SECTION TWO 



PHOSPHOTUNGSTIC ACID-EOSIN-HAEMATOXYLIN 



(After Massignani and Malferrari, 1961) 



For Negri bodies 



Solutions required: 



A. Harris haematoxylin, without acetic acid, or 

 Carazzi haematoxylin. 



B. HCl, cone. . . . . . . . . I ml. 



Distilled water . . . . . . 200 ml, 



C. Lithium carbonate, saturated, aqueous. 



D. Solution C . . . . . . . . i ml. 



Distilled water . . . . . . 200 ml. 



E. Eosin, yellowish, water soluble . , i gm. 

 Phosphotungstic acid . . . . 07 gm. 



Grind together with a mortar and pestle, then add 10 ml. dis- 

 tilled water and grind into a paste. Pour in 90 ml. of absolute 

 alcohol, in lots of about 10 ml. at the time, and grind until all 

 the solid has gone into solution. Pour the solution into a 250 ml. 

 measuring cylinder. Rinse the dye off the pestle into the mortar 

 with not more than 50 ml. absolute alcohol. Transfer the washings 

 to the dye solution in the cylinder. Now rinse out the mortar 

 with 50 ml. absolute alcohol and add the rinsings to the cylinder. 

 Make up the volume of the stain in the cylinder to 200 ml., then 

 transfer it to a beaker and add two drops of solution C. Stir for ten 

 minutes with mechanical stirring (a domestic type (kitchen) mixer 

 will serve the purpose). Restore the volume of the mixture to 

 200 ml. with absolute alcohol. 



This preparation may then be used for preparing either solution 

 F or G as below: 



F. Solution E, merely filtered. 



G. Centrifuge solution E for forty minutes at 1,500 

 revolutions per minute, in a refrigerated centrifuge. 



H. As solution E but distilled water is used instead of 

 absolute alcohol. Centrifuge the suspension so ob- 

 tained, then dissolve the precipitate in absolute 

 alcohol. Add this alcoholic solution to the super- 

 natant to produce the staining solution. 



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