332 Microorganisms (chap. 20) 



chloride with 0.0 IM phosphate, pH 7.0) and the excess saline wiped off 

 except over the sections. Labeled antibody is pipetted over the section; 

 the slides, covered with a petri dish with moist cotton or filter paper 

 attached to its undersurface, are allowed to stand, room temperature: 

 20-30 minutes. The slide is wiped dry except for the section and 

 mounted in buffered glycerol (anhydrous glycerol, 9 parts, buffered 

 saline, I part), then covered with a cover glass. Examination is made 

 under the fluorescent microscope. 



INDIRECT METHOD (layering). 



A tissue section containing antigen is covered with unlabeled specific 

 antiserum to allow the antibody molecules to react with it and be fixed 

 in situ (humid environment): 20 minutes. The slide is rinsed off in 

 buffered saline: 10 minutes, then wiped dry except for the section, and 

 fluorescein-labeled antiglobulin serum (prepared against the species 

 which furnished the specific serum) added: 20 minutes. The slide is 

 washed in buffered saline and mounted in buffered glycerol. Cells that 

 reacted with the antiserum will be covered with antibody (globulin) 

 and will have reacted with the labeled antiglobulin. This has been 

 termed layering because the bottom layer is antibody, the middle layer 

 the antigen, and the labeled antibody lies on top. 



Coons (1958) recommends control slides and uses a blocking tech- 

 nique. Unlabeled specific antiserum is added to a slide and allowed to 

 react for 20 minutes. Then, if washed with buffer solution and treated 

 with a drop of labeled antibody, only a faint fluorescence, if any, 

 should be exhibited. 



Silvers tein (1957) described a means of applying contrasting fluores- 

 cent labels for two antibodies using rhodamin B with fluorescein. 



The Coons methods have been applied for the detection of viruses, 

 rickettsiae, epidemic typhus, mumps, influenza, canine hepatitis, 

 chicken pox, canine distemper, measles, psittacosis, and poliomyelitis. 



This description is intended as only a brief resume of Coons' technics 

 in order to acquaint the technician with their potentialities. The fol- 

 lowing references contain extensive and pertinent discussions of the 

 procedures: Coons (1956 and 1958) and Mellors (1959). 



A tremendous bibliography is developing in this field; the following 

 references will lead to many others: Buckley et al. (1955); Cohen et al. 

 (1955); Coons et al. (1942, 1950, 1951, 1955); Coons and Kaplan (1950); 

 Kaplan et al. (1950); Lacey and Davis (1959); Leduc et al. (1955); Mel- 

 lors et al. (1955); Noyes (1955); Noyes and Watson (1955); Tobie (1958); 

 Watson (1952); and White (1955). 



