Chemical Fixation

From Ruzin, 1999. Plant Microtechnique and Microscopy

COAGULATING FIXATIVES

ACID FIXATIVES

The result of stabilizing tissues with these types of fixatives produces the so-called acid fixation image (cf. Basic fixatives, this chapter). These fixatives are good for preserving chromatin, nucleoli, and spindles. The cytoplasm is preserved as a stringy, coagulated mass, but some organelles are dissolved (e.g., mitochondria.)

Formalin–Acid–Alcohol: FAA and FPA

Formalin–acetic acid–alcohol (FAA) and formalin–propionic acid–alcohol (FPA) are good general-purpose fixatives. Compared to other fixatives such as acrolein, tissue penetration is not particularly fast and, due to the presence of alcohols, shrinkage may occur. You may vary the amount of acetic or propionic acid from 2 to 6% to modulate shrinkage and better preserve chromatin structure. Increase concentration of acetic or propionic acid to induce greater tissue swelling and to counteract alcohol shrinkage. Generally, tissues are killed and hardened within 18–24 h when treated at RT. The fixative is stable and does not induce hardening, so tissues may be stored in these solutions indefinitely. FPA is considered by some to yield better preservation than FAA, but this may be a tissue-specific phenomenon. Johansen (1940) and Brooks et al. (1966) consider FPA to be an excellent fixative for anatomical and morphological studies. Johansen (1940) and Bruni and Tosi (1980) specifically recommended FPA for preserving laticifers. FAA loses effectiveness with storage.

Carnoy’s fixative

Carnoy’s fixative is a chloroform-containing fixative. It penetrates tissues extremely rapidly and can fix small tissue pieces in minutes rather than the hours required for other fixatives (Chamberlain, 1932; Sass, 1958). Delicate tissues can be damaged when transferred from aqueous solutions to this fixative, due to the extreme hydrophobicity of chloroform and resultant rapid tissue dehydration. Reserve Carnoy’s for more hearty samples. This fixative has traditionally been used for cytological structures.
Fix small tissue pieces approximately 1 h, wash several times in absolute ethanol, infiltrate, and embed immediately.

Farmer’s fixative

Farmer’s fluid is an anhydrous fixative solution that causes rapid dehydration and fixation. As with Carnoy’s fixative, the rapid exchange of tissue water for fixative can cause extreme cellular disruption. However, these two fixatives are excellent for cytological investigations (Sass, 1958; Golubovskaya, 1994).