

 of each no difference ever arose as to whether a bird was or was not a 

 reactor. Hens Nos. 267, 8, 1, 10, 6, 2096, 10, 18, 6, 2, 48, 7, 13, 53, 792, 

 714, 464 and 61 were all proven by all three technicians to be infected hens, 

 having the agglutinin present, varying in its powers to cause agglutina- 

 tion of Bacterium pullorum. From tables 7 and 8 it can be seen that 

 with the results in the agglutination work, especially the is made with 

 the blood drawn on July 19, and with the serum of hens Nos. 267, 2, 10, 

 2096, 5, 2, 48, 52, 53, 452, 792, 714 and 464, the serum reactions were 

 consistent with the three technicians until the seventeenth or eighteenth 

 days, when the reactions began to vary considerably. This is indeed 

 an interesting feature in favor of the test, and it is possible, under better 

 conditions of preservation, that it may be kept longer. After the hun- 

 dreds of tests made in this laboratory it would be safe to state that prop- 

 erly preserved and cooled agglutinative sera may be retained in a good 

 state for subsequent tests for as long as two weeks. On the other hand, a 

 carefully prepared test fluid, made from newly incubated cultures of Bad. 

 pullorum, and suspended in 0.85 per cent, physiological saline solution 

 containing 0.5 per cent, phenol, if retained on ice will remain in good 

 condition for making the tests even after two months. 



In some instances a serum retained for three weeks, when used by one 

 technician on the 6th of August reacted, and had lost its agglutinative 

 powers on the 7th when used by another technician. At the beginning 



