534 METHODS FOR INVERTEBRATES. 



best diluted for use about one in five, and films are stained for from five 

 to thirty minutes, washed, dried and mounted in balsam or euparal. 

 The hsemamoebae are stained purple, nuclei blue, red cells faint blue or 

 grey. Films may be differentially stained by means of eosin-methylene 

 blue, Borrel's blue, or still better, by one of the Romano wsky methods. 



1005. Eosin-Methylene Blue. The film is prepared and fixed either 

 by alcohol-ether or methyl alcohol, and is then stained for thirty seconds 

 in a 0-5 per cent, solution of eosin, and is then washed and stained in a 

 saturated solution of methylene blue for thirty seconds. In a successful 

 preparation the red ceUs are stained pink and the nuclei of leucocytes 

 and parasites blue. 



1006. Ronald Ross's Thick Film Method. Frequently the parasites in 

 these cases are only present in very small numbers, and in the examina- 

 tion of the ordinary thin films their presence may be overlooked. To 

 meet such cases Ross recommends about 10 to 20 cubic mm. of the blood 

 to be spread in a thick film on a slide, which is dried by waving gently 

 over a flame and is then washed in water. By such treatment the 

 haemoglobin is dissolved out from the erythrocytes. The film may 

 now be stained for a minute in 0-5 per cent, to 1 per cent, aqueous 

 eosin, followed by a 1 per cent, aqueous solution of methylene blue, 

 made alkaline by the addition of 0-5 per cent, sodium carbonate and 

 heated. For this may, with advantage, be substituted a solution of 

 Unna's poly chrome methylene blue diluted 1 in 2 or 3 with water. Films 

 after staining are washed, dried, and mounted in balsam. Only the 

 leucocytes and the parasites are stained by this method. 



1007. Borrel's Blue. This method depends upon the formation 

 of an oxidation product of methylene blue. Silver oxide is prepared 

 by dissolving the nitrate in distilled water and precipitating the 

 oxide with a 10 per cent, solution of sodium hydroxide. The 

 precipitate obtained is washed thoroughly in distilled water and a 

 saturated solution of methylene blue is added. The mixture is well 

 shaken and allowed to stand for about a fortnight. The super- 

 natant fluid is then pipetted ofT and constitutes Borrel's blue. 

 Films are prepared and fixed for half an hour in either alcohol, ether- 

 alcohol or absolute alcohol, and are then stained with Laveran's 

 solution prepared as follows :- 



Borrel's blue ...... 1 c.c. 



0-1 per cent, aqueous eosin . . .5 



Distilled water . . . . . .4 



The slides are placed film side downwards in this fluid and allowed 

 to stain for from five to fifteen minutes, and should then be washed 

 in distilled water treated with a 5 per cent, aqueous solution of 

 tannic acid for one to two minutes, washed and dried. Red cells 

 are stained pink, nuclei of leucocytes purple violet ; whilst the 



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