Image cytometric method for quantifying the relative amount of DNA in bacterial nucleoids using Escherichia coli

An image cytometric method for quantifying integrated fluorescence was deve loped to measure the relative DNA contents of bacterial nucleoids. Image an alysis was performed with newly developed macros in combination with the pr ogram Object-Image, all downloadable from http://simon.bio.uva.nl/object-im age.html. 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 meth od was tested with a narrowly distributed Escherichia coli population, whic h was obtained by growing cells into stationary phase, The width of the ima ge cytometric distribution was approximately 6%, in good agreement with res ults obtained by now cytometry. (iii) The error contribution of manual focu sing could be kept below 2%, although a strong dependency between integrate d fluorescence and focus position was observed. (iv) The results were verif ied with a now cytometer, which gave similar distributions for the DNA cont ents per cell expressed in chromosome equivalents (4.8 fg of DNA). We used the presented method to evaluate whether the DNA conformation had any effec t on the total fluorescence of bacterial nucleoids. Experiments using nucle oids with the same amount of DNA in either a dispersed or a compact conform ation showed no significant difference in integrated fluorescence, indicati ng that it is possible to determine the DNA content per nucleoid independen tly of the actual organization of the DNA.