Microscope Activities, 21: Simple Staining of Microscopic Specimens

In the past, Hooke College of Applied Sciences offered a microscopy workshop for middle school and high school science teachers. We thought that these basic microscope techniques would be of interest not only for science teachers, but also for homeschoolers and amateur microscopists. The activities were originally designed for a Boreal/Motic monocular microscope, but the Discussion and Task sections are transferable to most microscopes. You may complete these 36 activities in consecutive order as presented in the original classroom workshop, or skip around to those you find interesting or helpful. We hope you will find these online microscope activities valuable.

EXPERIMENT 21: Simple Staining of Microscopic Specimens

Goal

To learn how to stain live materials to increase their visibility, and to accomplish this with inexpensive and commonly available colored materials.

Level

Basic

Materials Needed

Food colors, egg dyes, fountain pen inks, textile dyes, methylene blue, variety of color pencils, skin markers, etc.

Procedure/ Discussion

Since so many microscopic objects that we wish to observe are colorless and transparent, the desire to stain them with colors to increase their visibility goes back to the beginnings of the science. Robert Hooke (after whom the Hooke College of Applied Sciences is named), in his book, Micrographia, published in 1665, describes the use of numerous stains, including Logwood, which is still used today. Over the years, staining has evolved from merely coloring specimens to increase their visibility, to differentiation. What is meant by this is that the chemical makeup of the various structures within a specimen are considered, and specific dyes are developed to stain only those structures with that specific chemical makeup, thus differentiating them from all other structures in the specimen. Combinations of dyes may be used, either together or in sequence, to stain different structures different colors; triple stains are commonly used. In recent years, ultraviolet fluorescent materials have been developed to a very high degree. The science of histochemistry has produced many hundreds of highly specialized differential staining techniques.

Many, if not most, of the stains in common use produce variable results; from lot to lot the staining results are variable, and may, in some cases, not work, or work poorly. As a result, a Stain Commission was formed, wherein members of the Commission actually test every lot of dye submitted, and issue a certificate for those dye lots that produce the expected results. Such Certified Stains are absolutely reliable, but expensive, because there is a surcharge added to cover the cost of testing.

Most stains today are used on tissues which have been fixed, embedded, and sectioned; the stains may be alcohol soluble, or soluble in solvents other than water. None of these can be used on live materials, because solvents other than water will deform or kill live specimens. There are some water soluble stains which can be used to color live specimens without killing them, and these are termed “vital” stains.

The current litigious social climate regarding all chemicals now makes it not only expensive, but difficult or impossible to purchase any chemicals; even schools have imposed restrictions; for the amateur microscopist the situation is dire. What can be done about this? For one thing, it is still possible to obtain useful stains and chemicals from online sources, but the most interesting challenge is to be resourceful enough to test commonly available colored materials intended for entirely different applications.

Figure 21-1 illustrates a number of different colored materials that are water soluble and effective in coloring live microscopic specimens without distorting or killing them.

Some of the colored materials include food coloring and egg dyes; these may be in powder form (far left in the figure) or liquid form (center in the figure). These all incorporate dyes which are approved by the Food and Drug Administration for human consumption. Different brands of food coloring may use different dyes, so each needs to be tried. But note: not all organisms will be stained by a particular dye; what works on one species may not work as well, or at all, on another specimen.

Textile dyes are another wonderful source of stains which are available at all supermarkets. The clothes dyes, or textile dyes, are available in both powder and liquid form (a liquid form of blue dye is illustrated at left rear in the figure); there is even a de-coloring agent available in the same section.

Fountain pen inks are another commonly available, inexpensive source of coloring, but be careful, some of the blacks are particulate, not mixtures of three dyes; that is, they consist of colloidal or very finely divided carbon black particles; in general, go for washable black inks; blue inks are particularly effective coloring agents.

On the right side of the figure are what appear to be three pencils, together with a slide showing three drops of water that each pencil has colored; these represent another possible source of stains. The pencil on the left, with the red top, is an Eagle 858 Perfection Copying pencil, made by Eagle Pencil Company, New York. The lead in this pencil incorporates a very intense reddish-purple dye. All one has to do is briefly touch the drop of water containing the live material with the pencil tip, and it is instantly colored; it may take a few minutes for the organism to take up the stain—generally the longer they are exposed, the deeper they are stained.

The middle pencil in the figure is an Eberhard Faber NOBLOT INK PENCIL 705, made during the late 1960s to the early 1970s; you can see from the water drop in front of this pencil that it provides a source of blue dye; again, all one has to do is touch the drop of water holding the live specimen with the point of the pencil, and the color is instantly added.

On the far right in the figure is what looks like a pencil, but is not; it is a skin marker used by surgeons to mark the line of incision on the skin prior to making the cut. This particular product is labeled: DEVON SKINMARKER with Ruler™; it is manufactured in Japan for DEVON IND., INC., and is #150. This is a highly effective source of microscopical stain, as it appears to be gentian violet based, or a member of the crystal violet family.

A general use stain is methylene blue, water soluble. Again, because methylene blue is a chemical, it may be difficult to purchase. However, methylene blue is readily available in either solid or liquid form in almost any aquarium shop, because it is used to treat fungal infections in fish. Methylene blue is also available in almost any quantity from online sources.

Task

Assemble sources of dyes from around the school and home, similar to those suggested here, and try them out on live organisms in a drop of water taken from the aquarium. Try some marking pens and transparentwater color paints. Go to the art section in a crafts and hobby store, and select some colored inks to try; there are scores of choices. Try stamp pad ink. Make small vials of the most effective stains to add to your laboratory bench or field kit.