STANDARD METHODS AND REAGENTS 467 



Flemming's,^ or Zenker's ^ solutions. Schaudinn's solution warmed 

 to a temperature of about 50° C. is the most popular fixative. The 

 smear is left in the fixing fluid for about ten minutes and is then 

 stained. 



The iron-alum-hematoxylin method of Heidenhain, or some one 

 of its numerous modifications, is the staining method of choice for 

 protozoological material. The following procedures are routine 

 in this laboratory for the staining of intestinal protozoa in general. 



Smears are fixed as above in Schaudinn's fluid, after which they must not 

 be allowed to dry during the subsequent transfers. The fixed smears are 

 placed in 95 per cent alcohol to harden. This requires about five minutes 

 after which they are washed for five minutes in tap water and placed in a 

 4 per cent solution of ferric alum for ten minutes. They are then rinsed off 

 with tap water and placed immediately in the stain which is a 0.5 per cent 

 solution of hematoxylin in water. From ten to twenty minutes is usually a 

 sufficiently long time to stain the organisms. After they have been taken 

 out of the stain they are again rinsed in tap water and decolorized in a 2 per 

 cent solution of ferric alum. This is the most critical procedure of the entire 

 process. Until the action of a particular combination of stain and decolorizer 

 has been ascertained the progress of the destaining should be frequently 

 checked by observing the slide (washed previously in tap water to tempo- 

 rarily check the decolorizing) under the microscope. After a few experi- 

 ences with the material in hand it will be possible to time the exposure to 

 the destaining fluid so that it will not be necessary to observe the slides 

 under the microscope. When the contrast of cytoplasm and nuclear material 

 is sufficiently great the process of destaining is brought to an end by washing 

 thoroughly with tap water. If the slide is to be kept for a long time it is 

 best to wash it for from two to several hours but if it is made for the 

 purpose of immediate diagnosis ten minutes is sufficient time. After washing, 

 the slide is immersed in 95 per cent alcohol for from two to four minutes, 

 in absolute ethyl alcohol for five minutes, and in xylol for five minutes, after 

 which a clean coverslip is mounted over the preparation with neutral balsam. 



^ Flemming's solution (strong) 



Chromic acid (1% aqueous solution) 15 parts 



Osmic acid (2% aqueous solution) 4 parts 



Add just before use: 



Glacial acetic acid i part 



^Zenker's solution 



Potassium bichromate 2.5 gms. 



Mercuric bichloride 5.0 gms. 



Sodium sulphate i.o gm. 



Distilled water loo.o c.c. 



Add just before use: 



Glacial acetic acid 5.0 c.c. 



