PUBLIC P5
P5 .
RS Medium - Nutrient Medium (NM) Component
User-provided medium: This medium was provided by one of our users. Please be aware that we do not curate or test the media that are composed by our users. Be cautious when trying this medium, especially since no valid reference is given.
Culture medium recipe
Vol.
×
Equipment needed: Autoclave
Autoclavable bottles
0.22 µm vaccum filters
0.22 µm syringe filters
50 ml conical tubes
| Compound | Amount | Unit | Conc. [g/L] | Conc. [mM] | |
|---|---|---|---|---|---|
|
Na2SiO3 x 5 H2O
(Single stock in H2O. Autoclave and store at RT.) |
0.4500 | g/50ml | - | - | |
|
FeCl3 x 6 H2O
(Single stock in H2O. Autoclave and store at RT.) |
0.0600 | g/50ml | - | - | |
|
Na2EDTA x 2H2O
(Single stock in NNM. Autoclave and store at RT.) |
0.0090 | g/50ml | - | - | |
|
NaNO3
(Single stock in NNM. Filter and store at RT.) |
1.5000 | g/50ml | - | - | |
|
NaH2PO4 x H2O
(Single stock in NNM. Filter and store at RT.) |
1.0000 | g/50ml | - | - | |
|
K2HPO4
(Single stock in H2O. Filter and store at RT.) |
2.0000 | g/50ml | - | - | |
|
NH4Cl
(Single stock in NNM. Autoclave and store at RT.) |
0.0500 | g/50ml | - | - | |
|
Thiamine HCl (B1)
(Single stock in H2O. Filter and store at -20C.) |
0.0500 | g/ml | - | - | |
|
Biotin (B8)
(Single stock in H2O. Filter and store at -20C.) |
0.0001 | g/ml | - | - | |
|
Cyanocobalamin (B12)
(Single stock in H2O. Filter and store at -20C.) |
0.0010 | g/ml | - | - | |
|
Folic acid (B9)
(Single stock in H2O. Filter and store at -20C.) |
0.0010 | g/ml | - | - | |
|
Pyridoxine (B6)
(Single stock in H2O. Filter and store at -20C.) |
0.0010 | g/ml | - | - | |
|
Riboflavin (B2)
(Single stock in H2O. Filter and store at -20C.) |
0.0010 | g/ml | - | - | |
|
Niacin (B3)
(Single stock in H2O. Filter and store at -20C.) |
0.0010 | g/ml | - | - | |
|
D-pantothenate acid hemicalcium salt (B5)
(Single stock in H2O. Filter and store at -20C.) |
0.0010 | g/ml | - | - | |
|
Myo-Inositol (B7)
(Single stock in H2O. Filter and store at -20C.) |
0.0010 | g/ml | - | - | |
|
4-Aminobenzoic Acid
(Single stock in H2O. Filter and store at -20C.) |
0.0010 | g/ml | - | - | |
|
L-Glutamine
(Single stock in H2O. Autoclave and store at -20C.) |
0.6000 | g/50ml | - | - | |
|
Sodium pyruvate
(Single stock in NNM. Autoclave and store at 4C in the dark.) |
1.9800 | g/50ml | - | - | |
|
D-Glucose
(Single stock in NNM. Autoclave and store at 4C.) |
1.9800 | g/50ml | - | - | |
|
N-acetylD-glucosamine
(Single stock in H2O. Autoclave and store at -20C.) |
1.5900 | g/50ml | - | - | |
|
D-Ribose
(Single stock in NNM. Autoclave and store at 4C.) |
2.0000 | g/50ml | - | - | |
|
Sodium succinate
(Single stock in NNM. Autoclave and store at RT.) |
2.7200 | g/50ml | - | - | |
|
y-aminobutyric acid
(Single stock in NNM. Autoclave and store at -20C.) |
1.5500 | g/50ml | - | - | |
|
Sodium acetate
(Single stock in NNM. Autoclave and store at RT.) |
1.2300 | g/50ml | - | - | |
|
Decanoic acid
(Single stock in H2O. Autoclave and store at RT.) |
0.0070 | g/50ml | - | - | |
|
L-Arabinose
(Single stock in NNM. Autoclave and store at 4C.) |
1.3200 | g/50ml | - | - | |
|
L-Rhamnose
(Single stock in NNM. Autoclave and store at 4C.) |
1.8100 | g/50ml | - | - | |
|
Fucose
(Single stock in NNM. Autoclave and store at 4C.) |
3.6100 | g/50ml | - | - | |
|
D-Mannose
(Single stock in NNM. Autoclave and store at 4C.) |
3.9600 | g/50ml | - | - | |
|
Galactose
(Single stock in NNM. Autoclave and store at 4C.) |
0.9800 | g/50ml | - | - | |
|
D-Xylose
(Single stock in NNM. Autoclave and store at 4C.) |
8.0000 | g/50ml | - | - | |
|
Sodium alginate
(Single stock in H2O. Autoclave and store at 4C.) |
0.0900 | g/50ml | - | - | |
|
Methyl-cellulose
(See notes below) |
