Preparación de medios de cultivo

Colección de cultivos de algas y microalgas de la Universidad de Austin Texas

P-IV Metal Solution Component Recipe

Directions
For 1 L Total
1. To approximately 950 mL of dH2O, add the nutrients in the order listed while stirring continuously.
 Note: The Na2EDTA should be fully dissolved before adding other components.
2. Bring total volume to 1 L with dH2O.
3. Store in dark at freezer temperature.

Note final concentration listed is for the stock solution.

Stock Solution
# Component Amount Final Concentration
Concentration

1 Na2EDTA•2H2O (Sigma ED255) 0.75 g/L 2 mM

2 FeCl3•6H2O (Sigma F-1513) 0.097 g/L 0.36 mM

3 MnCl2•4H2O (Baker 2540) 0.041 g/L 0.21 mM

4 ZnCl2 (Sigma Z-0152) 0.005 g/L 0.037 mM

5 CoCl2•6H2O (Sigma C-3169) 0.002 g/L 0.0084 mM

6 Na2MoO4•2H2O (J.T. Baker 3764) 0.004 g/L 0.017 mM

F/2 Medium Recipe

Directions
For 1 L Total
1. To approximately 950 mL of non-pasteurized seawater ( 30-35ppt), add each of the components in the order specified (except
vitamins) while stirring continuously.
2. Bring total volume to 1 L with non-pasturized seawater.
 *For 1.5% (w/v) agar medium: add 15 g of agar into the flask; do not mix.
3. Cover and autoclave medium.
4. Add sterile vitamins to cooled medium.
 *For agar medium add vitamins, mix, and dispense before agar solidifies.
5. Store at refrigerator temperature.
 Final
#Component Amount Stock Solution Concentration
Concentration

NaNO3 (Fisher
1 1 mL/L 7.5 g/100 mL dH2O 880 µM
BP360-500)

NaH2PO4·H2O(MCIB
2 1 mL/L 0.5 g/100 mL dH2O 36 µM
742)

Na2SiO3·9H2O
3 1 mL/L 2 g/100 mL dH2O 70 µM
(Sigma 307815)

Trace Metals
6 1 mL/L
Solution

7Vitamin B12 Solution 1 mL/L

Biotin Vitamin
8 1 mL/L
Solution

Thiamine Vitamin
9 1 mL/L
Solution
Bristol Medium Recipe

H.C. Bold's modification of Bristol's recipe (Bold 1949). General purpose freshwater medium and as bristol's solution, an essential
component of other media-- see Bold 1NV Medium, Bold 3N Medium, Bristol-NaCl Medium, LDM Medium, Proteose
Medium, Soil Extract Medium, and Trebouxia Medium.

Directions
For 1 L Total
1. To approximately 900 mL of dH2O, add each of the components in the order specified while stirring continuously.
2. Bring total volume to 1 L with dH2O.
 *For 1.5% (w/v) agar medium: add 15 g of agar into the flask; do not mix.
3. Cover and autoclave medium.
4. Store at refrigerator temperature.

Final
#Component Amount Stock Solution Concentration
Concentration

NaNO3 (Fisher
1 10 mL/L 10 g/400mL dH2O 2.94 mM
BP360-500)

CaCl2·2H2O
2 10 mL/L 1 g/400mL dH2O 0.17 mM
(Sigma C-3881)
 MgSO4·7H2O
3 10 mL/L 3 g/400mL dH2O 0.3 mM
(Sigma 230391)

K2HPO4 (Sigma
4 10 mL/L 3 g/400mL dH2O 0.43 mM
P 3786)

KH2PO4 (Sigma
5 10 mL/L 7 g/400mL dH2O 1.29 mM
P 0662)

NaCl (Fisher
6 10 mL/L 1 g/400mL dH2O 0.43 mM
S271-500)
BG-11 Medium Recipe

Improved recipe as of March 2009

Directions
For 1 L Total
Liquid media:
1. To approximately 900 mL of dH2O, add the first nine (9) components in the order specified while stirring continuously.
2. Bring the total volume to 1 L with dH2O.
3. Cover and autoclave medium.
4. Allow to cool then store at refrigerator temperature.

Agar media:
1. To approximately 400 mL of dH2O, add the first nine (9) components in the order specified while stirring continuously.
2. Bring total volume to 500 mL with dH2O.
3. In a separate container add 15 g of agar to 500 mL of dH2O.
4. Cover and autoclave both solutions.
5. In a water bath, allow both solutions to cool to 45 - 50 °C.
6. Add sterile Sodium Thiosulfate (Component #10) to agar solution and mix well.
7. Combine both agar and liquid solutions, mix well. Note the agar can solidify quickly.
8. Allow to cool then store at refrigerator temperature.
 Stock SolutionFinal
# Component Amount
Concentration Concentration

1 NaNO3 (Fisher BP360-500) 10 mL/L30 g/200 mL dH2O 17.6 mM

2 K2HPO4 (Sigma P 3786) 10 mL/L0.8 g/200 mL dH2O 0.23 mM

3 MgSO4·7H2O (Sigma 230391) 10 mL/L1.5 g/200 mL dH2O 0.3 mM

4 CaCl2·2H2O (Sigma C-3881) 10 mL/L0.72 g/200 mL dH2O 0.24 mM

5 Citric Acid·H2O (Fisher A 104) 10 mL/L0.12 g/200 mL dH2O 0.031 mM

6 Ferric Ammonium Citrate 10 mL/L0.12 g/200 mL dH2O 0.021 mM

7 Na2EDTA·2H2O (Sigma ED255) 10 mL/L0.02 g/200 mL dH2O 0.0027 mM

8 Na2CO3 (Baker 3604) 10 mL/L0.4 g/200 mL dH2O 0.19 mM

9 BG-11 Trace Metals Solution 1 mL/L

Sodium Thiosulfate Pentahydrate (agar

10 1 mL/L 49.6 g/200 mL dH2O 1 mM
media only, sterile) (Baker 3946)
 Vitamin B 12 Solution Component Recipe
Directions
For 200 mL Total
1. Prepare 200 mL of HEPES buffer (50 mM).
2. Adjust the pH to 7.8.
3. Add Vitamin B12 (0.1 mM) wait until fully dissolved.
4. Sterilize by 0.45 µm Millipore filter. Store in dark at freezer temperature.
 The amount of vitamins added can vary from medium to medium so the final concentration is not listed.

Note final concentration listed is for the stock solution.

# Component Amount Stock Solution Concentration

1 HEPES buffer pH 7.8 (Sigma H-3375) 2.4 g/200 mL dH2O

2 Vitamin B12 (cyanocobalamin, (Sigma V-6629)) 0.027 g/200 mL dH2O

 Biotin Vitamin Solution Component Recipe

Directions
For 200 mL Total
1. Prepare 200 mL of HEPES buffer (50 mM).
2. Adjust the pH to 7.8.
3. Add biotin (0.1 mM) wait until fully dissolved.
4. Sterilize by 0.45 µm Millipore filter. Store in dark at freezer temperature.
 ** The amount of vitamins added can vary from medium to medium so the final concentration is not listed.

# Component Amount Stock Solution Concentration

1 HEPES buffer pH 7.8 (Sigma H-3375) 2.4 g/200 mL dH2O

2 Biotin (Sigma B-4639) 0.005 g/200 mL dH2O

 Thiamine Vitamin Component Recipe

Directions
For 50 mL Total
1. Prepare 50 mL of HEPES buffer (50 mM).
2. Adjust the pH to 7.8.
3. Add Thiamine (1 mM) wait until fully dissolved.
4. Sterilize by 0.45 µm Millipore filter. Store in dark at freezer temperature.
 The amount of vitamins added can vary from medium to medium so the final concentration is not listed.

Note final concentration listed is for the stock solution.

# Component Amount Stock Solution Concentration

1 HEPES buffer pH 7.8 (Sigma H-3375) 2.4 g/200 mL dH2O

2 Thiamine (Sigma T-1270) 0.067 g/200 mL dH2O

BG-11 Trace Metals Solution Recipe
Directions

For 1 L Total

1. To approximately 900 mL of dH2O add the components in the order specified while stirring continuously.

2. Bring total volume to 1 L with dH2O.

3. Store at refrigerator temperature.

# Component Amount Stock Solution Final Concentration
Concentration

1 H3BO3 (Baker 0084) 2.86 g/L 46 mM

2 MnCl2•4H2O (Baker 2540) 1.81 g/L 9 mM

3 ZnSO4•7H2O (Sigma Z 0251) 0.22 g/L 0.77 mM

Na2MoO4•2H2O (J.T. Baker

4 0.39 g/L 1.6 mM
3764)

5 CuSO4•5H2O (MCIB 3M11) 0.079 g/L 0.3 mM

Co(NO3)2•6H2O (Mallinckroft
6 49.4 mg/L 0.17 mM
4544)