Using cell cycle analysis to estimate in situ growth rate of the dinoflagellate Dinophysis acuminata: drawbacks of the DNA quantification method

In an attempt to use cell cycle analysis to estimate in situ gross growth r ate of the dinoflagellate Dinophysis acuminata, epifluorescence microscopy in combination with an image analysis system was used to measure the relati ve DNA content of DAPI stained D. acuminata nuclei. To be able to estimate growth rate with this method, the time it takes for a cell to synthesise a second DNA copy and complete mitosis (the duration of the terminal event in the cell cycle) must be known or measurable. The duration of the terminal event is normally estimated graphically from diurnal variations in the phas e fraction curves. No diurnal variation in the phase fractions was detected in this study, and consequently no reliable estimate of the duration of th e terminal event could be obtained. The main drawback was the difficulty in delineating the S phase in DNA histograms based on only a few hundred cell s. No dear S phase maximum could be obtained during our 48 h study. The pre sence of double-nucleated cells and a constantly high percentage (23 to 43% ) of cells with double genomes (G2 + M phase cells) suggests, under the ass umption that no cells can arrest in the G2 or M phase, that the population was actively dividing, but not clearly in phase with a diurnal cycle. Chang & Carpenter (1991) previously estimated the duration of the terminal event (the duration of the S + G2 + M phases) in this species to be 11 to 13 h. A 12 h duration of the terminal event in this study would yield specific gr owth rates of 0.69 to 0.75 d(-1). We conclude that the number of cells that can be measured using epifluorescence microscopy (a few hundred per sample ) is too low to allow detection of a low degree of synchronisation, especia lly with regard to the S phase. Estimations of in situ growth rate of poorl y synchronised populations of phytoplankton using the cell cycle technique will require DNA measurements on several thousand cells per sample, e.g. us ing flow cytometry or automated image cytometry.