TRIPLE-STAINED SECTION OF AMPHIOXUS 129 



for the 1 per cent phosphomolybdic add specified in the original method. The 

 sections, when they have been washed thoroughly after the mordanting in 

 mercuric chloride, are placed in the 1 per cent solution of acid fuchsin for a 

 period of about two minutes. This time is not critical; it is necessary only to 

 make sure that the entire section is thoroughly stained. On removal, the sections 

 are rinsed rapidly in water for the purpose of removing the surplus stain, and 

 then are placed in the 1 per cent phosphotungstic acid until such time as the 

 red stain has been removed entirely from the connective tissues. This may be 

 judged partly by the cessation of the color clouds which rise from the section 

 and partly by an examination under the lower power of the microscope to 

 make sure that the septa between the myotomes are free from color. The speci- 

 fied time of 2 minutes is usually sufficient, and the sections will not be dam- 

 aged however long they may be left. On removal from this solution, they are 

 rinsed quickly in water and placed in the acid-methyl blue-orange G solution 

 where they should remain for at least 15 minutes. The mistake is often made 

 of leaving them for too short a time in this stain, for they will have the appear- 

 ance of being deeply stained after an immersion of only a few moments. It 

 does not matter how long they remain; it is the author's experience that soak- 

 ing for at least 15 minutes discourages the subsequent removal of the blue 

 from- the tissues. After they are removed from this rather thick staining mix- 

 ture, the slides are washed thoroughly in water. This wash is designed to 

 remove not only the whole of the adherent stain from the slide, but also to 

 permit the oxalic acid to be leached out of the tissues. No "differentiation" 

 of the stain should take place in water, since the necessary differentiation is 

 produced by the absolute alcohol used for dehydration in the next stage. It is 

 quite impossible to take sections stained by this method up through the com- 

 mon graded series of alcohols, but no grave damage will be occasioned by the 

 omission of this step. If, however, the operator is one who insists that his sec- 

 tions pass through a graded series, mixtures of acetone and water should be 

 substituted for alcohol and water. When the sections reach absolute alcohol, 

 they should be watched very closely, while being moved continuously up and 

 down in the alcohol. The blue color will leave them in great clouds; these 

 clouds will taper off quite 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 



