CHAP, ii PRACTICAL HINTS 33 



of any size osmic acid forms a crust of hardened imperviable proto- 

 plasm which prevents the penetration of the reagent into the interior. 

 It is, therefore, a reagent eminently suited for the preservation of 

 minute larvae and the permeable tissues of calcareous sponges. 



For the denser tissues of siliceous sponges other reagents would 

 be more suitable ; such, for instance, as a mixture of 3 parts 

 concentrated solution of corrosive sublimate in water and 1 part 

 of glacial acetic acid. This is one of the best and most universally 

 employed preservatives : many investigators use, however, a smaller 

 proportion of acetic acid (often as low as 5 per cent) than that just 

 mentioned ; it is to be remembered that acid reagents are unsuitable 

 for calcareous sponges and for other organisms which contain much 

 calcareous matter, because the evolution of carbonic acid gas dissolves 

 the calcareous matter, and so causes the formation of blebs in the 

 tissues and of artificial rents and cavities which have no counterpart 

 in the living animal. When it is desired to decalcify, this is best 

 accomplished when the organism is in strong alcohol. If a drop 

 or two of nitric acid be added to a small bottle (of two fluid ounces) 

 full of strong alcohol and well shaken, a solution is produced which 

 will decalcify so slowly that the resulting gas is at once dissolved and 

 never forms bubbles. 



A different method of decalcifying organisms which have been 

 preserved in osmium tetroxide may be mentioned here. If, after 

 being blackened by immersion in the solution and then rinsed in 

 clean water, the specimens be immersed in Midler's fluid, not only 

 will the calcareous matter be slowly removed but also the excess of 

 metallic osmium, and the tissues will be rendered less brittle. 

 Mliller's fluid is a mixture of bichromate of potash, which contains 

 unsaturated chromic acid and sulphate of sodium. Flemming's 

 fluid, which is a very favourite preserving medium, is really an 

 attempt to combine the advantages of osmium tetroxide and chromic 

 acid, for it is a mixture of these two fluids with acetic acid. It is an 

 excellent preservative, but is intensely acid and open to the same 

 objections as other acid reagents. The same remarks apply to 

 Hermann's fluid, which is a mixture like Flemming's fluid, in which 

 acid platinic chloride replaces the chromic acid. 



When it is desired to make whole mounts of minute forms it 

 will generally be found that osmium tetroxide, corrosive sublimate, 

 etc., render them too opaque. Strong formalin that is a 40 per cent 

 solution of the gaseous formic aldehyde in water is a splendid 

 reagent for this purpose. It kills small larvae instantaneously, with- 

 out any shrinkage. It is apt, however, to become acid by the oxidation 

 of the aldehyde into formic acid ; it is therefore advisable to carefully 

 neutralize the solution before employing it. Further, the compound 

 which it forms with protoplasm is soluble in water. Therefore, after 

 a few minutes' sojourn in the formalin solution, the specimens must 

 be instantly transferred to absolute alcohol, and in this they must 

 be stained. Eosin or methyl green dissolved in absolute alcohol are 



VOL. i D 



