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IMPORTANT:  Certain stain formulations and reagents can be hazardous.  Do not spill stains on clothing, skin, or important objects or papers.  Treat all stain formulations as non-edible.  Information on this site is provided "as-is";  use at your own risk.

Some Common Biological Stains

I.  Introduction

  Professionals have an apparently endless array of microscopy reagents available, including so many biological stains and dyes that it's difficult to know where to start.  This article will focus on some of the common ones, including a few materials readily available to the school teacher or amateur microscopist.
 There are a number of common substances which can be used for stain, such as methylene blue, bromothymol blue, Congo red, iodine tincture, mercurochrome tincture, and others.  India ink, as commonly used by artists, is even suitable for some applications.  Many common food dyes are entirely adequate for use in staining samples.
 Dyes have differing degrees of toxicity, with some of them (food dyes) being essentially non-toxic (but please read this;  see also the entry for  Erythrosine).  Either way, handling dyes in a lab situation does not normally include eating them.  Three simple rules can avoid a problem altogether:
• Do not ingest microscopy reagents.    
• Do not get them in your eyes.  
• Do not inhale them.  
Common sense is in order:  wash your hands, wear safety goggles, and use other safety equipment (e.g., lab apron, gloves) as appropriate.  Also, stains do exactly what their name suggests:  they stain objects, sometimes irreversibly.  Do not get them on important papers, clothing, and so forth.
 Stains have different uses depending on their affinity for certain organelles or cellular components.  That often depends on the cell components' pKa, reactivity, surrounding pH, and other properties.  Iodine, for example, will react with and highlight areas containing starch or glycogen in a cell;  methylene blue tends to concentrate around areas of a cell having acidic pH.  
Within some narrow band of pH or in the presence of the right compound at the right concentration, a dye that seems to stain everything can suddenly be very specific.  Being able to dye different cell components properly may seem unimportant to a beginner, but it took generations of scientific research to figure out much of this.  Much of what we've been able to learn about cells is actually the result of this science.

II.  The Dyes

Let's talk about some common dyes and their properties for microscopy.

Brilliant Blue FCF (C.I. Acid Blue 9; FD&C Blue #1; Alphazurine), is used as blue food-coloring in the USA.  It is a large molecule (mol. wt. nearly 800).  It is well-suited for staining proteins2 and for bacteria.  This dye is the same one used in a common biochemistry protocol for measuring protein content.  The preparation has a few percent total concentration of acetic acid in a 50/50 water/alcohol solution.  Note that the lowering of pH (in this case, by the acetic acid) is important to make this stain bind to proteins.  pH-dependency is not uncommon for microscopy stains.  Brilliant Blue is not all that specific in its protein binding unless the conditions are adjusted carefully;  the typical cell contains quite a bit of material that will end up blue. 
To prepare just enough of this stain for a couple of temporary slides, you can add 2 drops of vinegar, 1 drop of 95% ethanol, and 1 drop of FD&C Blue #1 food coloring to a clean spot plate well.  Mix it with a clean glass rod or dropper.  The end result will be roughly 2.5% in acetic acid and 25% in alcohol.


Figure 1.  Why you should floss regularly.  Shown here, bacilli (rod-shaped bacteria) from food remnants that accumulated on human teeth. Magnification is 400x, demonstrating that certain bacteria can be viewed without an oil-immersion lens.  
Stain used
:  Brilliant Blue FCF in 5% acetic acid.     Fixing method:  None.

Photograph taken with a Mini-VID USB camera on an Observer III microscope at 400x.  This is an old picture;  the newer Mini-VID has higher resolution.

Carmine is obtained from the cochineal insect. The most important component of carmine is carminic acid, which is also useful in industry and analytical chemistry;  it can be used for determining the presence of certain metal ions, such as aluminum.
A dry powder is often prepared in the form of carmine aluminum calcium lake ("carmine alum lake").  A biological stain known as borax carmine is prepared by dissolving the carmine lake powder in water with sodium borate (borax), boiling this solution for at least 15 minutes to deepen the color, and mixing the solution 1:1 with alcohol.  If one started with 1 g of the carmine powder, the other ingredients would be 2 g of borax, 50 mL of water, and finally 50 mL of neutral grain spirits.


