Method 9

Growth conditions:

Temperature: 15oC

Light conditions: 50-100μmol/m/s2 14 hours light:10 hours dark or 16 hours light:8 hours dark

Salinity: 35 ppt

Culture upkeep and transfer:

1. Swirl each culture gently once daily.

2. Grow in autoclaved 250 or 125mL glass Erlenmeyer flasks with autoclaved cotton or Styrofoam stoppers.

3. Transfer cultures to new media every 5-6 days.

Add 10mL old culture to 40mL of chilled media or 10mL old culture to 15mL of chilled media, cultures can be

diluted out, so do not add too little started culture to new media

Work in the biosafety hood to prevent contamination and flame media bottles and flasks after each transfer or

use. For pippetting cultures, use no smaller than a 25mL pipette.

Media:

1:1 mixture of f/2 and ESAW (Harrison et al. 1980, modified according to Cottrell & Suttle 1991). Add 500mL f/2 media to 500mL ESAW media, mix, and autoclave in 500mL aliquots in 1L containers.

Note: Several solutions must be autoclaved ahead of time, allowed to sit, then combined and autoclaved again. Begin preparations at least 4 days prior to need of completed media.

f/2 media – from the CCMP website

1. To 950mL filtered seawater or combined ESAW solutions 1&2 without nutrients, trace metals, vitamins, or enrichments added, add the following:

Quantity Compound Stock Solution Final Concentration in Media

1mL NaNO3 75g/L dH2O 8.83 x 10-4 M

1mL NaH2P04 * H2O 5g/L dH2O 3.63 x 10-5 M

1mL Na2SiO3 * 9 H2O 30g/L dH2O 1.07 x 10-4 M

1mL f/2 trace metal solution

1mL f/2 vitamin solution

2. Bring the final volume up to 1L with ESAW or filtered seawater and autoclave.

f/2 media stock solution preparation:

NaNO3 - dissolve 37.5g in 500mL MilliQ water

NaH2P04 * H2O – dissolve 2.5g in 500mL MilliQ water

Na2SiO3 * 9 H2O – dissolve 15g in 500mL MilliQ water

f/2 trace metal solution

1. To 950mL dH2O add the following:

Quantity Compound Stock Solution Final Concentration in Media

3.15g FeCl3 * 6H2O 1 x 10-5 M

4.36g Na2EDTA*2H2O 1 x 10-5 M

1mL CuSO4 * 5H2O 9.8g/L dH2O 4 x 10-8 M

1mL Na2MoO4 *2H2O 6.3g.L dH2O 3 x 10-8 M

1mL ZnSO4 * 7H2O 22.0g/L dH2O 8 x 10-8 M

1mL CoCl2 * 6H2O 10.0g/L dH2O 5 x 10-8 M

1mL MnCl2 * 4H2O 180.0g/L dH2O 9 x 10-7 M

2. Bring final volume up to 1L with dH2O and autoclave.

f/2 trace metal stock solution preparation:

CuSO4 * 5H2O – dissolve 0.98g in 100 mL MilliQ water

Na2MoO4 *2H2O – dissolve 0.63 g in 100 mL MilliQ water

ZnSO4 * 7H2O – dissolve 5.5g in 250mL MilliQ water

CoCl2 * 6H2O – dissolve 1g in 100 mL MilliQ water

MnCl2 * 4H2O - dissolve 18g in 100mL MilliQ water

f/2 vitamin solution

1. To 950 mL dH2O add the following:

Quantity Compound Stock Solution Final Concentration in Media

1mL Vitamin B12 1g/L dH2O 1 x 10-10 M

10mL Biotin 0.1g/L dH2O 2 x 10-9 M

200mg Thiamine*HCl 3 x 10-7 M

2. Bring final volume up to 1L with dH2O and autoclave (or filter sterilize). Store in refrigerator (4oC).

f/2 vitamin solution stock solution preparation:

Vitamin B12 – dissolve 1g in 1 L MilliQ water – will be dark red in color, filter sterilize, and store stock in fridge.

Biotin – dissolve 10mg in 96mL MilliQ water (4mL for 4% water of crystallization), store stock in fridge.

ESAW media – from Harrison et al. 1980 with modifications from Cottrell & Suttle, 1991

1. Prepare artificial seawater base solutions 1 and 2 separately as below.

2. Autoclave the 2 solutions separately, allow to sit for 48hours for gaseous exchange, then combine in a 1:1 ratio.

3. Add 10mL ESAW nutrients and trace metal solution and 1mL f/2 vitamin solution to 950mL of combined solutions 1 and 2.

4. Enrichments (according to Cottrell & Suttle, 1991), 5mM of pH 7.7 Tris-HCL and 10nM Na2SeO3.

a. Add 5mL of 1M Tris-HCL to 950mL ESAW from #3 or 2.5 mL to 500mL.

b. Add 100 uL of stock Na2SeO3 to 950mL of ESAW from #3 or 50uL to 500mL. Na2SeO3 stock solution – dissolve 0.0043g in 250 mL MilliQ water = 100,000 nM stock solution. Caution: This compound is highly toxic, measure and mix in biosafety hood, wear gloves and lab coat when working with the solution and powder.

5. Bring final volume up to 1L and autoclave.

ESAW solution 1 – anhydrous salts – these amounts are doubled from the original paper, so that when added to 1L final volume and mixed with 1L final volume of solution 2 the resulting 2L of salt solution is about 35ppt. Alternately, add half the following volume to 500mL, and when mixed with half volumes added to 500mL for solution 2, one would get 1L total of 35ppt.

Compound g/L solution Final mmol/L in combined Sol. 1 & 2 mixture

NaCl 41.516g 362.661

Na2SO4 6.954g 24.993

KCl 1.174g 8.038

NaHCO3 0.34g 2.066

KBr 0.169g 7.249 x 10-1

H3BO3 0.045g 3.715 x 10-1

NaF 0.0054g 6.570 x 10-2

ESAW Solution 2 – hydrous salts

Compound g/L solution Final mmol/L in combined Sol. 1 & 2 mixture

MgCl2 * 6H2O 18.79g 47.176

CaCl2 * 2H2O 2.632g 9.139

SrCl2 * 6H2O 0.0428g 8.200 x 10-2

ESAW nutrients and trace metal solution – Autoclave when complete and store at 4oC.

Compound g/L solution Final umol/L in ESAW

NaNO3 4.667g 549.09

Na2SiO3 * 9H2O 3.000 105.60

Na2glycerolPO4 0.667 21.79

Na2EDTA 0.553 14.86

H3BO3 0.380 61.46

Fe(NH4)2(SO4)2*6H2O 0.234 5.97

FeCl3 * 6H2O 0.016 5.92 x 10-1

MnSO4* 4H2O 0.054 –add 1mL stock solution 2.42

ZnSO4* 7H2O 0.0073 - add 331.8uL stock solution 2.54 x 10-1

CoSO4 * 7H2O 0.0016 - add 135uL stock solution 5.69x 10-2

Stock ESAW nutrients and trace metal solution preparation:

MnSO4*4H2O – dissolve 2.0449g MnSO4*H2O in 50mL MilliQ water = 40.898g/L solution, add 1mL add same amount of Mn as should be in the solution.

ZnSO4* 7H2O – same stock solution as for f/2 media – add 331.8uL to get correct concentration for ESAW media

CoSO4 * 7H2O – same stock solution as CoCl2 * 6H2O for f/2 media – add 135 uL to get correct concentration of Co for ESAW media.