112 



LABORATORY MANUAL OF MICROBIOLOGY 



Method A. Flemming^s Triple Stain. — For general cytology 

 of the nodule. Vascular system, bacteroid area, shape and 

 position of infection threads, starch grains. 



Remove paraffin from the sections in xylol 1 minute; rinse in 

 95 per cent alcohol 1 minute. 



Stain 2 minutes in safranin (saturated solution in equal parts 

 of 95 per cent alcohol and anilin water). Rinse in water. 



Fig. 8.- 



-Cross-section of alfalfa nodule showing infection threads, 

 triple stain. 



riemming'a 



Stain 3 minutes in crystal violet (saturated aqueous solution). 

 Rinse in water. 



Treat with Orange G (saturated aqueous solution) from a 

 medicine dropper. Time should be as short as possible. Rinse 

 with absolute alcohol. Destain with clove oil until the desired 

 differentiation of infection threads and bacteroid tissue is reached; 

 observe under microscope. Rinse quickly in absolute alcohol. 



Clear in xylol 5 to 10 minutes. 



Mount in Canada balsam. 



Method B. Heidenhain's Haematoxylin. — For bacteria within 

 infection threads, internal structure of bacteroids. 