Crystal Violet  (Gentian Violet;  hexamethyl-p-rosaniline chloride) is useful for a number of stain recipes, including Gram stain and Sternheimer-Malbin stain.  The latter is good for staining polymorphonuclear leukocytes;  it can allow one to distinguish between aged and fresh leukocytes.  The fresh ones absorb only a very light amount of the stain (Sternheimer and Malbin, 1951).  Furthermore, red blood cells do not take up the stain much, if at all (1951).
Crystal violet is sometimes used alone, as a ca. 0.5% solution in distilled water.  "Named" formulations (Gram, for example) may use different concentrations of crystal violet, in conjunction with other additives.   For example, a Gram stain recipe in Locquin and Langeron (1983) calls for a water solution having about 5% crystal violet (w/v), roughly 10% ethanol (v/v), and 2% of aniline (v/v).

Eosin Y (also called C.I. Acid Red 87) is a fluorescein dye having four bromine atoms on the main fluorone structure.  Eosin Y is a very useful stain in microscopy and may be used by itself (in aqueous or alcoholic solution) or as part of one of the various staining protocols such as "H&E" (or "E&H";  eosin and hematoxylin), the Papanicolaou method, or Giemsa and its many variants.  Giemsa stain uses methylene blue, chemically transformed by heat and high pH, to form other dyes which are then combined with the eosin Y.  The resulting "neutral" precipitate is redissolved in alcohol and used as the actual stain.
If a microscopy kit were to have only a couple of stains, eosin Y should be one of them.  However, erythrosine (see below) can often be used in its place.

binucleated epithelial cell stained only with eosin
Figure 2.  A binucleated "umbrella cell" having polygonal character, even in its isolated state.  Notice the purplish-stained nuclear material (chromatin) and the prominent nucleoli.

Stain used:  Eosin Y (aqueous)
Magnification:  400x

Photo taken with Mini-VID USB on an Observer III microscope.


Erythrosine B or FD&C Red #3, is used to color "Maraschino cherries" and other edible products.  
Common red food coloring ("baking needs" aisle at your local store) is usually a mixture of Erythrosine and Allura Red AC (aka FD&C Red 40, an azo dye).   The writer did some experiments with this mixture and found something interesting).
Whereas eosin Y has four bromines on the fluorone structure, erythrosine has four iodines (see, for example, the Merck Index or the respective Wikipedia entries for these dyes).  In other words, erythrosine is very similar to eosin Y.  
Erythrosine stains are often prepared as a 1% w/v solution in water or alcohol.

Fast Green FCF is, in fact, FD&C Green #3 and is (or was) a common food coloring. This writer hasn't yet been able to find any food coloring kits that contain it, perhaps because "green" can be made by mixing blue and yellow dyes. Fast Green FCF stains are about 0.5% w/v of the dye in water or alcohol.  
Fast Green is used primarily as a counterstain.


Hematoxylin (Logwood) comes from a tree that grows in Central America (especially Belize).  It is extremely useful for biological stains.  This is a must-have for the laboratory, along with eosin Y, crystal violet, and methylene blue.  
Hematoxylin should in theory be pronounced "he-mat-o-ZY-lin", not "he-ma-TOX-uh-lin".  That is because the root words are the ancient Greek hematos, for "blood" and xylon, for "wood".
To be technically correct, logwood is the hematoxylum from which the dye hematoxylin is extracted;  the famous Chemcraft sets contained shavings of this wood as a ready source of the dye.
Some hematoxylin recipes use hazardous reagents such as mercuric salts or chloral hydrate, but these are not part of the core preparation:  hematoxylin, alum3, distilled water, alcohol, and sometimes glycerin. 

