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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 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 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.
Safety:
Chromium trioxide is toxic and is probably a human carcinogen. Wear gloves, preferably a double-layer, when working with it. Wear safety goggles. Work with as small an amount as necessary at a time.
Wastes containing hexavalent chromium should be saved for reclamation / recycling. It is not recommended to work with Cr(VI) without knowing how to do this.
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. CrO3 must be kept away from alcohol, papers, and other easily-oxidized materials. 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 it must not contact paper or combustible materials.
Materials
CrO3 (chromium trioxide, "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 were obtained from a parotid gland infection with which the author awoke one morning. This was an unplanned event, but it was not allowed to go to waste.
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. These washings were saved for later reclamation of the Cr(VI) by evaporative concentration et seq.
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 finally 100% IPA that had been dried on a bed of 3A molecular sieves.
Finally, the specimen was treated with xylene. The excess was removed 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. 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. Leukocytes obtained after the infection had cleared. The fixing
and staining protocol gave no differentiation here, despite spectacular results
on 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 remarkable, at least on white blood cells from a bacterial infection (i.e., specimen F1). In this experiment, the CrO3 caused intense color differentiation on subsequent dye application 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.
We can conclude, simply by comparing the photos, 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.
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. However, its exact interactions 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 CrO3. 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.
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.
Though the color differentiation seen in F1 has a rational, cytochemical basis, it is entirely coincidental that the color scheme is suggestive of an "inflamed" or "angry" condition. Perhaps this "angry cell" response could have some practical application.
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).
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)
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).
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