PATHOGENIC BACTERIA. 377 



tion becomes manifest by the collection of the bacteria into visible masses or 

 flocculi, which form a sediment. Most investigators prefer to watch the results 

 under the microscope, using an ordinary slide, or, better, the hanging-drop. 

 Young cultures less than twenty-four hours old in bouillon, and kept in the 

 incubator, may be used, or, better, cultures kept at room-temperature for 

 twenty-four hours. Johnston and McTaggart recommend that the bouillon 

 cultures be freshly made each time from stock cultures on agar, which need 

 only occasionally be transplanted. Certain stocks of typhoid bacilli seem especi- 

 ally suited to this reaction, and such a stock should be secured. 



Blood-serum, blister-serum, fresh blood and dried blood have all been used 

 with success. Blood dried on unglazed paper or cover-glasses as proposed 

 by Wyatt Johnston is extremely convenient. To perform the test the drop 

 of dried blood is mixed on the paper with sterilized bouillon or normal salt 

 solution, and portions of the suspension of the blood-serum obtained in this way 

 are tested upon the typhoid cultures. The objection to this procedure lies in 

 the dificulty of securing an accurate dilution. An approximate knowledge of the 

 degree of dilution may be acquired by mixing drops of dried blood of known 

 volume with definite amounts of water, and observing the tints. These should 

 be kept in mind as standards. The dilution may be measured with the hemoglo- 

 binometer or with the pipette of the hemocytometer. The New York Board of 

 Health have found blister-serum satisfactory and easy to obtain. A little of the 

 diluted serum is mixed on the cover-glass with a definite amount of the fresh 

 bouillon-culture, and is examined as a hanging-drop. In a short time the 

 characteristic clumping and loss of motility occur. At the same time a drop 

 of the culture alone, and a drop of the culture mixed with normal serum, similarly 

 diluted, should be examined as controls. The dilutions finally resulting after 

 mixing with the drop of culture, when placed under the microscope, vary 

 from one part of serum in 30 to i in 50. The higher dilution at which clumping 

 takes place the more definite the result. The time within which the reaction 

 occurs varies from a few minutes to about one. With little dilution the time 

 should be short; with greater dilution it may be longer. Both cessation of 

 motility and clumping should take place. In a positive case the cessation of 

 motion and the clumping of the bacilli should be complete. Normal blood 

 sometimes exhibits agglutinative properties in some degree. Some cultures 

 show a tendency to clump without the addition of any blood serum, but 

 such sources of error are checked by the control tests described. If the 

 reaction in any case is not satisfactory, it should be tried with a higher dilu- 

 tion, i to 50 or more, and the result should be positive if the case is a genuine 

 case of typhoid fever. 



The agglutinating power usually appears in the blood between the seventh 

 day and the end of the third week of the disease; it may be seen earlier; it is 

 often delayed and appears late. The test frequently has to be repeated when the 

 first result is doubtful or negative. Reports indicate that the method is a great 

 aid in the diagnosis of typhoid fever, though not infallible. 



The use of dead cultures for the agglutination reaction has been recommended 

 by various authors, and Ficker has placed his so-called "Diagnosticum," a 

 suspension of dead typhoid bacilli, on the market. For this method of diagnosis 

 Ruediger* makes the following recommendation: Inoculate a large flask 

 100 or 1,000 c.c. with the typhoid bacillus and incubate at 36 C. for 24 hours. 

 At the end of this time add i c.c. of formalin for every 100 c.c. of broth. This 

 is now ready for use, and can be preserved for many months. It must be shaken 

 up every time before use to distribute the organisms. Ruediger further advises 

 that in making the test four drops of blood be mixed with 2 c.c. of a 2 per cent, for- 



*Journ. Inject. Dis. Vol. I. 1904. pp. 236-240. 



