126 RESEARCH IN PROTOZOOLOGY 



3. Making permanent slides 



For additional information about slide making the reader is 

 referred to McClung's (1929) Handbook of Microscopical Tech- 

 nique (Hoeber). The following suggestions may be of value: 



A. Wet films. Before making smears for fixation, cecal and 

 intestinal contents may require a little dilution in a saline diluent. 

 A thin, even smear is spread out on a slide or cover and then placed 

 in the fixative before drying can occur. 



a. Fixation. The writer has shown (1921) that different fixa- 

 tives give different results when subsequently stained in the same 

 manner. It is therefore desirable to use a number of fixatives if 

 a full knowledge of the animals is desired. For routine work, 

 Schaudinn's sublimate-alcohol-acetic employed at about 40° C. is 

 a satisfactory fixative. Comparable results may be obtained with 

 Worcester's fluid, or sublimate-acetic. Picric acid mixtures, espe- 

 cially Bouin's fluid and Hollande's fluid are very useful and the 

 writer has found the latter especially satisfactory for intestinal 

 flagellates. Osmic acid alone (one to two per cent), applied either 

 as liquid or as fumes, or chrom-osmic or chrom-osmic-acetic mix- 

 tures are all useful. Weak Flemming's fluid was found to preserve 

 the parabasal body of TrifricJwmonas muris better than other 

 fixatives, although one per cent chromic acid and Flemming's 

 stronger fluid without acetic were also useful. 



b. Staining. The most satisfactory stain for cytological details 

 is probably iron alum-hematoxylin. The most selective differ- 

 entiation is usually obtained when the preparations are allowed 

 to mordant and stain for from two to twenty-four hours. However, 

 shorter periods give workable results. For example, one may 

 mordant ten minutes in a two to four per cent solution of iron alum 

 at 30° to 40° C, rinse in water, then stain for ten minutes in 

 0.5% haematoxylin at the same temperature, then rinse in water, 

 destain in the iron alum, wash thoroughly in water, dehydrate, 

 clear and mount, and obtain usable slides. Delafield's hematoxylin 

 gives good results with some flagellates. The Feulgen method 

 (Cowdry, 1928) is supposed to stain only chromatin (thymonu- 

 cleic acid) and should be used to supplement other technique. 

 Counter-staining is sometimes desirable and may be carried out 

 with eosin, orange G, light green, acid fuchsin or Bordeaux red. 

 Other polychromatic effects can be secured by using Mallory's 



