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[DESCRIPTION]
Multiwell plates are convenient for isolating cyanophages from environmental samples using the liquid bioassay approach (Table 1). This protocol describes a typical procedure (96-well assay) used to detect and isolate cyanophages from marine samples that lyses <em>Synechococcus</em> sp. strain DC2 (also known as CCMP1334 or WH7803). Using this method, greater than 10<sup>5</sup> lytic phages per milliliter of seawater have been detected in the Gulf of Mexico (Suttle and Chan 1994) that lyses this permissive target host.

[GUIDELINES]
As mentioned earlier, there are some basic similarities between the isolation procedures for bacteriophages and cyanophages. However, the specific protocols for isolation of cyanophages differ substantially from the procedures presented in the previous section and are described in detail in the following sections. Figure 2 shows possible strategies one could follow, depending on the suspected titer of cyanophages in the sample of interest. These assays assume that the target cells are unialgal and clonal. If cultures are not unialgal or clonal, complete lysis of the culture may not occur or plaques could be obscured by contaminating bacteria. Axenic cultures of the host are preferred for the plaque assay, but not necessary for liquid assays. Many strains of marine cyanobacteria can be purchased from culture collections. Alternatively, new hosts can be isolated from the natural environment of interest. However, it can take a lot of time and effort to produce clonal cultures. As well, many cyanobacteria do not grow well on solid substrate. Unless the target host of interest is already cultured on solid substrate, the simplest method for isolation of novel cyanophages would be via the liquid bioassay. It is simple, inexpensive, and the host need not be axenic. <br /><br />In principle, a small volume of a water sample is added to the host culture and monitored over time for signs of infection. The treated cultures are compared with control cultures by eye for obvious signs of viral infection such as total lysis (clearing) of the culture, decrease or change in overall pigmentation of the culture, or clumping and settling of cells to the bottom of the culture vessel. This approach has been used to isolate and detect cyanophages from seawater as well as marine sediment samples (Suttle and Chan 1993; Waterbury and Valois 1993; Wilson et al. 1993; Suttle 2000). Multiwell plates (e.g., Corning brand 24- or 96-well polystyrene plates with lids) are the culture vessel of choice. They are conducive to the screening of many samples, require minimal culture volumes, and take up little incubator space. Glass culture tubes with screw caps (e.g., 13-mm or 25-mm diameter) may be substituted and are useful for screening larger sample volumes. These glass tubes allow nondestructive monitoring of the in vivo chlorophyll fluorescence of the cultures using a fluorometer (e.g., Turner Designs TD700) or similar. Any lysis of the cells would result in a decrease in relative fluorescence (rf) compared with the control cultures.<br /><br /> Liquid assays can be used for all aquatic cyanobacteria and for screening all types of samples, including sediments, and host cells need not be axenic. Samples to be tested are not subjected to the possibility of elevated temperatures encountered when using plaque assays.<br /><br /><strong>Table 1.</strong> Suggested volumes of target cells and sample to use for the liquid bioassay.<br /><br />
<table style="height: 149px;" width="590">
<tbody>
<tr>
<td width="126">Culture vessel</td>
<td width="81">Cell volume</td>
<td width="77">Sample volume</td>
<td width="61">Max. total volume</td>
</tr>
<tr>
<td width="126">Plate, 96-wells</td>
<td width="81">200-250 µL</td>
<td width="77">50-100 µL</td>
<td width="61">300 µL</td>
</tr>
<tr>
<td width="126">Plate, 24-wells</td>
<td width="81">2.5-3 mL</td>
<td width="77">0.2-0.5 mL</td>
<td width="61">3.5 mL</td>
</tr>
<tr>
<td width="126">Tube, 13 100 mm</td>
<td width="81">3.5-4 mL</td>
<td width="77">0.5-1 mL</td>
<td width="61">4.5 mL</td>
</tr>
<tr>
<td width="126">Tube, 25 150 mm</td>
<td width="81">30-35 mL</td>
<td width="77">2-10 mL</td>
<td width="61">40 mL</td>
</tr>
</tbody>
</table>
<br /><strong>Figure 2.</strong> A flowchart suggesting various strategies of cyanophage isolation depending on type of sample and characteristics. <br /><br />
<div><img id="s-mce-img" class="s-mce-img" style="display: block; margin-left: auto; margin-right: auto;" src="https://s3.amazonaws.com/pr-journal/baec2w6.jpg" alt="" width="300" data-src="https://s3.amazonaws.com/pr-journal/badc2w6.jpg" data-ofn="fig2.jpg" /></div>
<strong>Tips and Tricks:</strong><br /><br />1. Use neutral density screens (gray or black window screening) to attenuate the light. Low light levels enhance development of pigments, which allows for easier discrimination of lysed versus unlysed cultures in the wells. <br /><br />2. Condensation forming on lids can occur due to temperature changes in drafty incubators. Excessive condensation can make it difficult to visualize the wells. Sandwich the full plates between a layer of empty plates to insulate the cultures from temperature shifts. <br /><br />3. Sealing the lid to the bottom of the plate helps to slow down evaporation of well contents, particularly the ones located at the plate perimeter.

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