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CAUTION:  Chromium trioxide is toxic and is a powerful oxidizer.  If you choose to attempt any of the procedures or experiments mentioned on this web site, you do so entirely at your own risk.  In order to use this web site you must read and agree to the Terms of Use.

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Selective Staining of Infection-Mobilized Leukocytes by Common Food Dyes Using Chromium Trioxide as a Fixative / Mordant

by C. Thorsten
June 2007


Abstract:  0.2% chromium trioxide in 5% acetic acid was tried as a fixative / mordant for a specimen that was subsequently stained with the "5-minute food color stain" (i.e., Brilliant Blue FCF + Allura Red AC + Erythrosine).  The specimen consisted of exudate from a human parotid gland having a mild bacterial infection.  Color differentiation was poor in epithelial cells and ordinary leukocytes but excellent in infection-response leukocytes.  In the latter case, cell nuclei stained deep blue, while cytoplasm stained blood-red.  Specificity toward infection-related leukocytes was thought to be due to sialic acid (N-acetylneuraminic acid and derivatives) on cell-surface glycoproteins.


Introduction:

Differential staining is a well-established property in microscopy and histochemistry.   It is also well-known that the choice of fixative can affect the outcome of staining.  Depending on the fixative, the pH, and other factors, dye molecules can be caused to adhere more or less tightly to various cell structures and even to express marked color variations.  This latter phenomenon is called metachromasy and has been discussed in the literature for several decades (q.v. Kelly, 1958).   Indeed, it has been known since at least the 1870's that certain tissues and cell components stained a color different from that of the actual stain preparation used (Heschl, 1875;  Cornil 1875;  Ehrlich 1877;  etc.).

The use of multiple dyes after treatment of substrate with a transition-metal fixative (e.g., chromium, manganese, iron, copper, uranium, etc) would seem to present the most abundant opportunities for expression of differential staining.  Chromium especially would seem to be a good candidate, at least instinctively, since chromium compounds themselves can range from blue, green, and purple (Cr3+) all the way through yellow, orange, and red (Cr6+).  Cr(VI) would seem especially useful because of its reactivity toward oxidizable organic functional groups.

Although hexavalent chromium is toxic, it does not represent as much of a hazard as osmium tetroxide or mercuric chloride.  Traditional fixative recipes have often utilized one or both of these in addition to the chromium.  The author hoped to develop a fixative / dye protocol using only Cr(VI) without either of these other two substances.  Because the sample to be stained was of a thin, diffuse nature, it seemed reasonable that a lower concentration of Cr(VI) might work.  Finally, the author thought it worthwhile to try readily-available but overlooked dyes:  namely, food colorings available on the consumer market.


Safety Notes:   

Chromium trioxide is toxic and is probably a human carcinogen.  It is recommended to wear gloves when working with it.  Wear safety goggles.  Work with as small an amount as necessary at a time.   Do not get Cr(VI)-containing solutions on bare skin, as they may cause sensitization. 

Wastes (e.g., unused stock solutions) containing hexavalent chromium should be saved for reclamation / recycling.

CrO3 is a potent oxidizer and can cause spontaneous fires in contact with certain materials.  There are two main points to remember in this experiment:

1.) Acetic acid is not readily oxidized by CrO3, since it is already in the last stage of oxidation before CO2.  That last step (carboxylic acid ---> CO2) is very hard to attain in dilute solution.  There is the theoretical possibility of peroxyacetic acid (CH3CO3H), but in very dilute solution this was not deemed an acute hazard.  The use of dilute acetic acid as a solvent for CrO3 is a well-established practice.

2.) Alcohols (especially primary alcohols such as methanol and ethanol) are readily oxidized by CrO3 and are prone to spontaneous combustion on contact with solid CrO3.  Care must be taken during the experiment that this condition never exists.   Because of this fire danger it is crucial to make sure no particles of CrO3 become spilled on the bench or dropped on the floor.   Also, solutions of CrO3 must not contact paper or combustible materials.

This experiment makes use of a secondary alcohol under dilute conditions.  The final concentration of IPA in this experiment is only about 17%, with the concentration of CrO3 being only 0.2%.   Nevertheless, this solution should be kept away from all combustible materials.

