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[DESCRIPTION]
The following are media recipes necessary for isolating and maintaining <em>Synechococcus</em> isolates.

[GUIDELINES]
<strong>SN MEDIUM</strong> (used for maintenance of culture stocks)<br /><br />
<table>
<tbody>
<tr>
<td>75% Seawater   </td>
<td>(750 mL)</td>
</tr>
<tr>
<td>25% Milli-Q Water</td>
<td>(250 mL)</td>
</tr>
</tbody>
</table>
<br />SYN Stocks <br />
<table>
<tbody>
<tr>
<td>Reagent</td>
<td>Amount</td>
<td>Stock Concentration</td>
</tr>
<tr>
<td>EDTA<sup>*</sup></td>
<td>5 mL/L</td>
<td>1 g/L</td>
</tr>
<tr>
<td>K<sub>2</sub>PO<sub>4</sub><sup>1</sup></td>
<td>2.5 mL/L</td>
<td>6.1 g/L (anhydrous)</td>
</tr>
<tr>
<td>NaNO<sub>3</sub></td>
<td>2.5 mL/L</td>
<td>30%</td>
</tr>
<tr>
<td>Na<sub>2</sub>CO<sub>3</sub></td>
<td>0.5 mL/L</td>
<td>20 g/L</td>
</tr>
<tr>
<td>Cyano TMM<sup>*</sup></td>
<td>1 mL/L</td>
<td>see recipe</td>
</tr>
</tbody>
</table>
<sup>1</sup> Na<sub>2</sub>HPO<sub>4</sub> OK also<br />* Matt's solution uses 0.1 mL/L &amp; contains EDTA<br /><br />2/23/09: for WH6501 make up CEDO-PRO99 supplement w/ NH<sub>4</sub>Cl, NaNO<sub>3</sub>, Na<sub>2</sub>HPO<sub>4</sub>, and CTMM (100 µL/L) <br /><br />Notes: May need to add VA Vitamin mix as necessary if cultures are pure (and lacking the heterotrophic bacteria contaminants that might make and provide the vitamins necessary) - 1 drop/50mL flask<br /><br />May also choose to do SN/10 by dividing the recipe by 10 and leaving out the nitrogen source to obtain a medium useful for isolating clonal cultures via serial dilution from natural seawater samples. <br />
<table>
<tbody>
<tr>
<td>SNX:</td>
<td>nitrate PLUS ammonium as nitrogen source<br />add 1 mL/L of NaNO<sub>3</sub> stock, plus 0.5 mL/L NH<sub>4</sub>Cl stocks</td>
</tr>
<tr>
<td>SNY:</td>
<td>just ammonium as nitrogen source<br />add 0.5 mL/L NH<sub>4</sub>Cl stock</td>
</tr>
<tr>
<td>SU:</td>
<td>urea as the nitrogen source<br />add 1 mL/L of 1M urea stock (60g urea/L=1M urea stock)</td>
</tr>
</tbody>
</table>
<br />For plates add sodium sulfate (NOTE: will get ~40-50 plates per liter of medium)<br />   -Add 0.35g Baculovirus agar per 100 mL SN/10 to get about 10 plates<br /><br />Can add more Carbonate (~3x more) to obtain cultures of Syn that are better for DNA studies<br /><br /><strong>SNAX MEDIUM<br /></strong><strong><strong><br /></strong></strong>
<table>
<tbody>
<tr>
<td>75% Seawater</td>
</tr>
<tr>
<td>25% Milli-Q Water</td>
</tr>
</tbody>
</table>
<br />SN stock reagents as follows: <br />
<table>
<tbody>
<tr>
<td>Reagent</td>
<td>Amount</td>
<td>Stock Concentration</td>
</tr>
<tr>
<td>EDTA</td>
<td>0.5 mL/L</td>
<td>1 g/L</td>
</tr>
<tr>
<td>K<sub>2</sub>PO<sub>4</sub></td>
<td>0.25 mL/L</td>
<td>6.1 g/L (anhydrous)</td>
</tr>
<tr>
<td>NaNO<sub>3</sub></td>
<td>0.25 mL/L</td>
<td>30%</td>
</tr>
<tr>
<td>Na<sub>2</sub>CO<sub>3</sub></td>
<td>0.25 mL/L</td>
<td>4 g/L</td>
</tr>
<tr>
<td>Cyano TMM</td>
<td>0.1 mL/L</td>
<td>see recipe</td>
</tr>
<tr>
<td>Ammonium Chloride 1M Stock</td>
<td>1 mL/L</td>
<td>1M</td>
</tr>
</tbody>
</table>
 <br />For plates add sodium sulfate<br /><br /><strong>CYANO TRACE METALS MIX (Cyano TMM)</strong> for 'SN'. 'SNX', 'SNY', 'ASW', plates and slants <br /><br />
<table>
<tbody>
<tr>
<td>ZnSO<sub>4</sub>·7H<sub>2</sub>O</td>
<td>0.222 g/L</td>
<td>[free Zn++]=0.192 mg/L</td>
</tr>
<tr>
<td>MnCl<sub>2</sub>·4H<sub>2</sub>O</td>
<td>1.4 g/L</td>
<td>[free Mn++]=0.39 mg/L</td>
</tr>
<tr>
<td>Co(NO<sub>3</sub>)·6H<sub>2</sub>O</td>
<td>0.025 g/L</td>
<td>[free Co++]=0.005 mg/L</td>
</tr>
<tr>
<td>Na<sub>2</sub>MoO<sub>4</sub>·2H<sub>2</sub>O</td>
<td>0.39 g/L</td>
<td>[free Mo]=0.15 mg/L</td>
</tr>
<tr>
<td>Citric acid·H<sub>2</sub>O</td>
<td>6.25 g/L</td>
<td> </td>
</tr>
<tr>
<td>Ferric Ammonium Citrate</td>
<td>6.0 g/L</td>
<td>[free Fe+++]=1.05 mg/L</td>
</tr>
</tbody>
</table>
<br /><strong><strong>VA VITAMIN MIX<br /><br /></strong></strong>
<table>
<tbody>
<tr>
<td>Vitamins</td>
<td>Primary Stock</td>
<td>To make solution</td>
</tr>
<tr>
<td>B-12 </td>
<td>1 mg/mL</td>
<td>0.1 mL/100 total</td>
</tr>
<tr>
<td>Thiamine (fridge)</td>
<td>---</td>
<td>weigh 200 mg/100 total</td>
</tr>
<tr>
<td>Biotin (fridge)</td>
<td>0.1 mg/mL</td>
<td>1 mL/100 total</td>
</tr>
<tr>
<td>Paba</td>
<td>2 mg/mL</td>
<td>0.5 mL/100 total</td>
</tr>
<tr>
<td>Folic Acid</td>
<td>1 mg/mL</td>
<td>0.1 mL/100 total</td>
</tr>
<tr>
<td>Niacin</td>
<td>1 mg/mL</td>
<td>10 mL/100 total </td>
</tr>
<tr>
<td>Inositol</td>
<td>---</td>
<td>weigh 100 mg/100 total</td>
</tr>
<tr>
<td>Ca pantothenate</td>
<td>2 mg/mL</td>
<td>10 mL/100 total</td>
</tr>
<tr>
<td>Pyridoxine</td>
<td>1 mg/mL</td>
<td>10 mL/100 total</td>
</tr>
</tbody>
</table>
<br />NOTES: Keep stocks and vitamin solutions frozen. Filter sterilize through a 0.2 µm Nucleopore filter. Do purity test on 1 tube to ensure that it is sterile before adding to stocks. <br /><br />

[STEPS]