Fixation

Gregarine Fixation Protocols
(based on various sources, modified through painful experience)

Brasil & Duboscq’s Fluid (Alcoholic Bouin's), Hollande’s Fluid and Schaudinn's Fluid. All are excellent fixatives. Schaudinn's is best for iron hematoxylin staining procedures and preserves nuclear features with minimal artifact. AFA is best for acid carmine staining, but it can produce some small amount of nuclear distortion. On the other hand, AFA is the least toxic and poses the fewest disposal problems. Hollande’s Fluid is an excellent protist fixative and is best for protargol staining. It’s application for other stain protocols has not been fully investigated. In general, I stick to AFA or Bouin’s. AFA in the field, Bouin’s for material that will be sectioned at a later date.
Wet smears for microscope slides: All fixatives are used in a similar manner. Prepare a thin smear of host intestine (with gregarines) on a clean coverslip and drop them face down into a petri-dish of fixative. The slips should float on the surface with no air bubbles. This holds the specimens against the glass until they are affixed through fixation of gregarine and host protiens. In each case, allow the slips to fix for at least 5 minutes, unless the stain protocol specifies a different fixation time. Rinse excess AFA or Brasil & Duboscq’s Fluid from specimens with 70% EtOH. Rinse AFA fixed specimen's for 5 minutes; Bouin's fixed specimens until all traces of yellow are removed. After fixation in Schaudinn's, Bouin's, or Hollande’s Fluid specimens must be dehydrated through a graded alcohol series: 30% (optional) 50%, 70% (3 minutes each). Remove excess mercury from Schaudinn-fixed specimens with two washes in Lugol's Iodine working solution (2 washes, 5 minutes each). Specimens fixed in Bouin's, or Hollande’s Fluid should be washed in 70% EtOH until all traces of fixative color are removed, or at least until the fixatives no longer discolor the EtOH. After fixation gregarines may be stained and processed as permanent mounts. A similar technique may be used with microscope slides rather than coverslips EXCEPT that slides won’t float. They are simply immersed in the fixative.
Entire fixation for gamonts or host intestines: Bouin’s, Brasil & Duboscq’s Fluid, and AFA are all suitable fixatives for this technique (in that order of preference). All fixatives are used in a similar manner. Specimens or host intestines are immersed in a vial of fixative (the volume of fixative should be at least 10 times the volume of tissue). Fixation should proceed for at least 30 minutes, but specimens can be stored in fixative for several years without damage. Rinse excess fixative from specimens with 70% EtOH. Rinse AFA fixed specimen's for 5-30 minutes, depending upon size (at least 3 changes of 3 minutes each); Bouin's or Brasil & Duboscq’s fixed specimens should be rinsed until all traces of yellow are removed. (A few drops of lithium carbonate can be used to increase the solubility of the picric acid and speed up the rinse, but this technique often affects the staining properties of the tissue.) Bouin's fixed specimens should be dehydrated through a graded alcohol series: 30% (optional) 50%, 70% (3 minutes each). After fixation gregarines may be stored in alcohol or stained and processed as permanent mounts.
Intestines should be straightened by prefixation. Flood one half of a slide with fixative and drag the intestine through the fixative. The intestine will straighten as it is dragged across the glass and through the fix. Postfix in vial as usual.

General Procedures and Notes

Working Notes: Mix all reagents and use without dilution. AFA keeps well (up to one year). The formation of a cloudy precipitate or the distinct smell of formic acid indicates that the solution should be discarded soon and replaced with a fresh stock.

Reagents

Formalin (40% aqueous formaldehyde)
95% Ethanol
Glacial Acetic Acid
Distilled water

Working Notes: Saturated spirits of picric acid are prepared by adding picric acid crystals to 70% ethanol until no more will dissolve. Some precipitate should remain as evidence of saturation. There is some explosive hazard with picric acid. This hazard is reduced by storing the acid under 70% EtOH, also providing a ready saturated solution. Stores well for several months.

Brasil & Duboscq’s Fluid (Alcoholic Bouin's Fixative)

Picric acid (saturated spirits)
Formalin (40% aqueous formaldehyde)
Glacial Acetic Acid

Working Notes: This mixture keeps well, but some persist in making the solution up immediately before use.

Bouin’s Fluid (Aqueous Bouin's Fixative)

Picric acid (saturated aqueous)
Formalin (40% aqueous formaldehyde)
Glacial Acetic Acid

Working Notes: Prepare a saturated aqueous solution of Mercury Bichloride with distilled water and store in a glass bottle in a cool, dark place. Some precipitate should remain as evidence of saturation. Schaudinn'd Fluid does not storE for more than a few days; however, the mercuric chloride and ethanol may be mixed as a stock solution that will keep for several months. Add 5 ml of acetic acid to 100 ml of stock solution to prepare a working solution for use within 48 hours.

Schaudinn's Fluid

Mercuric Chloride (saturated aqueous)
95% Ethanol
Glacial Acetic Acid

Working Notes: Store in a cool, dark place. Keeps forever. Used to remove mercury from fixed material to avoid precipitation in specimen. Add to 70% ethanol to form a straw-colored working solution. (Extra Lugol's in the working solution doesn't hurt.) Add more Lugol's stock to the working solution and the color disappears.

Lugol's Iodine

Iodine (resublimed)
Potassium Iodide
95% ethanol

Working Notes: Dissolve cupric acetate in water, followed by the picric acid. Add the formalin and acetic acid after solution. After fixation, transfer by graded series to 70% EtOH and continue repeated, gentle fluid changes until picric acid and cupric acetate are removed. Stores well.

Hollande’s Fluid

Picric Acid, wet crystal
Cupric acetate
Distilled water
Formalin (40% aqueous formaldehyde)
Acetic Acid, glacial

4 g
2.5 g
100 ml
10 ml
1.5 - 5 ml

60 ml
500 ml
40 ml
400 ml

75 ml
25 ml
5 ml

66 ml
33 ml
5 ml

2 gm
3 gm
100 ml

AFA (Alcohol Formalin-Acetic Acid)