Effects of Ultrasonic Radiation in Escherichia coli B using Fluorochrome Acridine Orange as a Vital Stain

Abstract

STRUGGER¹, one of the pioneers who introduced acridine orange as a biological fluorochrome, observed that dead cells take up more of this dye, and under ultra-violet excitation fluoresce red, whereas living cells take up less dye and fluoresce green. This difference in the fluorescence is often used as a test for viability of cells and is referred to as ‘Strugger effect’². Loss of viability associated with red fluorescence has also been confirmed by Krebs and Gierlach³ in their work involving radiobiological action on Allium cepa cells. Later work has shown that green acridine orange fluorescence in normal cells is to be associated with cell constituents rich in deoxyribonucleic acid⁴. It is also known from the observations of Beers et al. ⁵, Bradley and Wolf⁶, and Ranadive and Korgaonkar⁷, that deoxyribonucleic acid can form two complexes with acridine orange. One of these complexes is formed when the relative concentration of acridine orange to deoxyribonucleic acid is low, and exhibits strong green fluorescence and the other occurs when this ratio is high and shows orange or red fluorescence. Shift of fluorescence from green to orange is a critical step in this transition, whereas further change from orange to deep red is not sharp and occurs with further increase in concentration of acridine orange, without involving formation of new complexes. ‘Strugger effect’ may be explained in terms of these observations as a kind of damage to structures rich in deoxyribonucleic acid in the inactivated cells. This results in their high uptake of acridine orange to such an extent that the corresponding fluorescence is copper red. Few observations, however, are available to indicate the nature and extent of damage near the critical point when the shift of fluorescence from green to orange just occurs. A study of this nature in relation to viability was carried out by us on E. coli using ultrasonic waves for irradiation and the findings are reported here.