3 



58 



METHODS AND FORMULAS 



DS 13.40 



adays to secure supplies of living amphi- 

 oxus and to fix them oneself, or to prevent 

 the supplier from whom one secures the 

 fixed material from using Bouin's picro- 

 acetic-formaldehyde fixative (Chapter 18, 

 F 5000.1010 Bouin 1897) in their prepara- 

 tion. If it is possible to secure the hving 

 lancelets, they should be fixed by one of 

 the methods recommended for fixing very 

 heavily muscularized material, for it is 

 otherwise almost impossible to secure un- 

 broken sheets of muscle in the transverse 

 section unless one is prepared to sacrifice 

 histological detail to the interests of mor- 

 phological demonstration. It is also to be 

 regretted that popular demand has forced 

 the biological supply houses to sell only 

 large specimens, because these are too 

 large to be viewed at one time in even 

 the lowest power commonly available on 

 a student microscope. If any selection can 

 be exercised, care should be taken to pick 

 a specimen of not more than 2.5 mm. 

 greatest thickness in order that it may be 

 seen as a whole. 



If, however, one is forced to use a 

 Bouin-fixed specimen, it may be sectioned 

 without too much difficulty provided that 

 it first be soaked overnight in 1% nitric 

 acid. This treatment destroys much of the 

 fine cytological detail and should not be 

 applied to any specimen in which it is 

 desired to demonstrate, say, the detailed 

 structure of the endostyle. The writer, 

 however, is always prepared to sacrifice 

 such detail as this in a section desired for 

 class demonstration, in order to avoid end- 

 less questioning as to what is tliis and 

 that cavity which will be seen in a section 

 of Bouin-fixed amphioxus handled by 

 routine methods. 



Apart from this question of fracturing 

 the muscular layers, no difficulty will 

 present itself in sectioning. As many 10- 

 micron sections as are required should be 

 accumulated. If it is desired to place on 

 the same slide a collection of sections from 

 different regions of the animal, reference 

 may be made to the description of this 

 procedure in Chapter 12. 



When the sections have been mounted 

 on a slide, deparaffinized, and run down 

 to water, it is recommended that they be 

 treated overnight in a saturated solution 



of mercuric chloride and then washed in 

 running water for at least six hours. This 

 process, though it takes a day, improves 

 the \dvidness of Mallory's stain almost 

 beyond belief when it is applied to a sec- 

 tion of Bouin-fixed material. The actual 

 staining procedure is simphcity itself and, 

 though the original stain of Mallory 

 (DS 13.41 Mallory 1900) has been selected 

 for demonstration, there is no reason why 

 any of the other stains in the same section 

 should not be employed. The solutions 

 present no difficulty in their preparation, 

 though it is recommended that 1 % phos- 

 photungstic acid be substituted for the 

 1 % phosphomolybic acid specified in the 

 original method. After the sections have 

 been thoroughly washed from the mor- 

 danting in mercuric chloride, they are 

 placed in the 1 % solution of acid fuchsin 

 for a period of about two minutes. Tliis 

 time is not critical, it being desired only 

 to make sure that the entire section is 

 thoroughly stained. On removal the sec- 

 tions are rinsed rapidly in water, to re- 

 move the surplus stain, and are then 

 placed in the 1 % 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 or by an examination 

 under the low power of the microscope to 

 make sure that the septa between the 

 myotomes are free from color. The speci- 

 fied time of two minutes is usually suffi- 

 cient, but the sections will not be damaged 

 however long they may be left. On re- 

 moval from this solution they are again 

 quickly rinsed in water, and then 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 tliis 

 stain, for they will have the appearance 

 of being deeply stained after an immersion 

 of only a few moments. It does not matter 

 how long they remain in the solution ; it is 

 the writer's experience that soaking for at 

 least 15 minutes discourages the subse- 

 quent removal of the blue from the 

 tissues. After they are removed from this 

 rather thick staining mixture the slides 

 are thoroughly washed in water. The wash 



