STAINS AND STAINING A PRIMER FOR AMATEUR MICROSCOPISTS By Glenn Shipley Preface: Biological stains are dye chemicals for enhancing the visibility of structures of plant or animal specimens seen under the microscope in thin sections or thin films or smears. To do that a stain needs to have vivid color and a selective affinity for some structures and not for others. Without stains some structures are difficult or impossible to see with a brightfield microscope, because light just passes right through them. A. Kinds of stains. (1) Acid and Basic stains. Early investigators found that basic stains (high pH) would bind to cell nuclei (like DNA), while acid stains (low pH) would bind to cytoplasmic structures. Two standard examples are hematoxylin and eosin -"H & E" - stains still used in all medical histology labs. Hematoxylin stains nuclei bluish, and eosin stains the cytoplasm reddish. Hematoxylin is extracted from logwood tree bark, and eosin is a synthetic fluorescein-source dye. As coal and oil chemistry developed, synthetic aniline dyes began to compete with extracted natural ones. Methylene blue is an example of a basic aniline dye. (2) Gram stain. Since bacteria do not have enclosed nuclei, they require a different approach. This is where the Gram stain comes in, which uses crystal (Gentian) violet as a primary stain, and safranin as a counter stain, with iodine as a mordant. Gram positive bacteria retain the crystal violet stain after flushing with alcohol and acetone, whereas gram negative bacteria lose it in that wash. Crystal violet stains the gram negative bacteria so that they are visible - and contrasting with the dark-blue gram positive cells. (3) Polychrome and special stains. There are combination stains like the "trichrome" or three-color stains - an example being Wright's stain which is used to stain blood white cells. There are also a whole bunch of specialized stains for fats, bone, nerve cells, etc some of them using heavier metals like silver, mercury, and cobalt. (4) Vital stains. The above stains are used on dead tissues and cells, but there is another whole class of dyes called "Vital stains" which are taken up by cells (particularly protozoons) without killing them, but making them more visible. Congo red is an example of a vital stain. (5) Fluorescent stains. In recent years huge gains have been made in rapid detection of very specific entities by using antibodies tagged with fluorescent dyes. The antibody binds to some very specific structure (called an antigen). A special fluorescent microscope with a light source in the UV range is needed to stimulate the fluorescent dyes to give off photons and become visible. B. Staining Technique / Procedures. Most staining technique involves either flooding the slide specimen with the stain, or dipping the slide in a dish with stain in it. Either way the staining step is timed - usually a matter of a few minutes - after which it is flushed, usually with water, but sometimes with alcohol or acetone. A counter-stain is applied, using the same procedure, and the slide is again rinsed. Some stains require buffering, heating, etc - but these are usually only for special procedures. C. Stain Sources & Difficulties. For most specimens a collection of about a half-dozen stains will handle 95% of what hobbyists and amateur microscopists want to look at. Here is a list of these common stains: Hematoxylin – basic (acidophilic) stain Eosin Y (or B) – cytoplasmic counterstain for Hematoxylin Methylene blue – general nuclear stain Crystal (Gentian) violet – primary Gram stain Safranin – counterstain for Gram stain Wright stain – polychrome used for blood smears The problem is that biological and scientific suppliers will generally sell chemical products only to institutions like educational and research institutions. So our challenge is to find equivalent stains that are not restricted - like food and clothing dyes, DIY plant extracts, etc. Here are some common stains and stain-related materials available from grocery stores, pharmacies, and hardware stores: Kool-Aid – grape and dark cherry – use as concentrate (package in 50 mL or less) Food coloring dyes – red, blue, green, yellow – use concentrated D. Alternatives to Staining. In recent years there has been much excitement and progress made by using various kinds of illumination to visualize intracellular and other structures by means of light alone, without the need for killing and fixing and staining cells before hand. These include phase contrast, DIC, Nomarsky, etc. And of course there is transmission and scanning electron microscopy (SEM and TEM). But it is unlikely that any or all of these will replace the need and usefulness of staining techniques in microscopy. E. Some Internet Resources. http://en.wikipedia.org/wiki/Staining#Common_biological_stains http://www.scuddlebutt3.co.uk/MicroscopyStains.htm - UK website with alphabetical listing with pricing.
