46 STAINING METHODS 



The drop of salt solution or water on the slide should be tinged a light violet-rose 

 color with a fraction of a loopful and the faeces or other material emulsified in this. 



Protozoa take a rose-pink color with a distinct differentiation be- 

 tween endoplasm and ectoplasm. 



Should the faeces be quite alkaline the neutral red will be decomposed with the 

 formation of bilirubin-like crystals. 



The Giemsa formalin method described under Blood-work is of value in certain 

 cases. 



Panoptic Method. Highly to be recommended for the staining of protozoa, 

 whether in smears or in sections, is the Panoptic method. 



1. Wright's or Leishman's stain for one minute. 



2. Dilute with water and allow dilute stain to act for three to ten minutes. Wash 

 in water and then 



3. Pour on dilute Giemsa's stain. Allow to stain from thirty minutes to twenty- 

 four hours. Differentiate with i : 1000 acetic acid solution until blue stain just 

 shows commencing diffusion into the acetic acid. Then wash in water, 95% alcohol, 

 absolute alcohol and treat with xylol and mount in liquid petrolatum. 



With preparations other than blood smears, as sections, it is better to go from 95% 

 alcohol to oil of origanum, then mount. 



Owing to the great value of a sharp nuclear picture in differentiating amoebae 

 it is of great importance to use some iron haematoxylin method. That of Weigert 

 is given in the appendix. 



Mallory's Phosphotungstic Haematoxylin. Fix moist smears, film surface down, 

 in Zenker's fluid for five to ten minutes. Wash in water, treat with Gram's solu- 

 tion and wash with 70% alcohol until all the yellow color is discharged. Wash in 

 water. Then stain with Mallory's phosphotungstic hcematoxylin for one-half hour. 

 Wash clear and mount. See appendix. 



Mallory's Differential Stain for Amoebae. Staining in saturated aqueous solution 

 thionin for from three to five minutes. Next differentiate in 2% aqueous solution 

 oxalic acid for one-half to one minute. Then wash in water, clear and mount. 

 Nuclei of amoebae are stained a brownish red. 



Rosenbusch Iron Haemotoxylin Stain 



Rapidly smear out with a toothpick a small particle of faeces or other material 

 containing protozoa and, while still moist, fix by Giemsa's method and, after getting 

 rid of the mercury with iodine followed by 95% alcohol, treat smears with a 3.5% 

 solution of iron-alum in distilled water for one-half hour or over night, then wash 

 thoroughly in distilled water. 



Then stain from five to twenty minutes in the following haematoxylin stain: (i) 

 i% solution of haematoxylin in 95% alcohol. It takes at least ten days to ripen. 

 (2) A saturated solution of lithium carbonate. Add to 10 c.c. of the haematoxylin 

 solution 5 to 6 drops of the lithium carbonate one. Next wash well and differentiate 

 with about a i% solution of the iron alum. Again wash in water, pass through 

 alcohols to xylol and mount in balsam. 



