10 SMITHSONIAN MISCELLANEOUS COLLECTIONS VOL. I40 



in a plastic funnel placed above the microtome blade and object carrier 

 was used to lower the temperature during the actual microtomy. Be- 

 cause of the minute size of the material, crooked ribbons were almost 

 useless. A device was designed to prevent this nuisance (Lorenzo, 

 1959), and it greatly improved the condition of the ribbon. Trans- 

 verse, horizontal, and longitudinal sections were cut at 10 micra. 



Albumenized water was used as an adhesive. One drop of Mayer's 

 Egg Albumen was added to ten milliliters of distilled water. The water 

 was first boiled to expel gases which might form bubbles under the 

 sections. Because of the consistency of the imbedding medium, 

 stretching of ribbons was kept to a minimum. The excessive adhesive 

 was drained ofif, the slides were chilled briefly under dry ice, and 

 gently blotted, face down, on filter paper. Drying was continued on 

 the warming table for about an hour and completed in a desic- 

 cator before staining. The slightest trace of moisture under the sec- 

 tions caused loss of material in the subsequent steps of deparaffinizing 

 and hydrating. After the paraffin had been removed from the tissue, 

 5-minute immersion in o.i -percent celloidin followed by a brief (no 

 longer than 60 seconds) drying in air was employed as an added pre- 

 caution against section loss. 



Hansen's trioxyhematein counterstained with picrofuchsin (Rich- 

 ins, 1938) was found to be a good general stain. Muscle tissue stains 

 yellow, connective tissue and neurilemma red, and nuclei dark brown. 



Several silver impregnation methods were tried without success. 

 Controlling the pH of impregnation solutions (Samuel, 1953a; Peters, 

 1955) gave inconsistent results with the silver nitrate method of 

 Holmes (1943). Both chilopod and vertebrate nervous tissue was af- 

 fixed to the same slide for comparison of results. While the verte- 

 brate nerve fibers were impregnated well with this technique, the chi- 

 lopod tissue was not. The protargol method of Bodian (1936) was 

 adopted and proved most effective. Several "protargol" products, 

 however, were erratic. The Chroma ^'^ silver protein, manufactured 

 especially for use in Bodian staining, produced consistent success and 

 was used throughout the present study. 



The procedure outlined by Bodian was followed with slight modifi- 

 cations. Best results were always obtained with gold toning in i.o- 

 percent aqueous gold chloride without acetic acid. In the toning proce- 

 dure, best contrast was given when slides were kept in 0.25-percent 

 aqueous oxalic acid four times longer than in gold chloride. The time 

 ratio, therefore, of gold chloride to oxalic acid was i 4. Fifteen sec- 



^o The registered trade mark for the West German products originally known 

 as "Gruebler" stains. 



