Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1965 Nov 1;27(2):313–326. doi: 10.1083/jcb.27.2.313

STUDIES ON NUCLEIC ACID METACHROMASY

I. The Effect of Certain Fixatives on the Dye Stacking Properties of Nucleic Acids in Solution

Michael E Lamm 1, Lillian Childers 1, Merrill K Wolf 1
PMCID: PMC2106731  PMID: 4160916

Abstract

The stacking coefficients (K's) of nucleic acids have been thought to influence the color contrast between DNA and RNA in tissue sections stained with metachromatic dyes. This idea was tested by titrating toluidine blue (TB) and acridine orange (AO) in solution against DNA and RNA, native or treated with formaldehyde, acrolein, or Carnoy's fluid. Absorption spectra at varying polymer-dye ratios were used to compute K values by the methods of Bradley and colleagues. Results with both dyes fit Bradley's stacking equations. Fixatives did not block dye-binding sites but markedly altered K values. K of DNA was low, unaffected by aldehyde fixative, increased by Carnoy's fluid or heat denaturation. K of RNA was higher than that of DNA and was increased greatly by formaldehyde, almost as much by acrolein, considerably less by Carnoy's fluid. Aldehyde effects were partially reversed upon removal of aldehyde by dialysis. These observations accord with known effects of aldehydes and denaturation upon nucleic acid conformation. Differences between K's of DNA and RNA were greater after aldehyde treatment than after Carnoy's, and were greater with AO than with TB. This is generally consistent with the magnitude of the color contrasts observed in tissues. Additional factors must contribute to the intense color contrast observed in acrolein-fixed tissues stained with TB.

Full Text

The Full Text of this article is available as a PDF (957.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. ARMSTRONG J. A., NIVEN J. S. Fluorescence microscopy in the study of nucleic acids; histochemical observations on cellular and virus nucleic acids. Nature. 1957 Dec 14;180(4598):1335–1336. doi: 10.1038/1801335a0. [DOI] [PubMed] [Google Scholar]
  2. BERGERON J. A., SINGER M. Metachromasy: an experimental and theoretical reevaluation. J Biophys Biochem Cytol. 1958 Jul 25;4(4):433–457. doi: 10.1083/jcb.4.4.433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. BRADLEY D. F., FELSENFELD G. Aggregation of an acridine dye on native and denatured deoxyribonucleates. Nature. 1959 Dec 19;184:1920–1922. doi: 10.1038/1841920a0. [DOI] [PubMed] [Google Scholar]
  4. Bradley D. F., Wolf M. K. AGGREGATION OF DYES BOUND TO POLYANIONS. Proc Natl Acad Sci U S A. 1959 Jul;45(7):944–952. doi: 10.1073/pnas.45.7.944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. DART L. H., Jr, TURNER T. R. Fluorescence microscopy in exfoliative cytology. Report of acridine orange examination of 5491 cases, with comparison by the Papanicolaou technic. Lab Invest. 1959 Nov-Dec;8:1513–1522. [PubMed] [Google Scholar]
  6. FRAENKEL-CONRAT H. Reaction of nucleic acid with formaldehyde. Biochim Biophys Acta. 1954 Oct;15(2):307–309. doi: 10.1016/0006-3002(54)90083-9. [DOI] [PubMed] [Google Scholar]
  7. Feder N., Wolf M. K. Studies on nucleic acid metachromasy. II. Metachromatic and orthochromatic staining by toluidine blue of nucleic acids in tissue sections. J Cell Biol. 1965 Nov;27(2):327–336. doi: 10.1083/jcb.27.2.327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. GROSSMAN L., LEVINE S. S., ALLISON W. S. The reaction of formaldehyde with nucleotides and T2 bacteriophage DNA. J Mol Biol. 1961 Feb;3:47–60. doi: 10.1016/s0022-2836(61)80007-7. [DOI] [PubMed] [Google Scholar]
  9. HASELKORN R., DOTY P. The reaction of formaldehyde with polynucleotides. J Biol Chem. 1961 Oct;236:2738–2745. [PubMed] [Google Scholar]
  10. MORTHLAND F. W., DE BRUYN P. P., SMITH N. H. Spectrophotometric studies on the interaction of nucleic acids with aminoacridines and other basic dyes. Exp Cell Res. 1954 Aug;7(1):201–214. doi: 10.1016/0014-4827(54)90055-5. [DOI] [PubMed] [Google Scholar]
  11. STAEHELIN M. Reaction of tobacco mosaic virus nucleic acid with formaldehyde. Biochim Biophys Acta. 1958 Aug;29(2):410–417. doi: 10.1016/0006-3002(58)90200-2. [DOI] [PubMed] [Google Scholar]
  12. STEINER R. F., BEERS R. F. Polynucleotides. V. Titration and spectrophotometric studies upon the interaction of synthetic polynucleotides with various dyes. Arch Biochem Biophys. 1959 Mar;81(1):75–92. doi: 10.1016/0003-9861(59)90177-8. [DOI] [PubMed] [Google Scholar]
  13. TAYLOR K. B. Chromatographic separation and isolation of metachromatic thiazine dyes. J Histochem Cytochem. 1960 Jul;8:248–257. doi: 10.1177/8.4.248. [DOI] [PubMed] [Google Scholar]
  14. VON BERTALANFFY L., MASIN M., MASIN F. A new and rapid method for diagnosis of vaginal and cervical cancer by fluorescence microscopy. Cancer. 1958 Sep-Oct;11(5):873–887. doi: 10.1002/1097-0142(195809/10)11:5<873::aid-cncr2820110503>3.0.co;2-b. [DOI] [PubMed] [Google Scholar]
  15. WOLF M. K., ARONSON S. B. Growth fluorescence and metachromasy of cells cultured in the presence of acridine orange. J Histochem Cytochem. 1961 Jan;9:22–29. doi: 10.1177/9.1.22. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES