Appendix 



VI. IMORMAL CHICKEN YOLK SAC (NYS) 



A normal cliicken egg yolk sac suspension is used in the indirect immunonuorescence test 

 for antibody to the Legionnaires' disease bacterium (see chapter by Wilkinson et al.). The sus- 

 pension is needed for the preparation of working dilutions of the bacterial antigens and for the 

 initial dilution in titration of sera. 



1. Harvest the yolk sac from 12- to 14-day-old embryonated hens' eggs. Separate as much 

 of the yolk material from the sac as possible. The average yield of yolk sac per egg is 4 g. 



2. Place the yolk sacs in an equal volume of 0.02 M phosphate buffered saline. pH 7.2 

 (PBS), containing 0.05% sodium azide. 



3. Homogenize in an Ominimixer or Waring blender, and filter through two layers of gauze. 



4. Dilute the suspension with PBS containing 0.05% sodium azide to 0.5% (w/v) for antigen 

 preparation or 3% for use as a diluent in the FA test. 



VII. McFARLAND IMEPHELOMETER DENSITY STANDARDS 



Add 1% barium chloride and 1% sulfuric solutions to 10 uniform tubes in the proportions 

 shown below. Seal to prevent any evaporation, and label the tubes. Before using standards, shake 

 tubes well to resuspend the precipitate. 



160 



