Hoyer's acetocarmine (HAC): a one-step staining and mounting technique for chromosome preparation

Hoyer's solution is a water soluble mounting medium; acetocarmine, a water soluble stain. Florence S. Wagner, at the University of Michigan, Ann Arbor, working with fern chromosomes, combined these 2 solutions in equal volumes, producing Hoyer's acetocarmine. She and her husband have used this-with excellent results for several years.

In preparing a chromosome squash, the HAC is used as a conventional stain. Chromosomes stain lightly, but can be seen clearly using phase contrast. To make a preparation permanent the slide is allowed to sit for 2-3 days at room temperature and in this time the HAC slowly hardens. Slides are then sealed using clear nail polish. The great asset of this technique is that the slide becomes permanent without having to remove the coverslip.
Hoyer's solution
60 gms gum arabic 100 mls distilled water
400 gms chloral hydrate 40 mls glycerine

Dissolve gum arabic in water for 24 hours. Add chloral hydrate. Let stand 24 hours. Add glycerine. Do not use heat. Bubbles should disappear after several hours.
45 cc glacial acetic acid 55 cc distilled water

Add 0.5 gm of carmine, boil gently for 5 mins in a reflux condenser. Shake well and filter when cool. A drop or two of 45% acetic acid saturated with iron acetate can be added.

The Wagners at Michigan use Newcomers solution as the fixative of choice for fern material before HAC preparations. Newcomers was originally developed for bird chromosome research. An initial study using maize has been made to compare this fixative with the traditional Farmer's fluid (absolute alcohol 3 : acetic acid 1) and 2.5% glutaraldehyde in pH 6.8 buffer. The latter is used in electron microscopy for plant material and is recommended as a non-coagulative fixative. Newcomer's without dioxane was also tested but it was not satisfactory because the sporocytes burst too easily and the chromosomes did not stain significantly. Material placed in Newcomer's is allowed to stand several hours at room temperature, before being stored in a freezer. Fern material fixed in Newcomer's has been stored for years without deterioration. Summary of initial results:


With material fixed in Newcomer's it was easier to get more mother cells with good chromosome spreads than with material from any other fixative, otherwise the results with Newcomer's and Farmers were nearly equivalent.

When glutaraldehyde was used as a fixative the cytoplasm fixed well, but the nucleolus appeared to shrink in size, leaving a gap that appears as a distracting halo around the structure. Glutaraldehyde penetrates tissues very slowly and this may result in osmotic shrinking of the nucleolus before fixation.

James Leuck and Robert Dummer

Please Note: Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with consent of the authors.

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