Natural and Induced Fluorescence in Microscopic Organisms

Darken, Marjorie A.

doi:10.1128/aem.9.4.354-360.1961

Cited by 22 publications

(4 citation statements)

References 81 publications

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“…In many cases, upon exposure to ultraviolet radiation for periods over 1 or 2 min, the glow began to decrease and was soon extinguished. The inherent difficulties in using fluorescent microscopy to observe cells have been reviewed by Darken (7) and deRepentigny (24).…”

Section: Resultsmentioning

confidence: 99%

Direct Measurement of Acetylesterase in Living Protist Cells

Medzon

Brady

1969

J Bacteriol

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The fluorogenic acetylesterase (acetic ester hydrolase EC 3.1.1.6.) substrate, fluorescein diacetate, was used to measure enzyme activity in living protist cells. The visual enzyme assay was done by monitoring fluorochromasia by fluorescent microscopy. Quantitative fluorogenic assays were done by measuring the evolved fluorescein in a fluorometer. Of 59 strains of bacteria, 35 were fluorochromatically positive. Eight of the fluorochromatically negative strains were fluorogenically positive. Of 22 strains of slime molds and fungi, all were fluorochromatically positive. Three out of 12 different algae were fluorochromatically positive. Several unidentified protozoa were also fluorochromatically positive. Four out of six protozoa were fluorochromatically positive. Structures of special interest showing acetylesterase activity were: the growing hyphal tips of fungi, the vacuolated areas of yeast and protozoa, newly formed bacterial spores or immature fungal spores, "mesosome-like" bodies in Bacillus megaterium, and the cell membrane and nuclear region of green algae. Yeast protoplasts and bacterial protoplasts and spheroplasts were fluorochromatically positive when derived from positive cells and negative when derived from negative cells. There was no correlation between the possession of a capsule and acetylesterase activity. There was no effect on the viability of bacterial cells incubated in the presence of fluorescein diacetate. Paraoxon inhibited bacterial and yeast enzyme at 10-5 M. Eserine (10-5 M) and Paraoxon (10-7 M) inhibited B. megaterium enzyme. Sodium acetate at 10-2 M did not inhibit bacterial enzyme. The implications of these findings on the location and expression of esterase activity in living cells are discussed.Esterases have been identified in bacteria (3, 33, 36), in bacterial spores (25,26,34), in fungi (5, 22, 23), in algae (27), and in protozoa (1,11, 19). The enzymes were identified in cell extracts by gel electrophoresis, kinetic assays, and cytochemical localization by light and electron microscopy. The use of a multitude of substrates makes it difficult to compare the enzyme activities of various organisms.Rotman and Papermaster (29) demonstrated the uptake and enzymatic hydrolysis of fluorescein esters by living animal cells. The released fluorescein was concentrated inside the cell, rendering it visible by fluorescence microscopy. This phenomenon was termed fluorochromasia. An eyepiece photometer was used to measure the evolved fluorescein, which followed Michaelis-Menten kinetics. The adjective fluorogenic was applied to this process. Fluorescein diacetate (FDA) was I Presented at the 17th annual meeting of the Canadian Society of Microbiologists, Hamilton, Ontario, June 1967. found to be the best fluorogenic substrate. The same substrate was used by Roberts and Rosenkrantz (26) to demonstrate acetylesterase (EC 3.1.1.6.) activity in Bacillus coagulans cells and spores by a fluorogenic assay, but no report of fluorochromasia was made.This communication is concemed with the search f...

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Section: Resultsmentioning

confidence: 99%

Direct Measurement of Acetylesterase in Living Protist Cells

Medzon

Brady

1969

J Bacteriol

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“…Closely following the initial description of fluorescence as a phenomenon in 1845 (20), the first synthetic fluorophores were developed (21). However, it was not until the arrival of microscopes compatible with these earliest fluorescent probes (22,23) that applications in different research fields of biology emerged (24)(25)(26)(27). These early fluorescent labeling strategies contributed to the initial understanding of the subcellular order in bacteria (2,28), but it was the advent of green fluorescent protein (GFP) as a live-cell molecular beacon that transformed live cell biology and provided an avant-garde approach to convert standing theories into visual experiments.…”

Section: A Fluorescent Toolbox For Bacterial Vivisection Fluorescent ...mentioning

confidence: 99%

Bacterial Vivisection: How Fluorescence-Based Imaging Techniques Shed a Light on the Inner Workings of Bacteria

Cambré

Aertsen

2020

Microbiol Mol Biol Rev

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SUMMARY The rise in fluorescence-based imaging techniques over the past 3 decades has improved the ability of researchers to scrutinize live cell biology at increased spatial and temporal resolution. In microbiology, these real-time vivisections structurally changed the view on the bacterial cell away from the “watery bag of enzymes” paradigm toward the perspective that these organisms are as complex as their eukaryotic counterparts. Capitalizing on the enormous potential of (time-lapse) fluorescence microscopy and the ever-extending pallet of corresponding probes, initial breakthroughs were made in unraveling the localization of proteins and monitoring real-time gene expression. However, later it became clear that the potential of this technique extends much further, paving the way for a focus-shift from observing single events within bacterial cells or populations to obtaining a more global picture at the intra- and intercellular level. In this review, we outline the current state of the art in fluorescence-based vivisection of bacteria and provide an overview of important case studies to exemplify how to use or combine different strategies to gain detailed information on the cell’s physiology. The manuscript therefore consists of two separate (but interconnected) parts that can be read and consulted individually. The first part focuses on the fluorescent probe pallet and provides a perspective on modern methodologies for microscopy using these tools. The second section of the review takes the reader on a tour through the bacterial cell from cytoplasm to outer shell, describing strategies and methods to highlight architectural features and overall dynamics within cells.

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“…The use of fluors for the expressed purpose of visualizing cells was initiated by Provazek (1914), but this technique remained essentially dormant for a quarter of a century. A review of research pertaining to induced fluorescence in microorganisms is available (Darken, 1961b). Although the toxicity and mutagenic action of fluorescent dyes have been studied, induced fluorescence in actively growing cultures of microorganisms was not investigated specifically for cytochemical or cytological purposes until Freifelder and Uretz (1960) reported that yeasts and bacteria could multiply indefinitely in concentrations of acridine orange sufficient to permit microfluorescent observations of dividing cells.…”

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Absorption and Transport of Fluorescent Brighteners by Microorganisms

Darken¹

1962

Applied Microbiology

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DARKEN, MIARJORIE A. (American Cyanamid Co., Pearl River, N.Y.). Absorption and transport of fluorescent brighteners by microorganisms. Appl. Microbiol. 10:387-393. 1962.-The absorption of brighteners by living cells and their transport to subsequent growth is described. Brighteners are highly fluorescent, ultravioletabsorbing compounds which appear to be essentially nontoxic, stable biological markers. They have been effectively absorbed by growing cultures of bacteria, yeasts, actinomycetes, and higher fungi, with active growth centers evidencing the greatest flourescence.

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Natural and Induced Fluorescence in Microscopic Organisms

Cited by 22 publications

References 81 publications

Direct Measurement of Acetylesterase in Living Protist Cells

Direct Measurement of Acetylesterase in Living Protist Cells

Bacterial Vivisection: How Fluorescence-Based Imaging Techniques Shed a Light on the Inner Workings of Bacteria

Absorption and Transport of Fluorescent Brighteners by Microorganisms

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