, Volume 53, Issue 1, pp 63–77 | Cite as

The acridine dyes: Their purification, physicochemical, and cytochemical properties

II. Purification of acriflavine and proflavine and their cytochemical behaviour regarding machine-oriented evaluation
  • H. W. Tiffe
  • K. H. Matzke
  • G. Thiessen


The present investigation was designed to allow a critical comparison of the cytochemical behaviour of commercially available acriflavine dye samples and pure acriflavine and proflavine dyes, regarding their application in automated cell analysis. Thin layer chromatography, NMR-spectroscopy and mass-spectrometry were applied for the identification of the dye composition.

This study includes (1) a column chromatographic technique for the purification of larger dye quantities, (2) the investigation of the photodecomposition of different dye samples, and (3) the evaluation of the influence of various acriflavine/proflavine dye concentrations (1.6·10−3−4·10−6 mol/l) on to the emission spectrum of stained unhydrolyzed and hydrolyzed chicken erythrocytes.

The commercially available acriflavine dye samples showed a much higher reduction in fluorescence intensity than the pure dyes, whereby proflavine faded less than acriflavine. Photodecomposition is markably influenced by dye impurities. Fluorescence emission spectra were registered at various acriflavine and proflavine dye concentrations for unhydrolyzed and hydrolyzed chicken erythrocytes in order to investigate the dye-dye interaction and the behaviour of the cellular DNA-dye complex. Proflavine showed a similar spectral behaviour as acriflavine. The dye concentration-dependent spectral behaviour of the DNA-dye complex of these fluorochromes seems to be a very critical factor. A comparison of quantitative fluorescence measurements can only be performed by staining cells with the same dye quality, because automated cytology requires reproducible information of cells in “machinesensible” terms.


Emission Spectrum Acridine Cell Analysis Fluorescence Emission Spectrum Chromatographic Technique 
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Copyright information

© Springer-Verlag 1977

Authors and Affiliations

  • H. W. Tiffe
    • 1
  • K. H. Matzke
    • 1
  • G. Thiessen
    • 1
  1. 1.Department Radiologie, Abteilung für Nuklearmedizin und spezielle BiophysikMedizinische HochschuleHannover 61Germany

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