Ehrlich's Hematoxylin recipe:
Dissolve 0.2 gram (3.1 grains) of hematoxylin in 10 mL of 95-96% ethanol (neutral grain spirits U.S.P.) or isopropanol.  (To make this preparation according to the traditional method, do not use methanol or denatured alcohol.) 
Add 1 mL of glacial acetic acid.
Dissolve 5 grams of potassium alum in 10 mL of glycerin and 10 mL of water.  Add this solution to the alcoholic hematoxylin and mix thoroughly.
Allow the preparation to stand for a couple of weeks in a loosely-stoppered bottle kept in the sun.  The color will turn deep red.

Instead of the 10 mL of water and 1 mL of glacial acetic acid, 10 mL of "100-grain vinegar" (10% acetic acid) can be used.

A few specks of potassium permanganate or iodate are often added to accelerate the "ripening" of hematoxylin preparations;  the oxidizer converts the hematoxylin molecule into hematein upon standing.  Too much oxidation will of course ruin the dye (as in bleaching, which destroys the molecule). 
Hematoxylin preparations, like many prepared stains, have a limited lifespan.  They tend to develop tiny crystals or sediments, which of course are highly visible on the microscope slide.  Thus, they must be filtered or centrifuged periodically.


Indigo Carmine (Indigotine) is also known as FD&C Blue #2, another common food coloring.  Like many dyes, it is derived from coal tar.  Nevertheless, indigo carmine is a food dye;  this may explain its use in microscope kits for beginners.  (A passable stain assortment could be put together, at least in part, from food colors if minimal toxicity were a requirement.)
Like carmine, indigotine has a great variety of uses in the analytical / chemical industries.


Iodine:  the tincture (i.e., alcohol solution) can be used directly on specimens, or it can be diluted first.  The dark color of the starch-iodine complex is easy to visualize.  It will remain after the excess iodine is washed from specimen.  These darkly-colored regions correspond to glycogen or starch in a cell.  Iodine is therefore used for one of the simplest (and probably longest-known) histochemical methods.
Certain micro-objects will, when left in iodine tincture, draw up so much iodine that they look completely black.   This seems to happen especially with natural fibers.  These do not necessarily contain starch.


Malachite Green is one of many triarylmethane dyes.  By itself, this dye is not all that good for general microscopy, being perhaps in the same category as tartrazine.  Like tartrazine, malachite green is primarily a counterstain;  this means it gives general coloration to areas that have either failed to take up some other, more specific stain or which have been subjected to de-staining.  However, malachite green does have one or two highly specialized uses for which it is good, even preferable, as a primary stain.  It has sometimes been used for staining erythrocytes (Conn, 1929).
As is common with stain preparations, malachite green solutions develop tiny crystals or sediments on standing.  The solution must be filtered or centrifuged occasionally.


Methylene Blue
is often used for treating aquariums to prevent fungal infections in fish. It is also a widely used biological stain and indicator. Methylene blue preparations are typically 0.5 to 1.0% w/v solutions of methylene blue in an alcohol / water solution, made very slightly alkaline by addition of a few drops of dilute NaOH or KOH.  Loeffler's methylene blue is 0.5 g methylene blue powder dissolved in a solution of 100 mL distilled water, 30 mL ethanol4, and 1 mL dilute (1%) KOH (See http://www.hoslink.com/histo/histo_recipes2.htm).  
See the entry for "eosin Y" for Giemsa stain.
Methylene blue is good for staining bacteria and cell nuclei. 
While methylene blue can be toxic under some special conditions (medication interactions), it is probably not otherwise hazardous, and certainly not to the lab worker.  It's important to understand that toxicity is largely a question of context.  Working with any of these dyes as microscopy reagents is not the same as eating them. 
If a microscopy kit were to contain only three or four stains, methylene blue should be one of them.