Materials:

CrO3 (chromium trioxide;  chromic anhydride;  "chromic acid")
5% Acetic acid
Blue food color (FD&C Blue 1)
Red food color (FD&C Red 40 and Red 3)
Droppers
Isopropyl alcohol (IPA), 70%
Isopropyl alcohol, 90-95%
Isopropyl alcohol, 100% (anhydrous)
Xylene
Mounting Medium
Cover slips
Slides

Methods & Observations:

Reagent Preparation:
Fixative solution containing approx. 0.2% (w/v) of CrO3 in 5% acetic acid was prepared fresh. 

A version of the "5-minute food color stain" was prepared from the following (expressed as number of drops):

Red................1
Blue...............1
Water..............5
5% HOAc............2
IPA (91%)..........2

"Red" was standard FD&C red food coloring (Allura Red AC + Erythrosin B).  "Blue" was standard FD&C blue food coloring (C.I. Acid Blue 9). 

Specimen Collection:
The white blood cells originated from a mild parotid gland infection.

A thin, mildly-pustulent discharge of serous fluid was noted periodically from both Stensen's ducts, especially the right-hand one.  One such discharge was collected in a clean 10 mL micro beaker. 

Fixing:
A drop of this specimen was placed on a clean slide and treated with the 0.2% CrO3 / 5% HOAc fixative.  Fixing time was only 15 minutes due to the diffuse nature of the specimen;  after this time the fixative was removed via two rinsings with distilled water, followed by a final soaking in distilled water. 

Staining:
The fixed and rinsed preparation was treated with 3 drops of the "5-minute food-color stain" (prepared as detailed above).  Staining time was 15 minutes.  The excess stain was then rinsed off with tap water, followed by distilled water.

Dehydration:
The specimen was treated for 10 minutes each in 70% IPA, 91% IPA, and then 100% IPA that had been dried on a bed of 3A molecular sieves.
Finally, the specimen was treated with xylene.  The excess was drained off and the slide allowed to dry in the air for about 10 minutes.

Mounting
A clean coverslip was treated with a drop of synthetic mounting medium, of the same kind used in our slidemaking kits.  The specimen itself was also treated with a drop of the medium.  The coverlip was pressed down on the specimen until no air bubbles remained.  The mount was allowed to dry and then viewed with a microscope. 

Figure 1 shows the procedure's outcome on white blood cells from the parotid gland infection.  Figure 2 shows the outcome on ordinary epithelial cells that were also present (as incidental components) in the saliva sample.  Figure 3 shows what happened to leukocytes not involved in acute infection.  

We'll call these specimens F1, F2, and F3, respectively.

Figure 1  ( Specimen F1).   Leukocytes responding to acute infection;  fixed with CrO3;  stained with the "5-Minute Food Color Stain".  The intense color differentiation is readily apparent.

Magnification ca. 400x.
Figure 2  (Specimen F2).  Epithelial (cheek) cells.  It's obvious that the protocol worked much better on F1 than it did on these epithelial cells (F2).  This kind of specificity is very desirable. 

 Magnification ca. 100x.



Figure 3  (Specimen F3):   The "control" sample for white blood cells.  These were leukocytes obtained after the infection had cleared.  Notice the marked difference between this and the F1 specimen.  This suggests the protocol targeted not leukocytes in general, but only those involved in infection-response.  

 Magnification ca. 400x.



Discussion:


While various iterations of the Five-Minute Food-Color Stain showed promise as a supravital stain, the performance with dilute CrO3 / acetic acid fixative was truly remarkable on white blood cells from an infection (i.e., specimen F1).   In this experiment, the CrO3 caused intense color differentiation on subsequent  application of "Five Minute Food Color Stain" to those cells;  hence, there was a strong "pre-mordant" effect.

Judging from the observed colors, the Acid Blue 9 concentrated in the nuclei, while one or both of the red dyes concentrated in the cytoplasm.  The dyes assumed hues that had not been observed in the preliminary experiments with this stain by itself.  

