54 FIXING AND HARDENING AGENTS. 



This is a very moderate and delicate fixative, extremely useful for 

 objects that are to be studied in as fresh a state as possible in aqueous 

 media. Objects fixed in it stain instantaneously and perfectly with 

 methyl green. Osmic acid may be added to the liquid to increase 

 the fixing action. For cytological researches a valuable medium. 



91. Nitrate of Copper (G-ILSON, from GELDERD, La Cellule, xxv, 

 1909, p. 12). Nitrate of copper 200, formol 500, sea-water 200. 

 Seven parts of this solution to be diluted with 100 of sea- water. For 

 Crustacea. 



92. Acetate of Uranium (SCHENK, Mitth. Embryol. Inst. Wien, 1882, 

 p. 95 ; cf. GILSON, La Cellule, i, 1885, p. 141) has a mild fixing action, 

 and a high degree of penetration, and may be combined with methyl 

 green. 



FRIEDENTHAL (Sitzb. Ges. Nat. Freunde Berlin, 1907, p. 209) recom- 

 mends equal parts of saturated solution of the acetate and trichlor-acetic 

 acid of 50 per cent. 



93. Picric Acid. Picric acid in aqueous solution should be em- 

 ployed in the form of a strong solution whenever it is desired to make 

 sections or other preparations of tissues with the elements in situ, as 

 weak solutions, macerate ; but for dissociation preparations or the 

 fixation of isolated cells, weak solutions may be taken. Flemming 

 finds that the fixation of nuclear figures is equally good with strong 

 or weak solutions. The saturated solution is the one most employed. 

 (One part of picric acid dissolves in about 86 parts of water at 15 C. ; 

 in hot water it is very much more soluble.) Objects should remain 

 in it for from a few seconds to twenty-four hours, according to their 

 size. For Infusoria one to at most two minutes will suffice, whilst 

 objects of a thickness of several millimetres require several hours. 



Picric acid should always be washed out with alcohol, that of 70 per 

 cent, being mostly indicated. Staining should be performed by 

 means of alcoholic solutions, or if with aqueous, then with such as 

 are themselves weak hardening agents, such as hsemalum, carmalum, 

 methyl green. 



Washing out is facilitated by heat, the extraction being about 

 twice as rapid at 40 C. as at the normal temperature (FoL). 



It has been found by JELINEK (Zeit. iviss. Mik. } xi, 1894, p. 242) 

 that the extraction is greatly quickened by the addition of a base to 

 the wash-alcohol. He recommends carbonate of lithia. A few 

 drops of a saturated solution of the salt in water are added to the 

 alcohol ; a precipitate is formed. The objects are put into the 

 turbid alcohol, which becomes clear and yellow in proportion as the 

 picrin is extracted. Further quantities of carbonate are added from 

 time to time until the colour has been entirely extracted. 



