AGGLUTINATION yal 
Macroscopic Agglutination. By this procedure the presence of 
agglutinins is demonstrated without the use of a microscope. To carry 
out this procedure, a series of small tubes are necessary. Mixtures of 
physiological salt solution, a twenty-four-hour broth culture of B. typhi 
and serum are used. Dilutions of 1-20, 1-40, 1-80 and 1-160 should be 
prepared. These may be secured as follows: The dilution of serum 1-10 
may be secured by drawing the serum up to 1.0 mark on the white blood 
corpuscle pipette and then filling the bulb to the mark with sterile salt 
solution or bouillon. Transfer 1 c.c. of this 1-10 dilution to the second 
test tube and add 1 c.c. of the twenty-four-hour broth culture. This 
will give the 1-20 dilution. The other dilutions may be prepared in 
the same way. These tubes should be incubated at 37° C. and exam- 
ined at frequent intervals for the presence of a precipitate. 
Microscopic Agglutination. This requires the use of a microscope 
but has the advantage of requiring less time. The same dilutions 
should be prepared as were mentioned above under macroscopic agglu- 
tination. Instead of putting them into small test tubes hanging drop 
mounts are made which are examined for agglutination after thirty 
minutes. Agglutination in a dilution of 1 to 80 after thirty minutes 
constitutes a positive reaction. 
Identification of Bacteria by Agglutination. The technique is essen- 
tially the same as for the detection of the presence of agglutinins. In 
this case the bacteria are the unknown factors while the serum is known 
to be specific for a certain organism. The bacterium should be grown 
in broth for twenty-four hours and tested against dilutions of the serum. 
Coles’ Method for the Microscopic Agglutination of Bacteria. The 
author claims that the method is easy, rapid and requires less blood than 
some of the other methods. 
1. Draw a line across the middle of two slides at right angles to their 
long axes. 
2. Spread a film of blood to be examined on one-half of each slide, 
and, when dry, spread film of blood from a person who has not had 
typhoid fever or received protective inoculation as a control over the 
other half. Dry the smears carefully over a flame. 
3. By means of a platinum needle place a small drop of an emulsion 
of killed typhoid bacilli on the center of each half of both slides. Run 
the drop well over the smears taking care not to pass from one-half to 
the other without sterilizing the needle. 
4. On one slide carefully place a cover glass on each half of the slide 
taking care that they are well separated by a wax pencil mark. 
5. Place the other slide on a wet-piece of blotting paper and cover 
