enzyme carbonic anhydrase which is present in 

 \arious mollusks may be expected to accelerate 

 deposition of calcium carbonate, and the rate of 

 deposition is retarded by carbonic anhydrase 

 inhibitors. 



Complex metabolic cycles involved in shell for- 

 mation have been reviewed by Wilbur (1960), 

 and probable relations of carbon dioxide to shell 

 conchiolin and carbonate deposition are shown by 

 him in a summary diagram (fig. C, p. 25 of Willnu-'s 

 paper) . 



CYTOLOGICAL IDENTIFICATION OF CALCIUM 



Several methods for the identification and local- 

 ization of calcium salts in the oyster tissues are 

 available, but none are completely reliable. 

 Goniori (1939) suggests that soluble calcium could 

 be demonstrated by treating the frozen sections 

 with ammonium oxalate, the insoluble octahedral 

 crystals of calcium oxalate being easily recognized. 

 The use of a fixative consisting of formalin and 

 ammonium oxalate was also proposed (Rahl, 

 cjuoted from Gomori). Both methods tried in 

 my laboratory on sections of oyster mantle gave 

 unsatisfactory results. The diflRculty is the dis- 

 lodging of calcium-bearing granules and mucus 

 during sectioning, since the granules are easily 

 carried out by the knife's edge from their original 

 location inside the cells to the outside of the epithe- 

 lium. This difficulty can be avoided to a certain 

 extent by double embedding the tissue in colloidin- 

 paraffin. 



Indirect methods of Ca''"''' identification are 

 based on the use of heavy metals (silver, cobalt, 

 copper, and iron). Because almost all insoluble 

 calcium compounds in the tissues are either 

 phosphate or carbonate, any procedure which 

 would demonstrate the presence of these anions 

 may be considered specific for calcium. When the 

 sections are immersed in a solution of one of the 

 heavy metals the corresponding metallic salt is 

 formed at the sites of phosphate or carbonate. 

 The reduction may be effected by exposing to light 

 if silver nitrate is used, or by immersing in appro- 

 priate reducing reagents (ammonium sulfide, 

 acidified potassium ferricyanide). Identification 

 by staining of calcium is based on the formation of 

 insoluble lacs with several hydroxyanthraquinine 

 dyes (alizarin sulfonic acid, purpurin, anthrapur- 

 purin). Calcium deposited in the process of shell 

 formation may, however, contain substances which 

 interfere with the lac-forming reaction of alizarin. 

 Also, tlie dye frequently fails to stain old deposits 



and its color is affected by the presence of iron. 

 Although these complications limit the usefulness 

 of alizarin as a reagent for the determination of 

 calcium, I found that a 1 percent water solution of 

 alizarin S (sodium alizarin sulphonate) is probably 

 the best histochemical reagent for identification of 

 calcium in the oyster mantle. It readily reacts 

 with new deposits of calcium carbonate or calcium 

 phosphate and forms compounds resistant to both 

 acids and alkalies. 



To study the cytology of calcium secretion, 

 the deposition of conchiolin and its calcification 

 was stimulated by cutting oft" small pieces of shell 

 along the posterior margin of the oyster. Labora- 

 tory experience shows that such injury made 

 during the warm season is rapidly repaired. 

 Small pieces of the mantle border with the adhering 

 and partly calcified conchiolin were excised and 

 3 days later preserved in neutral formalin or 

 absolute ethyl alcohol. Sectioned tissues were 

 stained with alizarin S and other reagents for 

 demonstration of calcium. The preparations 

 showed a large number of alizarin stained globules 

 or granules, about 1 .5 /i or less in diameter adhering 

 to the surface of the mantle. Identical granules 

 were found inside the goblet cells of the epithelial 

 layer along both sides of the mantle (fig. 100). 



The results of the staining and other histo- 



IVlicrons 



Figure 100. — Calcium containing grannies discliarged by 

 the epitlielial cells at the cilgc of the mantle of C. 

 virginica. Neutral formalin 3 percent, alizarin. 



THE MANTLE 



101 



