1908.] SPICULES OP CALCAREOUS SPONGES. 667 



spicviles in distilled water a few drops of ordinary glycerine and 

 albumen-solution, such as is used for sticking sections on slides. 

 The spicules were shaken up in this and then centrifuged down, 

 a,fter which a drop or two of the fluid, with the spicules, was 

 drawn up with a pipette, spread out on a slide, and dried off on 

 the paraffin oven. When dry, the spicules were fixed on the 

 slide by plunging it into absolute alcohol, whereby the glycerine 

 is extracted and the albumen coagulated. The spicules can now 

 be decalcified and stained in any way that is desired. Other 

 methods of imbedding and fixing the spicules were also tried but 

 were not satisfactory. By means of the albumen solution, 

 provided that neither too much nor too little be used, good 

 permanent preparations of the decalcified and stained spicules can 

 be made and mounted in Canada balsam. 



In addition to this method, spicules in distilled water, without 

 addition of albumen, were treated with acids and stains and the 

 eflects of them watched under the microscope. Since the spicules 

 treated in this manner were not fixed to the slide, it was impos- 

 sible to wash out the stain and mount them permanently in 

 Canada balsam, but it was possible to observe in detail the effects 

 of the acids and stains upon the spicules, and there was the 

 advantage that the results were not complicated by the presence 

 of the albumen, which is itself stained by both nigrosin and 

 indulin. 



The Spicules o/CIathrina contorta*. 



We shall begin with an account of the results attained with the 

 spicules of Clathrina contorta, of which we had a very abundant 

 material ; the differences presented by other species will be noted 

 subsequently. Spicules fixed to the slide with albumen were 

 treated for about half-an-hour with a combination of an acid and 

 a stain in the following proportions : — 



|°/g to 1°/^ of acid in distilled water, except in 

 the case of picric acid of which a saturated 

 solution was used 9 vols. 



17q stain in distilled water 1 vol. 



In this way, picric, nitric, acetic, and hydrochloric acids were 

 combined, respectively, with either nigrosin or indulin as a stain. 

 After staining for 20 minutes or half-an-hour, the preparations 

 were washed with distilled water, absolute alcohol, oil of cloves, 

 and mounted in Canada balsam. The results in all cases were 

 the same. The spicule was completely decalcified, and left behind 

 a deeply stained sheath, and an axial filament in each ray (figs. 1- 

 10). The best and clearest results were obtained with the picric 

 acid combinations ; with the other acids the filaments were 

 stained just as deeply, but there was a frequent tendency to foi-m 

 a flaky deposit which obscured the result. 



* For an account of the spiculation and nomenclature of this sponge, see Minchin, 

 P. Z. S. 1905, ii. pp. 3-20. 



