468 . RESEARCH IN PROTOZOOLOGY 



The above method is recommended for purposes of diagnosis 

 only. If material is to be prepared for careful cytological study 

 the original method of Heidenhain should be more closely adhered 

 to, allowing from four to twelve hours' immersion in the mordant 

 (4 per cent ferric alum solution) and a similar length of time 

 in the stain. By doing this a much more critical decolorization can 

 be obtained. Sections may be stained in general in accordance with 

 the directions above except that the paraffin will have to be re- 

 moved either with xylol or chloroform before the staining is under- 

 taken. In general the process will go somewhat more slowly with 

 the sections than with the smears due to a greater thickness of the 

 former. 



Another method preferred by some protozoologists for routine 

 work is Mayer's hemalum staining technique. Procedure is as 

 follows : 



The stock solution of Mayer's hemalum" is diluted at least twenty times, 

 and the fixed smears are left in it for several hours or overnight. After 

 staining, the films are left in running tap-water, or in slightly alkalinized 

 distilled water until they are blue. They are then dehydrated in alcohol, 

 cleared in xylol, and mounted in acid-free balsam. If the slides are over- 

 stained, they may be decolorized in acid alcohol," washed, and reneutralized 

 in running tap water. 



A technique which^ it is alleged, is a specific test for chromatin is 

 becoming widely used by protozoologists. It has been devised by 

 Feulgen (Cowdry, 1928) and depends upon the presence of thy- 

 monucleic acid. The procedures are as follows: 



I. Fixation. 



Corrosive sublimate 6 per cent aq. sol 98 parts 



Glacial acetic acid 2 parts 



" Mayer's hemalum 



Hematein i.o gm. 



Ethyl alcohol (90% ) 50.0 cc' 



Alum 50.0 gms. 



Distilled water looo.o c.c. 



The hematein is dissolved in the alcohol with the aid of 

 heat, and is added to the water in which the alum has been 

 dissolved. Keep a crystal of thymol or some other fungi- 

 cide in the stock solution. 



^ Acid alcohol 



Ethyl alcohol (60%) 99.0 c.c. 



Hydrochloric acid .,.......:.. i.o c.c. 



