188 



METHODS AND FORMULAS 



F02 



adjusted so that it is not below that of the 

 isoelectric point of the proteins to be fixed. 

 He considered a pll in the general vicinity 

 of 4.2 to be the most desirable. His work 

 was pubhshed at the same time as that of 

 Gihei 1925 (3432, 39:164), who gives a 

 table showing the pH of a few fixatives, 

 none of which, however, were as alkaline 

 as the upper Hmit indicated by Cretin. 

 This work was followed in 1928 by the de- 

 tailed study of Zirkle 1928 (17191a, 4:201) 

 who came to the conclusion that there was 

 a critical pH between 4.2 and 5.2. Fixative 

 solutions more acid than this critical 

 point were required for nuclear fixation, 

 while those more alkaUne gave better 

 results in cytoplasmic studies. Jacquiert 

 1930 (6630, 104:483) considered that a 

 low pH with a suitable osmotic adjust- 

 ment would give the more generally desir- 

 able fixative. This last study confirmed 

 the work of Burchardt 1897 (6011, 

 12:337) who recommended alkahne di- 

 chromate for cytoplasmic fixation and acid 

 dichromate for nuclear fixation. This uni- 

 versal preference for acid fixatives must 

 not cause one to lose sight of the work of 

 Barnabo 1904 (3389, 13:198) and 1905 

 (3389, 14:139, 205) who stated that mer- 

 curic fixatives buffered with bicarbonate 

 gave, in general, very much better pic- 

 tures both of nuclear and cytoplasmic 

 fixation than did the more usual acid mix- 

 tures. Another characteristic which may 

 be mentioned is that the majority of mate- 

 rials used for fixation are either strongly 

 reducing or strongly oxidizing materials. 

 This was first brought forward as a basis 

 for the construction of formulas by Unna 

 and Golodetz 1912 (7175, 22:10). They 

 based their results on previous studies by 

 Unna 1911 (1739, Fest. Waldeyer, 78) on 

 the oxidizing and reducing properties of 

 hving tissues. This view, that oxidizing 

 and reducing qualities should first be 

 sought, is advocated very strongly by 

 Langeron in the 1942 edition of his Precis 

 de Microscopic. 



The Uterature is so confused that no 

 specific recommendations can be made for 

 the theoretical production of a perfect 

 fixative. The confusion which exists, 

 however, with regard to the desirable 

 qualities of a formula is very httle com- 



pared to the confusion which has arisen 

 ill the jiractical i)reparation of these solu- 

 tions. Authors have, in many instances, 

 made no attempt to check the literature 

 for the existence of a solution before they 

 have invented one of their own. This 

 duplication is all the more difficult to 

 check for the reason that authors prepare 

 their own solutions from any stock solu- 

 tion wliich happens to be on their shelves 

 at the time. Thus various authors have 

 recommended a few milUhters of a 2%, 

 3%, 4%, or 5% solution of potassium 

 dichromate to be mixed with a few other 

 milhhters of 0.25%, 0.5%, 1%, or 2%, 

 osmic acid. One of the most laborious 

 features of the preparation of this work 

 has been the reduction of all these form- 

 ulas to a standard volume as though they 

 had been prepared from dry salts. These 

 standardized formulas are offered in two 

 ways. First, together with the full citation 

 to the original appearance, have been 

 given the ingredients required to prepare 

 250 milUhters of solution from standard 

 materials. This figure of 250 milhliters, 

 rather than 100, has been taken as a 

 matter of convenience since this is the 

 quantity most usually made up. The 

 names of chemicals have been given in full 

 rather than in symbolic form for the reason 

 that it is difficult to know where to draw 

 the fine. It is to be presumed that any 

 biologist would not have the slightest 

 difficulty in preparing the solution made 

 up of HgClz and HNO3. It is felt, how- 

 ever, that the majority would be stopped 

 cold by the directions to mix (N02)3- 

 CeHiOH, HCHO and CH3COOH. To 

 avoid the usual confusion between formol, 

 formaldehyde, and formalm, the author 

 has used the term 40% formaldehyde 

 throughout, intending to indicate by this 

 the fluid which comes in the bottle so 

 labeled. 



Formulas are also presented in an 

 abbreviated, alphabetical fist, permitting 

 the preparation of the formulas referred 

 to from standard solutions, which act, as 

 it were, as the lowest common denomi- 

 nator of about 80% of fixatives employed. 

 The explanation of this method, together 

 with the list of fixatives, forms the second 

 half of the present chapter. 



