Histological dyes 1 - The world under the microscope

AD = aqua dest. = distilled water

ml = millilitre = 1/1000 litres = 1cm³

dr = drop

g = gram

Most dyes are offered in an aqueous or alcoholic solution to the section to be stained. Aqueous dyes are manufactured with neutral reacting (PH7) distilled water. To prevent possible fungal growth, 1-3ml Toluol can be added to 100ml dye solution. In this ratio it does not harm the dye solution. Other possibilities are: adding Formol or some Thymol crystals. Thymol harms the Thiazine dyes such as Toluidine O, Thiodine and Methylene blue a little. It is also possible to add some finely ground silver powder without adverse effects (oligodynamic effect of silver (van Walsem, 1932)). Solutions of Thiazine dyes are generally prepared: make a saturated solution in distilled water, allow to ripen for 1-2 days, mix with the same amount of 90% ethanol, shake well. This stock solution is very colorful and has a long shelf life. Just before staining dilute with AD (1:20 to 1:50).

Alcoholic dye solutions are made with pure Ethanol of 90-96%.

Old dye recipes often indicate inaccurate amounts such as: a large knife tip, a small knife tip, droplets instead of milliliters et cetera. It should be kept in mind that it usually does not improve to know the exact quantities when the quality (impurities) of the dyes used is not known. For example, 1gram of crystal violet in 100ml AD from the company Chroma can give a different staining than when the substance is involved from e.g. Merck.

Sometimes a recipe prescribes the use of Anilin water. This greatly improves the stainability. The effect of Aniline is based on the fact that Aniline dissolves the dye better.

- Add 5-10ml Aniline (Aniline oil or Aminobenzol) to 100ml AD and shake vigorously;

- Filter the solution through a moistened filter paper. (Aniline vapours are toxic: fume cupboard!)

For most recipes it is important to keep the order exactly. For some dyes such as haematoxylin, it is necessary to allow the prepared solution to ripen before it can be used. In an artificial way this ripening can be accelerated by adding an oxidant such as Potassium dichromate (K2Cr2O7), Potassium permanganate (KMnO4) of Sodium iodate (NaJO3).

By far the most widely used dyes and dye combinations are offered ready to use by the chemical industry. It would be naive to think that own production is better. Most manufacturers deliver a high quality and maintain a standard that is almost impossible to achieve in a home laboratory.

Dyes have a reasonable shelf life in a well-sealed glass packaging. Some can be kept for years, others for months and still others need to be freshly made again and again. It is advisable to keep all dyes in a dark bottle, in a dark cool ventilated area. The size of the bottle should match the amount of dye solution. Make sure there is as little air above the liquid as possible. The oxygen present may react with substances in the dye. Plastic bottles can also be used only if strongly alkaline dye is used, this will not harm the plastic. Never pour used or too much poured dye back into the storage bottle. This to prevent contamination.

When dyes and chemicals are made themselves:

- Prepare all necessary materials;

- work as cleanly as possible, take a new test tube if in doubt;

- do not deviate from the recipe;

- never add water to acid; always add acid to water. Do this slowly and carefully[3].

- Work safely, lab coat, safety goggles and possibly fume cupboard.

In case of your own production, always label the bottle with: type of substance and production date[1].

Some general rules for handling and cleaning glassware[3].

- Rinse with AD before use and allow to dry;

- Rinse all glassware with water immediately after use;

- clean with detergent and hot water;

- do not use abrasives;

- after using sulphuric acid dichromate for cleaning, rinse very well (chromic acid is toxic to living organisms);

- a good cleaning agent is also concentrated nitric acid (HNO3) in AD 1:9;

- clean bottles with dye with a little dilute hydrochloric acid (HCL); use sodium hydroxide (NaOH) if necessary;

- stop rubber first boiling in dilute NaOH, then rinse with water and then boil in dilute HCL. Rinse thoroughly with water.

Below is a list of the most common dyes used in histology[2].