12 PEACTICAL PABASITOLOGY 



which are acid-free. (Griibler and Co., Leipzig, supply a Canada 

 balsam which is quite acid-free.) 



Should the specimens be too deeply stained, the superfluous colour 

 may be removed by treating them with alcoholic solution of hydro- 

 chloric acid (a few drops hydrochloric acid to 100 c.cm. 70 per cent, 

 alcohol). The process of decolorization should be watched under the 

 microscope, and it should be allowed to proceed a little farther than 

 at first appears necessary, as the colour deepens again when the 

 specimens are blued in alkali. As soon as the desired shade is 

 obtained, the decolorized specimens are plunged into ammoniated 

 alcohol (1 drop of ammonia to 100 c.cm. 70 per cent, alcohol), when 

 the colour will turn blue. They should afterwards be rinsed in clean 

 70 per cent, alcohol. 



After the nuclei have been stained in hsematoxylin, it may be 

 found expedient to counter-stain the surrounding plasma in eosin or 

 erythrosin. In this case, the specimens should be soaked, either in 

 a concentrated watery solution of eosin before they are transferred to 

 alcohol, or in a weak alcoholic solution of eosin after they are taken 

 out of alcohol. 



Of other stains, safranin is useful for staining objects which have 

 been fixed by mixtures containing osmic acid. Borax-carmine and 

 alum-carmine are frequently valuable, as, for instance, in establishing 

 the presence of chromides. When using borax-carmine, the specimens 

 should be decolorized with alcoholic solution of hydrochloric acid and 

 afterwards rinsed in pure alcohol to free them from acid. Specimens 

 stained with alum-carmine should be rinsed in distilled water. 



Iron-hsematoxylin (Heidenhain) : Two and a half per cent, watery 

 solution of iron- alum (ferrous ammonium sulphate) as a mordant and 

 for purposes of differentiation ; and a ^ to 1 per cent, watery solution 

 of haematoxylin (which should, if possible, be at least four weeks old) 

 for purposes of staining. Cover-glass preparations and sections (the 

 latter should not exceed 6 //, in thickness) should be laid in the 

 mordant for four to twelve hours, or they may be put in over night. 

 After careful rinsing in distilled water they are allowed to remain in 

 the colour solution for six to twenty-four, or even, under certain con- 

 ditions, thirty-six hours. They are then again rinsed in water and 

 decolorized in the mordant, the precise degree of differentiation being 

 carefully controlled by the microscope, which should be furnished 

 with a strong dry lens. The specimens must once more be carefully 

 rinsed, this time in running water for half an hour, and, after passing 

 through the alcohol stages, they are transferred to xylol and, last stage 

 of all, to Canada balsam or cedar wood oil. 



The degree of differentiation will depend largely upon the purpose 

 in view. As a general rule, however, the process should be continued 



