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Image cytometric method for quantifying the relative amount of DNA in bacterial nucleoids using Escherichia coli
Author(s) -
Norbert O. E. Vischer,
P. G. Huls,
R.I. Ghauharali,
G. J. Brakenhoff,
N. Nanninga,
Conrad L. Woldringh
Publication year - 1999
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1046/j.1365-2818.1999.00597.x
Subject(s) - nucleoid , dna , fluorescence , population , escherichia coli , flow cytometry , cytometry , microbiology and biotechnology , biology , fluorescence microscope , resolution (logic) , chemistry , analytical chemistry (journal) , physics , chromatography , genetics , optics , gene , computer science , artificial intelligence , demography , sociology
An image cytometric method for quantifying integrated fluorescence was developed to measure the relative DNA contents of bacterial nucleoids. Image analysis was performed with newly developed macros in combination with the program Object‐Image, all downloadable from . Four aspects of the method were investigated. (i) Good linearity was found over a ten‐fold range of fluorescence intensity in a test with a calibration kit of fluorescent latex spheres. (ii) The accuracy of the method was tested with a narrowly distributed Escherichia coli population, which was obtained by growing cells into stationary phase. The width of the image cytometric distribution was approximately 6%, in good agreement with results obtained by flow cytometry. (iii) The error contribution of manual focusing could be kept below 2%, although a strong dependency between integrated fluorescence and focus position was observed. (iv) The results were verified with a flow cytometer, which gave similar distributions for the DNA contents per cell expressed in chromosome equivalents (4.8 fg of DNA). We used the presented method to evaluate whether the DNA conformation had any effect on the total fluorescence of bacterial nucleoids. Experiments using nucleoids with the same amount of DNA in either a dispersed or a compact conformation showed no significant difference in integrated fluorescence, indicating that it is possible to determine the DNA content per nucleoid independently of the actual organization of the DNA.