Create semi-permanent microscope slides of gametophytes using a mixture of a stain in a combination clearing – fixative – preservative agent.
Semi-permanent slides suitable for later observations or archival documentation can be prepared by mounting gametophytes in a Hoyer’s/Acetocarmine mixture. Hoyer’s Medium clears and preserves tissue; acetocarmine is one of the few stains stable in the presence of Hoyer’s Medium.
Materials:
- Hoyer’s Medium
- 0.5% Acetocarmine stain
- Plastic dropper bottle
- Slides
- Coverslips
- A source of rust, such as an iron dissecting needle
Prepare mounting mixture fresh by mixing 4 parts Hoyer’s Medium and 1 part Acetocarmine Stain. (1 mL of this mixture should be sufficient for 7-8 slides, using 22x22mm coverslips. ) For the acetocarmine to stain properly a small amount of iron oxide should be added to the Hoyer’s/Acetocarmine mixture. Do this by dipping an old rusty dissecting tool (e.g. forceps) or rusty iron nail into the mixture until a sufficient amount of iron oxide has leached off. Some experimentation is necessary to obtain the correct amount of iron — too little gives very weak staining; too much yields excessive precipitation and an excessively colored background.
Place 4-10 drops of the Hoyer’s/Acetocarmine mixture onto a slide. (For best results be sure slides are very clean.) Transfer gametophytes from the culture dish to the mixture. Submerge gametophytes under the mixture. Put coverslip in place. If gametophytes are large or convoluted it may be difficult to avoid trapping some air bubbles. Place completed slides on a tray to dry undisturbed for several days. Full clearing and staining of gametophytes may take more than several days. Retain the remaining mixture to add to those slides that dry out excessively. Slides prepared with a sufficient amount of Hoyer’s/Acetocarmine can keep for well over 10 years.
Hoyer’s Medium
| Distilled H₂0 | 50 mL |
| Arabic gum (acacia) | 30 g |
| Chloral hydrate | 200 g |
| Glycerin | 16 mL |
Dissolve Arabic gum completely in the distilled H₂O. Then, and only then, completely dissolve in Choral hydrate, then add glycerin and mix well. The medium may be diluted when needed with small amounts of distilled H₂O. Before using Hoyer’s medium, let it stand undisturbed for several days in order to clarify. Store mixture at 4 °C.
CAUTION: Choral hydrate is a controlled substance and used as a hypnotic and sedative. Abuse may lead to habituation or addiction. Some individuals are extremely sensitive to the vapor’s of Hoyer’s medium and develop headaches or other maladies.
Acetocarmine
| 45% Glacial acetic acid | 100 mL |
| Carmine | 0.5 g |
Boil acetic acid and carmine gently for 5 minutes in a beaker covered with a watch glass. Do this in a fume hood! Shake while the mixture cools. Filter cooled solution through #2 Whatman paper. Filtration takes a long time and several changes of paper are usually necessary. Store stain in brown glass at room temperature.
