INTRODUCTION. 47 



stain or Giemsa's stain. The action of all such stains depends 

 on certain loose chemical combinations. Medicinal (not 

 pure) methylene-blue contains a number of oxidation products, 

 the most important of which is methylene-azur. When watery 

 or alcohohc solutions of methylene-blue and of eosin are mixed 

 together a series of loosely combined chemical bodies are formed. 

 These different compounds possess different affinities for 

 different cell-structures, and thus differential staining results. 

 The red blood- corpuscles stain a transparent pink or orange 

 colour ; the nuclei of leucocystes, shades of violet ; eosinophile 

 granules in the coarsely granular leucocytes, red ; the cyto- 

 plasm of malaria and other blood-inhabiting protozoal parasites 

 a bright " Cambridge " blue, and their chromatin a bright 

 ruby -red. 



The Original Eomanowsky's Stain. — In large laboratories the 

 original Romanowsky's stain is still employed, as it is more 

 economical than Leishman or Giemsa. Two stock solutions are 

 needed. For preparing Solution A take medicinal (not pure) 

 methylene-blue 1 grm., pure sodium carbonate 0-5 grm., 

 distilled water 100 c.c, dissolve and place the stain either in 

 the 37° C. incubator or in full sun-Hght for 2 or 3 days. The 

 solution should acquire a deep purple colour before it is ready 

 for use. For Solution B, dissolve eosin, extra B.A., water 

 soluble 0-1 grm. in 100 c.c. of distilled water. 



Fix the film for 5 minutes in methyl alcohol, or for 10 minutes 

 in ordinary alcohol. Wash with distilled water and transfer 

 to a clear Petri dish. Dilute 1 part of solution A with 19 parts 

 of distilled water. Similarly dilute 1 part of solution B with 

 19 parts of distilled water. Mix equal parts of the diluted 

 solutions A and B, and pour the mixed stain, immediately 

 after mixing, into the Petri dish to cover the shde. Stain for 

 half an hour or longer. On tilting the Petri dish a red stain 

 should be seen at the edge, which will indicate that the 

 staining is proceeding properly. Wash off the stain with 

 distilled water and differentiate in a bath of distilled water. 

 When the film commences to turn pink, remove from the 

 bath and allow it to dry by slanting it against a vertical 

 surface. 



Leishman' s Stain. — To prepare the stain thoroughly clean 

 a glass (not porcelain) pestle and mortar and rinse out the 

 mortar with a httle pure methyl alcohol. Weigh out 0-15 grm. 

 of Leishman's stain powder (Merck's or Griibler's). Measure 

 into a perfectly clean glass cylinder 100 c.c. of methyl alcohol, 

 purissimum, acetone-free (Merck's). Add a httle of the alcohol 

 to the stain in the mortar and grind. Add more alcohol and 

 grind. Drain off the dissolved stain into a stoppered bottle. 

 Continue to add the alcohol in portions and grind until every 

 particle of the stain has gone into solution, and use the fuU 



