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Exercise 37. The Agglutination Test 
MATERIALS: 
Agar slant cultures of Alcaligines abortus 
Physiological salt solution 
Sterile hypodermic syringe 
Diluted immune and normal rahhit serum 
Graduated sterile 0.1 cc. pipettes 
Case 1 ce. sterile pipettes 
Rack for small test tubes 
10 small test tubes 
Rabbits 
The agglutination test is a means of detecting one group of antibodies in the 
blood serum of animals naturally or artificially inoculated with bacteria. 
These substances are called agglutinins. The test is highly specific, and con¬ 
sists of a clumping of a suspension of the bacteria in salt solution when they 
are brought in contact with their specific antiserum. The content of the ag¬ 
glutinins in a given serum is determined by dilution of the serum with 0.85 
per cent sodium chloride solution. The highest dilution of a serum which will 
agglutinate or throw the bacteria out of suspension determines the titer of the 
serum. The test may be made macroscopically in test tubes or microscopic¬ 
ally with a hanging drop. 
1* Immunize a rabbit against Alcaligines abortus as follows: 
(a) First inoculation. Inject, intraperitioneally 0.5 cc. of a sus¬ 
pension of the bacteria washed off an agar slope with salt 
solution. 
(b) Second inoculation. Five days after the first inoculation 
inject the animal as before with 1 cc. of a suspension of the 
same culture. 
(c) The third inoculation may be made five days after the second 
using a suspension of 3 cc. of the culture. 
2. One week after the last injection the animal should be bled. A prelimin¬ 
ary test may be made with a few drops of blood from an ear vein. In case the 
blood gives a sufficiently high titer, 1 TOO or above, a larger quantity of blood 
may be drawn. 
3. With the help of the instructor bleed the animal directly from the heart. 
About 8 or 10 cc. of blood should be taken with a sterile syringe. After draw¬ 
ing the blood is stored in a sterile test tube at ice box temperature, for 48 
hours. 
4. The clot, which soon forms, should be separated from the walls of the 
tube after 24 hours with a sterile glass rod. The contracting clot will squeeze 
out the serum, which should be clear and of a light yellow color. The clear 
serum should be removed with a sterile fine capillary pipette and stored in a 
sterile test tube for use. 
5. Use fresh 4 days old cultures of Alcaligines abortus and prepare salt 
solution suspension as was done for the inoculation of the animal. 
6. Prepare a stock solution of 1:10 from both immune and normal sera. 
This may be done by diluting 0.1 cc. of serum in 1.0 cc. of sterile salt solution. 
The other dilutions can be made from this stock dilution. 
