396 



METHODS AND FORMULAS 



DS 21.20 



coloration begins to spread over the skin. 

 Leave the frog for about ten minutes and 

 then inject another milhhter of stain into 

 the abdominal cavity. An hour later, 

 check to see if the nervous system has 

 picked up the methylene blue. This can be 

 done by dissecting the hind leg to disclose 

 the sciatic nerve, which is easily found by 

 slipping the handle of a scalpel between 

 the gastrocnemius and the posterior tibi- 

 alis muscles to disclose the nerve lying 

 alongside the bone. If the preparation has 

 been a successful one, the nerve will be 

 stained light blue, and as a final check the 

 nerve may be severed and the cut end ex- 

 amined under a hand lens. If the cut end 

 has a darker stain than does the outer 

 sheath, the impregnation may be con- 

 sidered successful, and one may proceed 

 with preparations to remove and fix the 

 brain. Pin the frog belly down in the 

 bottom of a dissecting pan. Remove the 

 skin from the whole of the head, and 

 scrape the frontoparietals clear of their 

 attached muscles. Break away the pos- 

 terior portion of the cranium so as to leave 

 the way clear for the removal of the upper 

 half to expose the brain. The brain can be 

 removed by inserting the sharp point of a 

 scalpel under the nasal bone, which is then 

 Ufted up and pulled away. This leaves the 

 end of the frontoparietal hanging free so 

 that it may be lifted very carefully T\dth 

 the edge of a scalpel. Do not jiermit it to 

 l)reak away, or the other end will drive 

 down and destroy the cerebellum. As soon 

 as it has been lifted, however, it may be 

 grasped in a pair of blunt-nosed forceps 

 and twisted with a rotary motion toward 

 the center of the skull. Tliis will result in 

 its coming away free without damaging 

 the brain. The same technique may now 

 be emplo3^ed on the other frontoparietal, 

 thus leaving clear the whole anterior 

 region of the brain. The brain will now be 

 exposed save for those portions which are 

 covered by the prootic bones, or by such 

 parts of the frontoparietal as remained 

 attached to the prootic bone when they 

 broke. Both the prootic and the remains 

 of th'e frontoparietal may be lifted off 

 carefully and the upper surface of the 

 brain fully exposed. 



It is almost impossible to remove the 



brain from its bed without damaging it. A 

 pair of heavy scissors should now be used 

 to cut through the remains of the nasal 

 and vomer bones, to sever the connection 

 of the parasphenoid bone from the pre- 

 maxillae, and to cut through the prootic 

 bones at the point of attachment of the 

 squamosals and the pterygoids. The en- 

 tire brain may now be lifted out, resting, 

 as it were, on a platter consisting largely 

 of the parasphenoid bone. 



It will be seen by reference to the tech- 

 nique under discussion (DS 21.211 Bethe 

 1896) that six alternative fixative solu- 

 tions are suggested. There is no doubt 

 that the solution which contains osmic 

 acid and phosphomolybdic acid is the 

 best, but the very high cost of osmic acid, 

 and the danger of working with this ir- 

 ritating reagent, may lead the operator to 

 choose in preference alternative formula 

 No. 2, which contains ammonium molyb- 

 date and chromic acid. The results are 

 nearly, but not quite, as good with this 

 reagent as with the osmic-sodium-phos- 

 phomolybdate mixture. Ammonium phos- 

 phomolybdate can, of course, be substi- 

 tuted for sodium phosphomolybdate in 

 the solution selected. 



The first fixation is in a saturated aque- 

 ous solution of ammonium picrate, which 

 is prepared by taking a saturated solution 

 of picric acid in water and adding to it a 

 considerable excess of undissolved picric 

 acid. Ammonia is now added drop by drop, 

 with constant shaking, until the surplus 

 picric acid at the bottom of the container 

 starts to dissolve. Further ammonia 

 should now be added until the solution 

 smells of free ammonia, and it may then 

 be shaken once or twice to complete the 

 saturation. A very emphatic warning must 

 be given at this point that ammonium 

 picrate is a highly explosive compound 

 which can be detonated without diffi- 

 culty by vibration alone. Under no cir- 

 cumstances whatever should dry am- 

 monium picrate ever be allowed in a 

 laboratory, and the fixative solution under 

 discussion should be prepared immediately 

 before use, and then thoroughly washed 

 down the sink as soon as the period of 

 fixation is finished. To leave the vessel 

 containing the ammonium picrate about 



