8i8 AGGLUTININS AND THEIR APPLICATIONS 



The macroscopic methods may conveniently be classified as "qualitative" and "quantita- 

 tive." In the former category are those methods which are for the most part designed to save 

 time and trouble, where a prompt laboratory opinion is desired as an aid to clinical diagnosis 

 or when a large number of colonies or cultures are to be provisionally identified. 



Essentially, a drop of highly potent agglutinating serum is brought into contact on a slide 

 with a homogeneous suspension of micro-organisms and rocked gently to and fro upon a 

 longitudinal axis. If the suspension and serum are homologous, a clumping, visible to the 

 unaided eye, occurs in from three seconds to two minutes. A control of diluent and micro- 

 organisms must remain uniformly turbid. The density of suspension should be heavy, ap- 

 proximately 5,000 to 10,000 million per cubic centimeter. Two additional controls should be 

 used; namely, normal serum from the same animal species as that from which the test serum 

 was obtained, used in the same dilution as in the test serum, and the test serum against its 

 homologous culture. The dilution of serum used in the test must be determined by prelimi- 

 nary trial. In addition, it must be sufficiently dilute so that group agglutination will not be 

 manifest and yet sufficiently concentrated for rapidity of action. As a rule, a serum of titre, 

 I in 4,000, may be used at i in 100. It is frequently desirable to make use of a serum which 

 has been "absorbed" by suspensions of closely related species. Such a serum may be pre- 

 pared in bulk after the method recommended by Andrewes.^ The authors of the Medical 

 Research Council, "Special Report Series," No. 51, comment upon this rapid method as 

 follows: "In any case it must be understood that this rapid method is merely one for pre- 

 liminary orientation, and may be wholly misleading; it cannot replace the cultural tests 

 which are requisite for establishing the nature of a bacterium. Neglect of this principle may 

 easily lead to serious errors in diagnosis." Bass and Watson^ applied this slide method as an 

 aid to the diagnosis of typhoid fever. 



Other macroscopic methods are those of Garrow,^ Neisser,'' and Wright.^ 



The macroscopic tube agglutination test is certainly to be chosen whenever possible since 

 with this method only is one able to determine with any degree of exactness the actual 

 measure of the agglutinins present in a serum. In the identification of species of micro- 

 organisms its use is obligatory. A series of dilutions are prepared, and to unit volumes of 

 these dilutions contained in tubes of uniform caliber is added a unit volume of bacterial 

 suspension. A control tube receives saline and suspension only. After thorough mixing, the 

 tubes are placed in a water bath or incubator at the desired temperature for a definite time. 

 Readings may be made after allowing the test to stand in the icebox or room, usually over- 

 night. Suspension of formalin-(o.i per cent) killed micro-organisms diluted to standard 

 opacity are commonly emploj^ed. Within limits, the lower the density the more accurately 

 the titre of a serum may be determined. From 300 to 1,000 million bacilli per cubic centi- 

 meter is a useful working range for the enteric group. The suspension may be made from a 

 broth culture or of a young agar culture. Repeated subculturing in broth for a period of two 

 or three weeks may be resorted to in the case of strains refractory to agglutination. Spon- 

 taneous agglutination^ may in certain instances be overcome by employing a buffered broth 

 for the culture and suspension of micro-organisms. Mudd' has described a method of detect- 

 ing the binding of agglutinins by suspensions which are agglutinated with difficulty. 



' Andrewes, F. W.: J. Path. &" Bad., 25, 507. 1922. 



' Bass, C. C, and Watson, J. A.: Arch. Int. Med., 6, 717. 1910. 



^ Garrow, R. P.: Lancet, i, 262. 1917. 



'' Neisser, M.: (Proscher) Centralbl.f. Bakteriol., Abt. II, Orig., 31, 400. 1902. 



s Wright, A. E.: "Medical Research Council, Special Report Series," No. 51, p. no. 1920. 



* Bliss, W. P.: /. Exper. Med., 36, 575. 1922. ' Mudd, S.: J. ImmunoJ., 13, 113. 1927. 