Printer Inks (as used in modern inkjet printers) may be a rewarding area of investigation.  The idea of inkjet pigments as microscopy stains occurred to this writer while browsing a 1990's text on dye chemistry.  
At first glance, it appears some inkjet dyes are structurally close enough to well-known stain pigments to have promise.  More information will appear on this website after some messy experiments involving broken-open ink cartridges and economy refill kits.


Safranin O (Cotton Red) is the major dye component of Sternheimer-Malbin stain, not to mention other formulations.  Safranin is also a counterstain for Gram.  Structurally, the safranin dyes are reminiscent of the fluorescein dyes.  
Safranin is not in any consumer products of which this writer knows, but it is among the half-dozen or so dyes that should be on every microscopist's shelf.
Occasionally the term "safranine" is used to describe safranin O (which itself is a mixture of two different safranins- confused yet?).  There was a time when "safranine" was supposed to mean a specific one of those two safranins:  the pure N-(2-methylphenyl) congener, not the mixture of the N-phenyl and N-(2-methylphenyl) congeners.  Conn (1929) points out that "tri-methyl phenosafranin" (i.e., the N-(2-methylphenyl) congener) is deeper red than the N-phenyl one.  Though a mostly academic point, it illustrates one reason why different lots of dye can vary in color and intensity even when free of adulterants.


Tartrazine, an azo dye, is also known as FD&C Yellow #5 and is a common food coloring.  Stains are prepared as a 1% to 2% w/v solution in water or sometimes hydrophilic organic solvents. Tartrazine is a counterstain;  it isn't that good as a primary stain for cell components.  Like other counterstains, though, it can add visual contrast, such as by providing a background for structures that have taken some other color.



References:

Baldwin, W. M. "Mercurochrome-220 soluble as a histologic stain".  Anat. Rec. 39:229, 1928.

Conn, H.J.  Biological Stains.  Geneva, NY:  Commission on Standardization of Biological Stains, 1929.

Locquin, M. and Langeron, M.  Handbook of Microscopy.  London:  Butterworths, 1983.

Merck & Co., Inc.  The Merck Index of Chemicals and Drugs, Sixth Edition.  Rahway, NJ:  Merck & Co., Inc., 1952.

Sternheimer, R. and Malbin, B.  "Clinical Recognition of Pyelonephritis, with a New Stain for Urinary Sediments".  Am. J. Med. 11:312-323, 1951 (September).








Notes:


1 Histochemistry focuses on reagent and dye interactions with specific enzymes, molecular side chains, structural proteins, lipids, etc.   The study of stain interactions with the gross structures of a cell (e.g., nuclei, vacuoles, nucleoli) falls somewhere under the umbrella of  cytochemisty, histology, and / or cytology.  In practice  the disciplines have some overlap.  There is "histocytochemistry", "histocytology", "cytohistochemistry", and so forth.   This writer does not pretend to understand the distinctions once they reach this level.
Back to Article

2 The negatively-charged sulfonate groups of this dye molecule will be attracted to positively-charged groups on proteins, especially when the pH is just acidic enough to protonate NH2 groups to form NH3+.  Certain amino acids (such as lysine) have these NH2 groups on side chains, which are still available even when the amino acid itself is involved in a peptide bond.  Back to Article

3 The alum can be the NH4, K, or Na variety. 
Aluminum sulfate will also work.  Al+++ is the mordant, which forms a complex and helps the dye adhere.  Certain other  metal ions (Fe+++, Cr+++, etc) are used as the mordant for alternate preparations.  Many stain formulae call for iron alum or chrome alum.  Aluminum is toxic by injection or ingestion, but it doesn't present much of a hazard, if any, to the lab worker.  Back to Article

4 Recipes for this stain call for "absolute ethanol", but in this case it doesn't really have to be anhydrous-- the alcohol is being dissolved in a large amount of water anyway.  95% ethanol will serve well.  The presence of methanol (as in denatured alcohol) should also make little difference for this stain.  Back to Article.




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