The intense color differentiation in F1 did not extend to specimens F2 or F3.  In fact, treatment of F2 and F3 with chromic acid and the "Five Minute Food Color Stain" gave virtually the same results as if there had been no CrO3 at all;  the stained color in both cases was the same purplish-pink hue as the dye bath.

It is clear that the cells in specimen F1 (infection-mobilized neutrophils, monocytes, lymphocytes, etc) had some chemical features that lent themselves to chromic acid binding, while ordinary leukocytes and epithelial cells had no special affinity or reactivity toward the reagent. 

Something about the infection-responding white blood cells, as well as the associated debris from the infection, seems to have invited stronger binding of the chromic acid and subsequent coloration by dye molecules.  This was most likely a function of the cytochemical changes associated with inflammation response, specifically glycosylation.  Neutrophils, monocytes, lymphocytes, and other responders to infection have an increased level of binding affinity toward certain molecules and chemical functional groups.  This is mediated by chains of sugar molecules attached to proteins and lipids on cell surfaces.  Sialic acid, a term that refers to N-acetylneuraminic acid and its congeners, is a crucial component of these immune-related oligo- and polysaccharides (Matthews and van Holde, 1990).   For example, an experiment by Miller-Podraza et al. (1999) has shown that neutrophil binding to Helicobacter pylori depends on sialic acid in the glycoproteins and glycolipids of the neutrophil cell surface. 

Chromium trioxide yields H2CrO4 (chromic acid) in aqueous solution.  At pH much above 0, the predominant species would be HCrO4- and/or HCr2O7- (due to dimerization).  The exact interactions of chromic acid ionic forms with various cytochemical features are still not completely understood.  There appears to be a combination of effects that depend on both oxidation and complex formation.  Chromic acid is a strong oxidant;  it can convert e.g. R-CH2-OH into R-COOH, and in the process it is reduced into e.g. Cr2O3.  The various oxidation states of Cr, including the uncommon +4 state, may play some role in the histochemistry of chromic acid (Roozemond, 1970);  these would arise through partial reduction of Cr6+ as it oxidized chemical functional groups in cells.  Lillie (1961) has demonstrated that CrO3 can oxidize the R-CH2-OH groups of cell polysaccharides to R-CHO (as opposed to going all the way to R-COOH, which is more typical in the organic chemistry lab), in the same manner as periodic acid.  These numerous aldehyde groups can then participate in other reactions such as Schiff base formation. 

Sialic acid is rich in functional groups for binding and possible chemical action by chromic acid / chromate species.  Sialic acid is most often incorporated into oligo- or polysaccharides as a terminal residue via an alpha-2-6 or 2-3 linkage.  In other words, the carbon #2 forms a glycosidic bond with the OH attached to the carbon #3 or carbon #6 of another sugar.  In this form, sialic acid presents an N-acetyl group, three secondary alcohol groups, a primary alcohol group, and a carboxylic acid group.  The primary alcohol (at carbon 9) can be oxidized to aldehyde or carboxylic acid.


Sialic acid
Figure 3.  Sialic acid. The glycosidic linkage forms at carbon #2, since it is part of a hemiacetal. This reacts with the OH group of another sugar molecule. Because the #3 and #6 carbons of sialic acid have no OH groups and thus do not lend themselves to glycosidic bond formation, sialic acid normally attaches only as a terminal residue in the saccharide chains (the OH at carbon 8 does, however, sometimes participate in glycosidic linkages). Its terminal positioning gives it a crucial role in immune system recognition and cell adhesion.

A study by Materazzi and Ferretti (1961) suggests that the carboxylic acid group of sialic acid plays some role in chromic acid binding;  the investigators found that acetylating these COOH groups blocked the metachromasy that would normally have resulted from chromic acid treatment (1961).

The surfaces of leukocytes and serum proteins have been shown to undergo increased sialylation (attachment of sialic acid molecules) during inflammation response (q.v. Chavan et al., 2005;  Delmotte et al., 2001).    This is consistent with what was observed in the present experiment:  chromic acid had the most marked effect on inflammation-related cells and debris.  Leukocytes and their targets, studded with sialic-acid-tipped polysaccharides, would seem perfect substrates for binding of (and possible oxidation by) chromic acid.

