STAINING, PRACTICAL AND THEORETICAL 



PHLOXIN - METHYLENE BLUE 

 Rapid smear technique for Negri bodies in brain tissue 



Solutions required: 



A. Phloxin 2% aqueous. 



B. Methylene Blue (Loeffler). 



Technique: 



1. The brain to be examined should be removed as quickly as 

 possible; then small segments, 3 to 4 mm. thick, are cut from 

 Ammon's horn perpendicular to its long axis and placed in a Petri 

 dish. Cut away adjacent tissue, leaving only the horn. 



2. Place a segment, cut surface downwards, on the small end 

 of a new one-inch cork ; then with a matchstick, wipe peripheral 

 tissue downward and outward, so that the segment is more firmly 

 attached to the cork and the grey matter containing the pyramidal 

 cells bulges upwards. Press this gently against a scrupulously 

 clean slide, and make a smear by repeating this process along the 

 whole length of the slide. This operation should be carried out 

 rapidly before the tissue commences to dry. 



3. Fix immediately by immersing in pure methyl alcohol for 

 five to ten minutes. 



4. Rinse in running water; then stain in the phloxin solution 

 from two to five minutes. 



5. Wash in running water; then stain in methylene blue 

 (Loeffler) for ten to thirty seconds. 



6. Decolorize in 80% alcohol; then dehydrate in 95% alcohol 

 and two changes of absolute alcohol. 



7. Clear in xylol and mount in balsam. 



Note: The slides should be handled with forceps throughout to 

 prevent the preparation being spoiled by coming into contact 

 with the fingers. 



Results: 



Pyramidal cells, blue; Negri bodies, bright red to reddish 

 brown. 



Reference: Dawson, J. R. (1934/5). 



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