viii„-6 MANUAL OF METHODS FOR PURE CULTURE STUDY 



heart can now be located with the needle which will be moved by the heart beat. Force 

 the needle into the heart. When it is in the heart, blood will flow into the syringe. 

 Slowly withdraw 50 ml. Quickly withdraw the needle and eject the blood into a 500 

 ml. Erlenmeycr flask, or into a large test tube and allow it to clot. The serum may be 

 obtained free of clot and cells by centrifugation. Five to six pound rabbits can be 

 bled monthly in this way. If the rabbit is to be sacrificed, another 50 ml. portion of 

 blood can be obtained in a similar manner, but preferably with another clean sterile 

 syringe and needle in order to avoid clotting in the syringe. 



PROCEDURE 



Procedure for Microscopic Agglutination Test: Dilutions of the 

 serum are prepared by diluting the immune serum with saline solu- 

 tion, care being taken to keep the serum twice the strength of the 

 final dilution desired, since the addition of an equal volume of the 

 antigen doubles the dilution of the serum on each cover slip. Upon 

 separate clean cover slips is placed a loop of the diluted serum. A 

 loop of the suspension of the organism is placed beside each drop of 

 diluted serum and the two mixed with a platinum wire. The cover 

 slips are then suspended over hollow ground slides as noted in the 

 technic for preparing a hanging drop preparation. The slides may 

 be held at room temperature for a short time, usually less than one 

 hour, and examined under a magnification of approximately 500 

 diameters. 



Some experience is necessary to discriminate between normal 

 reactions and false dumpings. In the true reaction all the organisms 

 in the field will be gathered into a few clumps and no organisms will 

 be found around the edges of the drop. In pseudo-reactions the 

 organisms may collect around small foreign particles, around the 

 edge of the drop, and in many small clumps containing a relatively 

 small number of cells. The beginner generally uses too heavy sus- 

 pensions. Much sharper readings can be made w^ith a very light 

 suspension of the organism being studied. 



Macroscopic Agglutination Test. Antigen: Wash off in saline the 

 growth from a 24-hour agar slant culture of the organism to be tested. 

 An emulsion which is too thick obscures the agglutination, while one 

 which is too thin does not provide enough bacteria for macroscopic 

 comparisons. The density of the emulsion of bacteria must be ad- 

 justed to meet the requirements of special conditions and to assure 

 constancy in the results. This adjustment can be made on the basis 

 of an actual count of the number of bacteria per ml. or by compari- 

 son with a standardized suspension of insoluble particles. The 

 latter method is usually more convenient, using the McFarland (1907) 

 nephelometer. A density of 0.5 on the McFarland nephelometer 

 scale is satisfactory for most purposes. The suspension should 

 be homogeneous, "smooth", and entirely free from particles. The 

 bacteria in the suspension may be killed by heat at 60°C. for 1 hour, or 

 living bacteria maybe used. Satisfactory preservatives for a suspen- 

 sion for the agglutination test are 0.5% phenol or 0.3% formalin. 



Some suspensions of bacteria tend to flocculate spontaneously, 

 necessitating as a control a suspension of the bacteria in saline which 

 is carried through the incubation period of the test. Spontaneous 

 agglutination may be due to many factors, such as surface tension, 

 electrical charges upon the surfaces of the bacteria and other un- 



