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[DESCRIPTION]
The modified dilution assay aims to partition phytoplankton mortality into virus- versus grazing-induced fractions and has previously been applied to several different environments to determine viral lysis rates of natural phytoplankton. The method involves creating a gradient of both grazing and viral lysis by dilution with different proportions of grazer- and virus-free filtrate, and assessing the subsequent impact on phytoplankton growth rates.<br /><br />Kimmance, S. A., and C. P. D. Brussaard. 2010. Estimation of viralinduced phytoplankton mortality using the modified dilution method, p. 65&ndash;73. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO.<br /><br /><br />

[BEFORE_START]
Prior to each experiment 0.2&ndash;0.45 &micro;m filters, silicon tubing, carboys, and bottles should be acid-washed in 5% HCL and rinsed thoroughly with Milli-Q water and then twice with the sample. The 0.2&ndash;0.45-&micro;m and kDa filters should be flushed prior to use with freshly prepared Milli-Q and the first few liters of both filtrates discarded. Ensure that all air bubbles are removed from the filter capsule and tubing during filtration by opening the filter valves. To speed up this process you may use two filters in parallel.

[GUIDELINES]
Experiments are set up based on the original protocol of the modified dilution method of Evans et al. (2003), and the following method is appropriate for both natural and cultured phytoplankton&ndash;virus systems (Fig. 1). The approach is based around combining mesoplankton-free seawater (whole water) with either grazer-free or virus-free diluents. To prevent contamination of seawater samples by handling, vinyl gloves are worn throughout the water collection and experimental setup. To create mesoplankton-free whole water for experiments, seawater is gently siphoned through 200-&micro;m mesh (or reverse sieved, Baudoux et al. 2006) using silicone tubing into clean, polypropylene carboys. Additional (reverse) prefiltration may be required, e.g., during sampling of colonial phytoplankton (Baudoux et al. 2006) or in productive, coastal, or sediment-filled waters, to prevent clogging during the later fine-scale filtration. Gentle filtration and avoidance of air bubbling are crucial to prevent destruction of the grazer, virus, and phytoplankton populations. To protect the phytoplankton communities within the seawater sample from excessive light (light-shock), experiments using natural phytoplankton populations are best set up predawn in dimmed-light conditions using light-proofed carboys. It is also recommended that experiments be performed at the same time of day because of the synchronicity of phytoplankton cell division and potential diel effects on viral infection processes. Filtration and handling should be conducted at in situ temperature. Handling time once the seawater has been collected should be kept to a minimum before the start of the experimental incubation. Ideally experimental setup should be within an hour of water collection if possible.<br /><br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/2ic2w6.jpg" alt="" data-src="https://s3.amazonaws.com/pr-journal/2hc2w6.jpg" data-ofn="Figure 1.jpg" /><br /><br /><strong>Figure 1</strong>:&nbsp;Modified dilution assay experimental design. Mesoplankton-free whole water (1) is combined with either &lt;0.45-&micro;m filtrate (2) or kDa filtrate (3) in the correct proportions to create the parallel t0 dilution series: &lt;0.45&micro;m series, with reduced grazing mortality (4) kDa series, with reduced grazing and viral mortality (5). Replicate sample bottles from the &lt;0.45-&micro;m and kDa dilution series are then created (6 and 7) and incubated under experimental conditions.<br /><br /><strong>References</strong><br /><br />Baudoux, A.-C., A. A. M. Noordeloos, M. J. W. Veldhuis, and C. P. D. Brussaard. 2006. Virally induced mortality of Phaeocystis globosa during two spring blooms in temperate coastal waters. Aquat. Microb. Ecol. 44:207-217.<br /><br />&mdash;&mdash;&mdash;, M. J. W. Veldhuis, H. J. Witte, and C. P. D. Brussaard. 2007. Viruses as mortality agents of picophytoplankton in the deep chlorophyll maximum layer during IRONAGES III. Limnol. Oceanogr. 52:2519-2529.<br /><br />&mdash;&mdash;&mdash;, &mdash;&mdash;&mdash;, A. A. M. Noordeloos, G. van Noort, and C. P. D. Brussaard. 2008. Estimates of virus- vs. grazing induced mortality of picophytoplankton in the North Sea during summer. Aquat. Microb. Ecol. 52:69-82.<br /><br />Evans, C., S. D. Archer, S. Jacquet, and W. H. Wilson. 2003. Direct estimates of the contribution of viral lysis and microzooplankton grazing to the decline of a Micromonas spp. population. Aquat. Microb. Ecol. 30:207-219.<br /><br />Kimmance, S. A., W. H. Wilson, and S. D. Archer. 2007. Use of the modified dilution technique to estimate viral versus grazing mortality of phytoplankton: limitations associated with method sensitivity in natural waters. Aquat. Microb. Ecol. 49:207-222.<br /><br /><br />

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