EAR 



106 



EAR 



centration while it takes longer to act 

 is less likely to overdecalcify. A few 

 investigators recommend the use of 3% 

 nitric acid in water and a constant tem- 

 perature (37^°C.) to hasten the proce- 

 dure. Degree of decalcification is us- 

 ually judged by probing with a needle, 

 or a simple test with an indicator such 

 as phenol red. Most small animal 

 bones are decalcified in about 4 to 10 

 days. However, human temporal 

 bones vary considerably and may take 

 as long as 6 to 8 weeks with weekly 

 change of the solution. After decalci- 

 fication, thorough washing for 24 hours 

 in running water is necessary following 

 which neutralization in 5% sodium sul- 

 fate may be used and washing in run- 

 ning water repeated. 



In order to preserve cytologic detail 

 attempts have been made to circumvent 

 decalcification in strong acids. Small 

 laboratory animals may be perfused 

 with Regaud's solution and following 

 fixation mordanted in potassium bi- 

 chromate for a considerable length of 

 time. The blocks can be embedded in 

 paraffin and sections made although de- 

 calcification is usually incomplete. 

 Mitochondria in hair cells, stria vas- 

 cularis and spiral ganglion cells can be 

 studied by this method. 



In his study of kittens, young rabbits, 

 dogs and rats, Van der Stricht, O., 

 Contrib. to Embryol., Carnegie Inst., 

 1920, 9, 109-142 fixed isolated cochleas 

 in 5% aq. trichlorlactic acid, Bouin's 

 and Zenker's fiuids, mordanted for 

 "many weeks" in 70% alcohol + a few 

 drops of iodine solution. After the last 

 2 fixatives he completed decalcification 

 in 2% nitric acid in 70% alcohol. Be- 

 fore imbedding in paraffin he stained 

 with Borax Carmine and he colored the 

 sections with Iron Hematoxylin, Congo 

 Red and Light Green. Directions will 

 be found in his paper for the demonstra- 

 tion of mitochondria in the sustentacu- 

 lar and hair cells. A differential stain 

 for hair cells is described by MacNaugh- 

 ton, I. P. J., and Peet, E. W., J. Laryng. 

 and Otol., 1940, 55, 113-114 with a fine 

 colored figure of the results. 



Celloidin is generally used for im- 

 bedding animal and human material. 

 It is not ideal since it is difficult to 

 handle, takes considerable length of 

 time to infiltrate and is expensive. 

 Various nitrocellulose samples have 

 been tried for small blocks of bone with 

 success but usually centers of large 

 blocks, particularly human temporal 

 bones, do not become sufficiently hard. 

 Concentrations of celloidin usually 

 ranging up to 15% are employed in suc- 

 cessive steps. While the material is in 



8%, 10% and 15% celloidin negative 

 pressure is used in moderate amounts 

 to insure infiltration of celloidin into 

 chambers of the inner ear. This should 

 be done with considerable caution as 

 rupture of the delicate membranes may 

 result. When the specimen is ready for 

 embedding it is amply covered with 15% 

 celloidin and allowed to remain in the 

 refrigerator (the lower temperature pre- 

 vents excessive bubble formation) until 

 it can be blocked. Blocking of the ma- 

 terial is important and depends upon 

 whether vertical or horizontal sections 

 through the cochlea are desired. This 

 is readily determined by such land- 

 marks as the eminentia arcuata, and 

 external and internal auditory meati. 



Sections of large blocks are usually 

 cut at 10 to 15 micra in thickness on a 

 sliding microtome. Every section is 

 numbered and kept for further study 

 if necessary, while every tenth or every 

 twentieth section is put aside for stain- 

 ing as a "tracer" series. 



The nerves of the tympanic mem- 

 brane were successfully stained intra- 

 vitally by the use of methylene blue; 

 Wilson, J. G., J. Comp. Neurol, and 

 Psychol., 1907, 17, 459-468. Peripheral 

 endings of the cochlear nerve were 

 stained with 1:5000 methylene blue in 

 isolated pieces of the fresh membranous 

 cochlea removed under the dissecting 

 microscope; Covell, W. P., Ann. Oto., 

 Rhino., Laryngo., 1938, 47, 62-67. 



The peripheral fibers of the cochlear 

 nerve have recently been demonstrated 

 by Fernandez, C. (Laryngoscope, 1951, 

 59, (in press)) using Bodian and Cajal 

 silver Methods. The course of the 

 fibers were traced in pieces removed 

 by dissection and mounted on slides. 

 A method for study of Wallerian de- 

 generation in the cochlear nerve by 

 use of the Swank-Davenport osmic mix- 

 ture is described by Rasmussen, G. L. 

 (Abstr., Anat. Rec, 1950, 106, 120). 

 Ross, E. L. and Hamilton, J. W. (Arch. 

 Otol., 1939, 29, 428-436) allowed mer- 

 curochrome to remain in the middle 

 ear cavities of dogs for 20 minutes to 

 2 hours, fixed the mercurochrome in 

 acid solution, decalcified the bones, and 

 studied the distribution of the dye in 

 frozen sections of the cochlea. The pas- 

 sage of fluorescein after arterial injec- 

 tion was observed by Gisselsson, L. 

 (Acta oto-laryng., 1949, 37, 268-275) in 

 the cochlea. Trypan blue has been 

 utilized to ascertain the effects of 

 trauma on scala media cells of the 

 cochlea (Lurie, M. H., Ann. Otol., Rhin., 

 Laryng., 1942, 51, 712-717). The capil- 

 lary areas of the cochlea have been 

 further studied in animals by Smith, 



