Disadvantages. Slowness; does not preserve division-figures; does not permit of staining for bacteria; does not give good results with many special staining methods (fibrin, elastic tissue, reticulum, etc.).
B. Erlitzky’s Fluid (Potassium bichromate 25.0 grms., copper sulphate 5.0 grms., water 1,000.0 cc.). Used for fixation of nervous tissue. The formation of pigment precipitates may lead to misinterpretation: the artefacts may be removed by hot water or dilute acetic acid.
C. Orth’s Fluid (Müller’s fluid 100 cc., formol 10 cc. Make fresh before using, as the mixture precipitates on standing.). Fix for 3-12 hours in the incubator, or for 24-48 hours at room temperature. Wash in running water for 12-24 hours; cut on the freezing microtome, or after-harden in acetone or alcohol and imbed.
Advantages. Combines the good features of Müller’s and formol fixations, and obviates some of the disadvantages. It is a good general fixing solution.
3. FORMOL OR FORMALIN (40 per cent solution of formaldehyde gas). Used in a ten per cent solution (water or physiologic salt solution nine parts, formalin one part), often incorrectly called 4 per cent formol or formalin, the mistake arising from the confusion with 4 per cent formaldehyde gas. For nerve-tissues it is better to dilute the formol with physiologic salt-solution than with water. Fix for 3-4-12 hours according to size of tissue. As it penetrates well large pieces can be used. Wash in water before after-hardening in alcohol, if tissue is to be employed for general work, otherwise it can be transferred directly to the alcohol without washing. Tissues can be kept in formol for some weeks, but after that time the staining-power is slowly affected, and the finer structures suffer.
Advantages. Probably the best fixing reagent for general pathologic work. It is cheap; easily made and kept in solutions of proper strength for fixing; hardens while it fixes; does not require after-washing; penetrates well; causes little shrinking; permits freezing directly from the fixing solution; preserves fat; permits the use of after-hardening with bichromate solutions for especial nerve-stains: preserves the red blood-cells: gives good results with nearly all stains, and differentiates bile-pigment from hæmatoidin. It is the best fixing reagent when tissues are to be sent some distance, as over-fixation occurs only after several weeks or even months.
Disadvantages. Affects many people unpleasantly, causing coryza, eczema of the hands and arms, and affections of the finger-nails, so that workers having acquired this idiosyncrasy cannot expose themselves to formol vapor; it dissolves glycogen and uric acid; does not fix cell-division figures as well as mercuric chloride or Flemming’s solution; causes the precipitation of diffuse hæmoglobin in the form of brown or black pigment-granules that may be taken for melanin, malaria pigment or hæmosiderin; causes a pseudo-ochronosis of cartilages; and, unless thoroughly washed from the tissues before after-hardening in alcohol, it makes carmine-staining difficult or unsatisfactory and affects also the specific staining-reactions for amyloid and mucin; it is not as good as alcohol or mercuric-chloride when the sections are to be stained for bacteria. In spite of these disadvantages it can be recommended as the best general fixing reagent.
For Orth’s fluid, formol-acetone and formol-alcohol see above.
4. FREEZING AND DRYING. The fresh tissue is frozen and dehydrated in a vacuum over sulphuric acid, at a temperature of 20-30°C.; when completely dried it is imbedded directly in paraffin. This method has been especially recommended by Altmann on the ground that the tissues are simply deprived of water without any change in volume.
5. HEAT. Physiologic salt-solution is heated to 80°C. Thin pieces of tissue are placed in the hot water for two minutes, and then after-hardened in alcohol. For larger pieces of œdematous tissues, cysts, etc., that cannot be cut into thin pieces, the salt-solution should be brought to 100°C and the tissues boiled for several minutes. This method is advised particularly for the coagulation of albumin in cysts, œdematous tissues, for the study of renal casts, etc.