FILTERS 



95 



FIXATION 



Elt'ord. Made of collodion. 



Fischler's modification of Benda's stain for 

 Jally acids and soaps (Fischler, F., 

 Zentralbl f. Allg. Path. u. path. Anat., 

 1904, 15, 913-917) has been severely criti- 

 cized by Lison (p. 203) who concludes 

 that it is of no microchemical value. 



Mallory (p. 120) has, however, given 

 a somewhat different description of the 

 technique. He explains that since the 

 Na and K fatty acid salts (soaps) are 

 soluble in formalin, it is necessary to 

 change them into insoluble Ca soaps by 

 saturating the 10% formalin fixative with 

 calcium salicylate. Comparison of 

 stained sections of such material with 

 others fixed simply in formalin shows the 

 presence and absence of the fatty acid 

 salts (soaps). Calcium soaps can be dis- 

 tinguished from fatty acids because they 

 resist solution in a mixture of equal parts 

 abs. ale. and ether or in hydrochloric 

 acid whereas the fatty acids are soluble 

 in this mixture and calcium in hydro- 

 chloric acid. The method, as detailed 

 by Mallory, is : Mordant frozen sections 

 of 10% formalin fixed material in sat. 

 aq. copper acetate (12.5%), 2-24 hrs. at 

 room temperature. Wash in aq. dest. 

 Stain 20 min. or more in Weigert's he- 

 matoxylin made up by mixing 1 gm. 

 hematoxylin dissolved in 10 cc. abs. ale. 

 with 1 cc. sat. aq. lithium carbonate 

 (about 1.25%) plus 90 cc. aq. dest. 

 several days before use. Differentiate 

 in Weigert's borax-potassium ferri- 

 cyanide, (2.5 gms.ferricyanide and 2 gm. 

 borax plus 100 cc. aq. dest.) much 

 diluted until red blood cells become 

 decolorized. Wash thoroughly in aq. 

 dest. Mount in glycerin jelly or glyc- 

 erin. Fatty acids deep blue black. Fe, 

 Ca and hemoglobin may also be stained. 

 To stain neutral fats inaddition stain with 

 scarlet red after washing out Weigert's 

 fluid, rinse in 70% ale. and in water and 

 mount in glycerin. 



Fixation by immersion is usually the first 

 step in making permanent preparations. 

 Compared with the direct microscopic 

 examination of still living cells removed 

 from the body and placed in approxi- 

 mately isotonic media, it has both ad- 

 vantages and disadvantages. Among 

 the first is the fact that the normal form 

 relations of the tissue components are 

 more faithfully preserved in large pieces 

 by fixation; because it is not necessary 

 to separate the tissue by teasing, or in 

 some other way, into sufficiently small 

 or thin pieces for microscopic study. 

 Moreover, by fixation, the cells are 

 suddenly and uniformly killed, so that 

 the changes resulting from unfavorable 

 fluid environment outside the body, 

 leading slowly or quickly to injury and 



death, are not encountered. The chief 

 objection to fixation is that the structure 

 is very definitely modified thereby and 

 care must be exercised in reaching con- 

 clusions as to living tissues from the 

 study of fixed ones. It is important to 

 restrict these structural changes to 

 those inseparable from the action of the 

 fixative itself, and of the subsequent 

 technique under the most favorable 

 conditions. 



P^educe to a minimum the time in 

 which these complicating alterations can 

 occur by prompt fixation. Remove the 

 tissue from an animal under general 

 anesthesia, or immediately after it has 

 l^een killed, by a method unlikely to 

 injure the tissues. In the case of human 

 tissues removed at operation one should 

 be on the look out for complicating 

 factors. If the tissue is collected at 

 autopsy the autopsy should be made at 

 the earliest possible mom.ent after death. 

 See Postmortem Changes. If delay is 

 unavoidable, keep the body, or the tissue, 

 inaniceboxto reduce the speed of chemi- 

 cal change. In case an excised tissue 

 cannot be immediately fixed, place it in 

 a covered glass container with some cot- 

 ton moistened with physiological saline 

 solution. Do not put it in the solution. 

 Keep the container likewise at a low 

 temperature. 



Carefully avoid injury to the tissue 

 from any cause. Letting its surface dry 

 during removal from the body, or at any 

 time before fixation, produces Artifacts. 

 So also does mechanical manipulation. 

 If forceps must be used, do not pinch the 

 part of which the preparation is to be 

 made. It is better to lift the tissues. 

 Scissors tend to squeeze the tissue, but 

 it is necessary to cut with them in some 

 cases. The ideal way is to cut with a 

 sharp razor blade. This is easy with the 

 liver, kidney, brain and other more or 

 less compact organs, but the sweep of a 

 razor blade tends to draw the tissue and 

 cause displacement, especially when the 

 specimen is heterogeneous, some parts 

 being loose connective tissue, others 

 muscle, others gland, etc. When feas- 

 ible, cut the tissues into slices and lift 

 them into the fixative. For fixatives that 

 penetrate easily (formalin, Zenker's and 

 Bouin's fluids, etc.) make the slices 4-6 

 mm. thick. For the poor penetrators, 

 in which osmic acid is the principal 

 ingredient (Bensley's A.O.B., Flem- 

 ming's fluid, etc.), the slices must be 

 not more than 2 mm. thick. In the case 

 of surface tissues (skin, gastric mucous 

 membrane, bladder wall, etc.) fix a strip, 

 flattened on the surface of a piece of 

 wooden tongue depressor or stiff paper 

 card. A volume of fixative at least 20 