The apparent cytochemical changes in the nuclei are also noteworthy.      Whereas the normal cell nuclei took up the usual deep purple of the "Five-Minute Food Color Stain", the nuclei of inflammation-response leukocytes stained deep, indigo blue or blue-black.  The specific mechanisms for this are currently unknown to this author;  certainly they are non-obvious, and it would take functional group blockades and other detailed studies to understand them. 

The microscopic result, seen together with the blood-red cytoplasm, presents a striking color clash that the author has nicknamed the "angry cell" response.  This staining response makes it stand out from other protocols with which the author is familiar.  The reader may note, in fact, that traditional staining methods have no such specificity for acute inflammation;   consider the numerous photomicrographs of more or less purplish-stained leukocytes from all different contexts.  The apparent specificity of the presently-studied protocol could make it useful for research and diagnostic applications.  Its relative simplicity could be valuable in remote locations and modestly-furnished laboratories.   Based on the best of his current knowledge and a search of the literature, the author believes this particular dye-fixative combination (and the application thereof) to be original.

Finally, further studies will be necessary to determine the protocol's tolerance to slight changes in composition and pH, as well as its response toward other, known conditions of inflammation. 



References

Chavan M., Kawle P., and Mehta N.  "Increased Sialylation and Defucosylation of Plasma Proteins Are Early Events in the Acute Phase Response". Glycobiology 15(9):838-848 (2005).

Cornil, V.  "Sur la dissociation du violet de méthylaniline et sa séparation en deux couleurs sous l'enfluence de certains tissu normaux et pathologiques, en particulier par les tissus en dégénérescence amyloide".  Compt. rend., Acad. Sc. 8: 1288 (1875)

Delmotte P., Degroote S., Lafitte J., Lamblin G., Perini J., Roussel P.  "Tumor Necrosis Factor Alpha Increases the Expression of Glycosyltransferases and Sulfotransferases Responsible for the Biosynthesis of Sialylated and/or Sulfated Lewis X Epitopes in the Human Bronchial Mucosa".  J. Biol. Chem. 277(1):  424-431 (2002 January 4)

Ehrlich, P. "Beiträge zur Kenntniss der Anilinfärbungen und ihrer Verwendung in der mikroskopischen Technik".  Arch. mikr. Anat. u. Entwicklungsmech. 13:263  (1877)

Heschl, R.  "Eine hübsche a vista-Reaktion auf amyloid degenerirte Gewebe".  Wien. med. Woch.  #32, columns 713, 715 (1875)

Kelly, J.W. "The Use of Metachromasy in Histology, Cytology, and Histochemistry". Acta Histochemica, Suppl. 1:85 (1958)

Lillie, R.D.  "The Histochemical Reaction of Aryl Amines with Tissue Aldehydes Produced by Periodic and Chromic Acids".  J. Histochem. Cytochem. 10(3):303-314 (1961).

Materazzi, G. and Ferretti, E.  "Histochemical and Histophysical Investigations of the Acetylation Blockade of Carboxylic Groups of Polysaccharides.  J. Histochem Cytochem. 18(7):504-509 (1970 July).

Mathews, C. and van Holde, K.  Biochemistry.  Redwood City, California:  Benjamin/Cummings Publishing Company, 1990.

Miller-Podraza H., Bergström J., Teneberg S., Milh M.A., Longard M., Olsson B.,  Uggla L.,  and Karlsson K.  "Helicobacter pylori and Neutrophils:  Sialic-Acid Dependent Binding to Various Isolated Glycoconjugates."  Infection and Immunity 67(12): 6309-6313 (1999 December).

Roozemond, R.C.  "The Staining and Chromium Binding of Rat Brain Tissue and of Lipids in Model Systems Subjected to Baker's Acid Hematein Technique".  J. Histochem. Cytochem.  19(4):244-251 (1971 April).



How to cite this article: 

Thorsten, C.  "Selective Staining of Infection-Mobilized Leukocytes by Common Food Dyes Using Chromium Trioxide as a Fixative / Mordant."  June 2007.  Retrieved from the CR Scientific LLC website:  http://www.crscientific.com/article-5-min-stain-CrO3.html



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