586 PROTOZOA 



1153. Methylen blue and eosin (Chenzinsky's formula, 

 p. 185) sometimes stains tissues, and especially blood, exquisitely, 

 but Trypanosomes only show blue nuclei and granules (Minchin, 

 Quart. Journ. Micr. Sci., liii, 1909, p. 785). 



1154. Mallory's eosin and methylen blue stain, recom- 

 mended for sections which have been fixed in Zenker's fluid. 

 § 342. 



A trace of colophonium should be present in the alcohol used 

 for differentiation in order to obtain the best results. 



1155. Giemsa's Stain (see §§ 875, 877). The preparation 

 supplied by Gurr, London, is quite satisfactory. 



1. For dried smears fixed with osmic vapour and absolute 

 alcohol. Dilute each drop of stain with 1 c.c. of neutral * distilled 

 water and place it in a clock-glass. Transfer slide from absolute 

 alcohol and place it, smear downwards, in the stain for twenty 

 to thirty minutes. Wash in distilled water, then tap-water, then 

 again in distilled, and allow to dry. Red blood corpuscles should 

 be bluish-mauve (they will be pink if fixed only in absolute, and 

 if too much osmic acid has been vised they will be blue or greenish- 

 blue). Parasites should be blue, with red nuclei and flagella. 



These dried smears are sometimes useful to compare with 

 films made by the better wet methods, especially when very 

 small flagellates, htemamoebae, etc., are being studied. 



2. For wet films or sections. Minchin obtained excellent 

 results with sections of fleas' stomachs fixed in Maier's modifica- 

 tion of Schaudinn's fluid. 



After washing, to remove all trace of fixative, transfer from 

 water to stain, diluted as above, for one hour. Then leave over- 

 night or for some hours in a weaker stain — 1 drop to 4 or 5 c.c. 

 water — rinse in water, and differentiate in acetone mixed with 

 different proportions of xylol, beginning with 95 per cent, acetone 

 for a very short time and ending with pure xylol. 



The mounting medium must be quite neutral. 



1156. The other Romanowsky stains (see § 878) are not suitable for 

 exact protozoological work, though Leishmann's stain is useful for 

 staining blood for diagnostic purposes, and if parasites are scarce it 

 may be necessary to make a thick film (Ross's method) and deha^mo- 

 globinise it in distilled water before fixing and staining (Carleton, op. 

 cil., 1926, p. 347). 



* The distiUed water should be stored over soda-lime or some other 

 substance to jirevent the absorption of C'Og, or it may be neutralised 

 by Giemsa's method with KgCOg, using haematoxylin as an indicator. 

 A 1 per cent, solution of the potassium carbonate is added drop by 

 drop to a measured volume of the distilled water containing a few 

 drops of a weak haematoxylin solution until the colour changes, after 

 well shaking, from yellowish-red to reddish-purple. In this way the 

 number of drops of the carbonate solution required to neutralise a given 

 volume of the distilled water is known. 



