Dilution technique is one the most commonly-used methods for studies of microzooplankton grazing and phytoplankton growth. Negative phytoplankton growth rates were usually observed in dilution experiments. We reviewed the factors may accounting for these negative values, and analyzed their impact on the results of dilution experiments. Measures can avoid the adverse effects of these factors were reviewed and analyzed. We conclude that it is not reasonable to simply take the negative phytoplankton growth rate as the failure symbol of dilution experiments. A lot of factors including the light and temperature regimes during incubation, sampling error, contamination of particle-free water and the added nutrients, and nutrient limitation can lead to the negative rates of phytoplankton growth, and impact the results of dilution experiments differently. Simultaneously, we demonstrate the effects of phytoplankton photo-acclimation, sampling error, contamination of particle-free water, and nutrient enrichment on dilution experiments based on our experiments. The results indicate that change of light conditions can lead to the changing of cellular pigment content of phytoplankton, and specific groups of phytoplankton responded differently, thus lead to the underestimation or overestimation of dilution experiment results based on pigment analysis; insufficient mixing before pigment sampling may cause the underestimation of the phytoplankton biomass after incubation, which lead to the underestimation of phytoplankton growth rate (even negative values), but may play no effect on the estimation of microzooplankton grazing rate; in addition, contamination from the added nutrients or particle-free water may inhibit phytoplankton growth, and even lead to phytoplankton death. In a word, the simulation of incubation conditions, and the control of man-made disturbance are critical factors for the success of the dilution experiment.
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