Workshop on stains and staining
Saturday 8th February 2025
Gordon Brown opened the workshop with a presentation explaining the different types of stains, how they work, how they can be prepared, and how to use them. He explained his modification of one of Robin Wacker’s 3-component stains that works in a few minutes and uses chemicals that are still available (Rhodamine B, Acriflavine and Astra Blue).
You can see the slides from Gordon’s PowerPoint presentation here:
Click the arrows to move through the slides. Click the symbol at bottom right for a larger version.
Gordon Brown’s presentation
Gordon provided a good selection of botanical specimens, slides, coverslips, brushes, tweezers and watch glasses. For blotting slides to remove excess stains and other liquids, he provided small pieces of kitchen paper towels.
Chemicals, specimens and equipment
Gordon Brown’s demonstration
Gordon brought several slides of botanical specimens that he had prepared using the techniques that he demonstrated to us.
Slides of plant sections
Gordon provided plant sections preserved in 70% isopropyl alcohol that we could stain and mount. The specimens were Scots pine leaves TS, holly leaf and stem, mistletoe stem, ash branch LS & TS, and ivy petiole and leaf. He had cut the sections in advance using his Reichert sledge microtome. For plant material, he usually cuts sections between 20 and 45 µm. To support specimens in the microtome, he uses blocks of cavity-wall insulation boards; you may be able to find offcuts in a skip!
Plant sections in 70% isopropyl alcohol
Gordon used a small paintbrush to transfer a section from one of the bottles onto a slide, and use a piece of paper towel to remove excess alcohol.
Gordon provided stains and reagents in small dropper bottles. They included his modification of a 3-component Wacker stain, isopropyl alcohol and Histo-Clear. The major components of Histo-Clear is d-limonene, which can be purchased cheaply as food grade and can be used as an alternative to Histo-Clear. He also provided a bottle of LOCA (used as a mountant) and larger wash bottles of deionised water. LOCA is not miscible with water or alcohols, so these have to be removed from the specimen.
Stains and reagents
Gordon added a drop of his Wacker stain from a dropper bottle to the section, and left it for a few minutes. Depending on the specimen, he leaves the stain for between 2 and 10 minutes.
Then he rinsed the specimen, first with deionised water and then with isopropyl alcohol, blotting at each stage. The final liquid is the Histo-Clear clearing agent. This also needs to be blotted, but leaving a film of liquid so that there are no cavities that would become air bubbles in the mountant.
Gordon’s preferred mountant is LOCA, which sets when exposed to UV radiation. He used a glass rod to transfer a generous amount from a bottle to the specimen, and then checked for air bubbles. If there are any, they can be removed with a needle.
For handling coverslips without breaking them, Gordon recommends plastic tweezers. He carefully lowered a coverslip onto the specimen, and pressed it down to squeeze out excess mountant.
Plastic tweezers for coverslips
The LOCA mountant that Gordon uses sets when exposed to ultraviolet radiation. It is possible to use sunlight or a UV torch, but Gordon uses lamps that are intended for setting nail varnish; they set the LOCA in 60 seconds.
Ultraviolet lamp
Once the LOCA has set, the excess around the coverslip can be removed by cutting and peeling, or by scrubbing in very hot water.
The slide is then ready to be labelled. Using the procedure that he demonstrated, Gordon can cut a section, stain it and mount it in 20 minutes.
Terry Hope checking his slide
Mistletoe stem (4× objective, slide and photo by John Gregory)
Report and most photographs by Alan Wood