8 MASS. EXPERIMENT STATION BULLETIN 323 



At the outset of the investigation a testing apparatus was employed which 

 was heated with tempered water. The temperature of the test plate was main- 

 tained between 80° and 95° F. The apparatus was not equipped with artificial 

 light. 



Tube Method. The technique of the tube agglutination method was identical 

 with that described in the Standard Methods (11) of Diagnosis for Pullorum 

 Disease. A diagnostic dilution of 1 :25 and an incubation period of 24 hours at 

 37° C. were employed. 



MISCELLANEOUS EXPERIMENTS 



Since standardization of the tube test has brought forth more uniform and 

 reliable results in pullorum-disease testing, it is only logical to assume that the 

 same would hold true if the technique of the whole blood test were perfected and 

 standardized. It is apparent that there is a great lack of uniformity in the manner 

 in which the whole blood test is conducted at the present time. Investigations 

 have revealed several factors that appear to play an important role in the accuracy 

 of the test. The need for further investigation of these influencing factors is 

 apparent, since various investigators disagree on certain phases of the whole 

 blood test. Increased efficiency of this method cannot be expected until each 

 problem has been analyzed and solved. This naturally requires a scientific ap- 

 proach in order to determine the true significance of the different factors influenc- 

 ing the accuracy of the test. 



In the development and standardization of the tube test, the serum-antigen 

 dilution factor was found to play an important role in the successful elimination 

 of infected birds. At first the fact was not appreciated that some infected birds 

 may exhibit low titres or intermittent reactions. Such birds would be overlooked 

 with a serum-antigen dilution above the serum agglutination titre. It follows that 

 with the use of the whole blood test the blood-antigen dilution should receive 

 further study. Measurements of blood and antigen have been lacking in uniformit v 

 and constancy in the methods described. 



In a few miscellaneous experiments an effort was made to determine what 

 variation in the quantities of blood and antigen might exist in the whole blood 

 test and its influence on the agglutination reaction. 



Antigen Measurements. A special pipette, simulating a medicine dropper, was 

 used for measuring the antigen by drops. Realizing that the size of the drops 

 might vary, a series of measurements was made to determine the amount of 

 antigen and its variation in the drops. Table 1 shows that in a total of 100 drops 

 the quantity of antigen varied from .042 to .055 cc. The majority of the drops 

 ranged from .044 to .047 cc, and the average quantity for the iOO drops was 

 .0470') cc. While these measurements were made in the laboratory under most 

 favorable conditions, it is likely that the variation might be even greater under 

 field conditions, when several operations are carried on simultaneously and at 

 greater speed. 



