Bouin’s solution

Strong Bouin’s fixative has been used successfully to preserve telophase figures in root tips and embryo sacs (Chamberlain, 1932). Weak Bouin’s was used to preserve phospholipids for subsequent identification using Orange G and Aniline Blue (Baker, 1946). Fix 12–24 h, rinse in 20% EtOH to remove residual picric acid,²⁶ then proceed with dehydration and embedding. Residual picric acid can interfere with subsequent tissue staining, so be sure to wash the tissue thoroughly before processing further. Tissues can be washed using a flowthrough system as described in the previous Notes.

²⁶Take care when handling pure picric acid as it may become explosive if it dehydrates over time.

Chemical Fixation

Allen’s solution

This cytological fixative is reported to be a replacement for Bouin’s solution (Emmel and Cowdry, 1964; Gray, 1964). Keep in mind that solutions containing picric acid may form insoluble precipitates that can interfere with staining, so be sure to wash the tissue thoroughly after fixation. As with other chrome-containing fixatives, mix just before using. Fix tissues overnight to 24 h, wash in 70% EtOH until the yellow picric acid is removed, and proceed with processing.

Water	75 ml
Formalin (37–40%)	15 ml
Glacial acetic acid	10 ml
Picric acid	1 g
Urea	1 g
Chromic acid (optional)	1 g

Flemming’s solution

Flemming’s solution (Chamberlain, 1932; Sass, 1958) is a good cytological fixative that contains both the coagulating agent chromium and the additive fixative osmium tetroxide. Osmium tetroxide fixation is restricted to the outer cell layers due to its slow tissue penetration, whereas the acetic acid thoroughly penetrates tissues. Thus, it is best to use Flemming’s solution on small tissue samples.

The “Stronger” solution (S) was one of the more highly regarded cytological fixatives in its day. Weaker solutions (W) are usually reserved for delicate tissues. Experiment to determine what formulation (or modification) is best for your particular material.

Make the fixative in two stock solutions: A containing the chromic and acetic acid and B containing only osmic acid (osmium tetroxide). Add osmium solution just prior to use. Fix very small pieces of tissue for 24–48 h, wash thoroughly, and prepare for sectioning the usual way. Osmium-fixed tissues need to be bleached before staining (see Bleaching tissues, Chapter 9).

Caution: Osmic acid is an extremely volatile and toxic compound. It can fix corneas and other tissues through vapor fixation. Use extreme caution and always use in the hood.

W			S
A	Chromic acid (1%) Acetic acid (1%) Glacial acetic acid	25 10	75 5
	DI	55
B	Osmic acid (2% aq)	10	20

Methanol–acetic acid (MAA)

Anthers have been fixed successfully for investigation of meiotic chromosomes at the pachytene stage using methanol–acetic acid (Makowski and Ruzin, 1994). Remove anthers from floral buds by dissecting in citrate buffer (0.01 M sodium citrate, pH 4.6). Fix anthers in fresh Y20°C (3:1) MAA fixative. Replace the fixative twice at 5 min intervals then store the anthers in the fixative at Y20°C for 2–24 h. Wash in buffer or DI before observation. Methanol–acetic acid preserves chromosome structure but mostly dissolves cytoplasm.