STAINING, PRACTICAL AND THEORETICAL 



Note: Dried films remain satisfactory for staining for many 

 months, if not indefinitely. During sigmoidscopy, the same 

 aspirate or curetted material (the author, Brooke, i960, states 

 that samples should not be obtained with swabs), which is exam- 

 ined in wet mounts, can be preserved in PVA-fixative. 



4. Immerse the preparation in 70% alcohol containing iodine 

 for ten minutes. 



5. Immerse for three to five minutes in each of two baths of 

 70% alcohol. 



6. Immerse in solution B (Wheatley's trichrome stain) for six 

 to eight minutes. 



7. Rinse in solution C for ten to twenty seconds. 



8. Immerse in 95% alcohol for five minutes. 



9. Immerse in carbol-xylol for five to ten minutes. 

 ID. Immerse in xylol for ten minutes. 



II. Mount Canada balsam in xylol, D.P.X., Permount, or 

 Cristalite or Clearmount. 



Results: 



The cytoplasm of thoroughly fixed and well stained E. histolytica 

 trophozoites and cysts is blue-green, tinged with purple. E. coli 

 cysts are slightly more purplish. Karyosomes of nuclei: brilliant 

 ruby red. Background material usually stains green, resulting in 

 a noticeable colour contrast with the protozoa. Organisms in the 

 thick portions of the smears take the more neutral shades of red 

 and green. In contrast with those stained with haematoxylin, 

 trichrome smears have a transparency which enables identification 

 of embedded protozoa. Helminth eggs and larvae can frequently 

 be identified in the stained smears, usually stained red and con- 

 trasting strongly with th^ green background. Non-staining cysts 

 and those staining predominantly red are most frequently associ- 

 ated with incomplete fixation. If unsatisfactory stained organisms 

 are obtained it usually indicates incomplete fixation associated 

 with poor emulsification. Thorough emulsification of preferably 

 soft stools (or those first mixed with saline saline) will give critically 

 stained cysts and trophozoites. Degenerate forms stain pale 

 green. Owing to the contrast between the organisms and the 

 background, larger protozoa can be seen with the one-sixth-inch 

 objective, but it is preferable to use the oil immersion objective. 



Reference: Brooke, M. M. (i960). 



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