Triple-stained Section of Amphioxus 237 



these clouds will taper off rapidly, leaving a terminal point at which no 

 color leaves for a moment or two before a slow stream again starts to 

 appear in the alcohol. As soon as the initial color clouds cease, the sections 

 should be removed to xylene, which instantly stops the differentiation. 

 If the operator is uncertain of this method or is trying it for the first 

 time, it is recommended that the slides be washed thoroughly in absolute 

 alcohol but removed to xylene before the color clouds have ceased to 

 leave the sections. Examination under the low power of the microscope 

 will show these preparations to have dull purple muscle tissue and in- 

 tensely blue connective tissue. A few trial sections should now be re- 

 turned to absolute alcohol for a few moments, put back into xylene, and 

 then reexamined. It is possible by this means to exercise the most perfect 

 control over the differentiation, which should be stopped when the 

 muscles and nuclei are clear red and the connective tissues are a clear 

 light blue. No attention should be paid, while differentiating, to any of 

 the structures, such as the gonad, which by this method acquire a violet 

 coloration that cannot be judged; the process should be controlled only 

 by apparent contrast between the pure blues and pure reds in the section. 

 The stains used in this preparation are alkali sensitive, and it is a cus- 

 tomary procedure in Europe to mount them in as acid a medium as pos- 

 sible. If one is using one of the synthetic resins, which are neutral, it 

 is strongly recommended that the coverslip, before it is applied to the 

 resin, should be dipped briefly in a strong solution of salicylic acid in 

 xylene. This salicylic acid will dissolve in the resin and provide a perma- 

 nently acid medium; with this precaution, the sections are permanent. 



SUMMARY 



1. Fix a small adult Amphioxus in any good fixative. If only a Bouin- 

 fixed specimen is available, soak it overnight in \% nitric acid. 



2. Cut 10-/x paraffin sections in the usual way, attach to slide, and grade 

 to water. 



3. If fixative does not contain mercuric chloride, soak sections over- 

 night in a saturated solution of this salt and wash for some hours in 

 running water. 



4. Stain in 1% acid fuchsin for about 2 min. 



5. Rinse quickly in distilled water and transfer to \% phosphomolybdic 

 acid until no more color comes away (about 2 min). 



6. Rinse quickly and place in Mallory's oxalic acid-methyl blue-orange 

 G mixture for 15 min. 



7. Wash thoroughly in water. Rinse in 95% alcohol to remove adherent 

 water. Differentiate each slide individually in absolute alcohol. Stop 

 differentiation with xylene and mount in balsam. 



