FIXATION 



96 



FIXATIVES 



times that of the tissue fixed is required. 

 Agitate the bottle slightly to prevent the 

 tissue from sticking to the bottom and 

 to ensure penetration from all sides. 



It may be desirable to inject the fixa- 

 tive via a large artery supplying the 

 tissue to be examined. This eliminates 

 mechanical injury to the tissue before 

 fixation, preserves gross form relations 

 better and is suggested when sections are 

 required of large specimens. Before in- 

 jecting the fixative wash out some of 

 the blood by Perfusion with physio- 

 logical salt solution, or at least let the 

 blood drain out from the veins, because, 

 if all is left in, it may clog the arteries 

 and block the entry of the fixative. 

 After fixation by vascular injection it is 

 customary to cut, with a razor blade, 

 suitable slices and to continue the fixa- 

 tion by immersion. Obviously such 

 tissues should not be employed for micro- 

 chemical analyses because there is a 

 danger of washing out chemical sub- 

 stances. Clearly, also, the speed of 

 fixation depends upon the degree of vas- 

 cularity. For avascular tissues such as 

 epidermis, cornea and cartilage fixation 

 by injection is not recommended. 



After the tissues have hardened a 

 little by immersion in or injection with 

 the fixative, it may be helpful to remove 

 them from the fixative and trim them 

 with a razor blade so that their size and 

 shape will be almost what is needed when 

 they are finally cut into sections. The 

 slices should have smooth upper and 

 lower surfaces including an area which 

 will yield sections that will fit nicely 

 under a 22 x 22 mm. cover glass unless 

 larger covers arc to be used. The shape 

 should be rectangular with opposite edges 

 parallel. In general it is well to have two 

 longer parallel edges and two shorter 

 ones, because a square surface is not so 

 convenient to section as an oblong 

 one. However one must bear in mind 

 exactly what one wishes to demonstrate. 

 This making of uneven surfaces smooth 

 does however introduce an experimental 

 error; because, where much is shaved 

 off, the fixation has penetrated less than 

 where little or no tissue has been re- 

 moved. After trimming return tissues 

 to a fresh supplj'^ of fixative. Tissues 

 fixed in poor penetrators should not be 

 trimmed. 



The time of fixation depends upon the 

 tissue, the fixative and the purpose in 

 mind. In general, 24 hrs. is suitable. 

 Some fixatives, particularly those con- 

 taining potassium bichromate and/or 

 osmic acid, are not very stable and for 

 this reason should be renewed. The 

 fixative deteriorates less quickly if the 

 fixation is carried out at a low tempera- 



ture in an ice box. The speed of fixation 

 is probably also somewhat diminished. 

 The effect of pH on chromium fixatives 

 lias been studied l)y Zirkle (C, Proto- 

 plasma, 1928, 4, 201-227). See results 

 obtained by adding Wetting Agents and 

 Hydroxybenzene Compounds to fixa- 

 tives. Fixation involving Decalcifica- 

 tion and Mordanting are special cases 

 described under these headings. For 

 choice of fixative see Fixatives. 



After fixation Washing may be neces- 

 sary, or Mordanting. The tissue may 

 be prepared as a Whole Mount, or Frozen 

 Sections may be made, or it may be 

 dehydrated, cleared and imbedded in 

 Paraffin or dehydrated and imbedded in 

 Celloidin for Sectioning. 

 Fixatives. The number from which to 

 choose is enormous but the number 

 actually employed is comparatively 

 small. Formalin unquestionably heads 

 the list as being used for a far greater 

 variety of purposes than any other fixa- 

 tive. It penetrates well and is an ex- 

 cellent preservative. It is the only 

 satisfactory fixative for use before the 

 cutting of frozen sections and as a pre- 

 liminary to certain microchemical re- 

 actions. Alcohol comes next in variety 

 of services performed but unfortunately 

 it brings about considerable shrinkage. 

 Both formalin and alcohol are frequently 

 combined with other ingredients. 



For routine purposes Zenker's Fluid, 

 either alone or with formalin, is perhaps 

 the most popular fixative. Tissues so 

 fixed give better contrasts of acidophilic 

 and basophilic components than are 

 obtained after fixation in formalin or 

 alcohol by themselves. Benin's Fluid 

 is also an excellent fixative for general 

 use and is being employed with increasing 

 frequency. It is particularly advocated 

 by dermatologists. Regaud's Fluid is 

 the fixative of choice for mitochondria 

 because it penetrates so much better 

 than Osmic Acid containing fixatives. 

 No important new fixatives have recently 

 been devised. 



In making the selection one is natu- 

 rally guided by data concerning the 

 structures which it is desired to demon- 

 strate (see Nerve Endings, Mitochon- 

 dria, etc.) or the substances to be re- 

 vealed (Lead, Copper, Oxidases, Lipids, 

 etc.) or the techniques that seem best 

 adapted to the purpose in mind (Mal- 

 lory's Connective Tissue stain, Wei- 

 gert's Method, etc.). Some of the more 

 important fixatives ai'e listed, further 

 data being given under each heading. 



Acetic osmic bichro- 

 mate 

 Alcohol (ethyl) 



Allen 



Barium chloride and 

 formalin 