1.0200 | g/50ml | - | - | |
|
Pectin
(See notes below) |
2.0000 | g/50ml | - | - | |
|
Guanosine
(Single stock in NNM. Autoclave and store at -20C.) |
0.0300 | g/50ml | - | - | |
|
Uracil
(Single stock in NNM. Autoclave and store at -20C.) |
0.1600 | g/50ml | - | - | |
|
50X Amino Acids solution (Sigma)
(Add directly to Mix solution 4 (Amino Acids)) |
2.2500 | ml | - | - | |
|
100X Amino Acids solution (Sigma)
(Add directly to Mix solution 4 (Amino Acids)) |
2.2700 | ml | - | - | |
|
Glycerol
(Add directly to Mix solution 5 (Carbon Sources)) |
45.0000 | µl | - | - | |
1
 |
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| 2 Single stock phase: Prepare the single stocks as reported above. Single stocks are prepared either in purified H2O or in the Non-Nutrient Medium (NNM) component of RS Medium (see accompanying protocol on MediaDive). Filtration is performed with a 0.22 µm pore size. | |||||
| 3 Single stock phase: Notes for Methyl cellulose: prepare the single stock in H2O. It is known that Methyl cellulose is soluble in cold water and insoluble in hot water. In order to prepare this stock, add the relative amount and shake the single stock from time to time for 20 minutes. Leave the stock in cold water overnight. Its color should be white. The day after, after autoclaving, it will result like a white solid, not liquid. Leave it at RT until it becomes liquid and transparent. Store at 4C. | |||||
| 4 Single stock phase: Notes for Pectin: prepare the single stock in H2O. To dissolve, use a hot plate heater with a magnetic stirrer for 20 minutes before autoclaving. Store at 4C. | |||||
| 5 Mix solutions phase: Mix solution 1 (Si and Fe+EDTA): add 2.27 ml each from the single stocks of Na2SiO3*5H2O, FeCl3*6H2O and Na2EDTA*2H2O. Fill to 50 ml with NNM. Filter via syringe through a 0.22 µm pore size. Store at 4C. | |||||
| 6 Mix solutions phase: Mix solution 2 (NO3-PO4): add 2.27 ml each from the single stocks of NaNO3, NaH2PO4*H2O, K2HPO4 and NH4Cl. Fill to 50 ml with NNM. Filter via syringe through a 0.22 µm pore size. Prepare this mix fresh every time. | |||||
| 7 Mix solutions phase: Mix solution 3 (Vitamins): add 4.5 µl each from the single stocks of vitamins (Thiamine HCl (B1), Biotin (B8), Cyanocobalamin (B12), Folic acid (Folate) (B9), Pyridoxine (B6), Riboflavin (B2), Niacin (B3), D-pantothenate acid hemicalcium salt (B5), Myo-Inositol (B7) and 4-Aminobenzoic acid). Fill to 50 ml with NNM. Filter via syringe through a 0.22 µm pore size. Store at 4C. | |||||
| 8 Mix solutions phase: Mix solution 4 (Amino Acids): add 2.25 ml from the 50X Amino Acids solution from Sigma and 2.27 ml from the 100X Amino Acids solution from Sigma. Add 2.27 ml of the single stock of Glutamine. Fill to 50 ml with NNM. Filter via syringe through a 0.22 µm pore size. Store at 4C. NOTES: The 50X AA solution contains Arginine (6.52 g/L), Cysteine HCl (1.56 g/L), Histidine (2.10 g/L), L-Isoleucine (2.63 g/L), L-Leucine (2.62 g/L), Lysine HCl (3.63 g/L), L-Methionine (0.76 g/L), L-Phenylalanine (1.65 g/L), L-Threonine (2.38 g/L), L-Tryptophan (0.51 g/L), L-Tyrosine (1.80 g/L) and L-Valine (2.34 g/L). The 100X AA solution contains L-Alanine (0.89 g/L), Glycine (0.75 g/L), L-Proline (1.15 g/L), L-Asparagine*H2O (1.50 g/L), Aspartate (1.33 g/L), Glutamate (1.47 g/L), Serine (1.05 g/L). | |||||
| 9 Mix solutions phase: Mix solution 5 (Carbon Sources): add 45 µl of Glycerol from Sigma (1.26 g/mL). Add 2.23 ml of each carbon source (Sodium pyruvate, D-glucose, N-acetrylID-glucosamine, D-ribose, Sodium succinate, y-aminobutyric acid, Sodium acetate, Decanoic acid, L-arabinose, L-rhamnose, Fucose, L-mannose, Galactose, Xylan, Sodium alginate, Methyl-cellulose, Pectin, Guanosine and Uracil). Fill to 50 ml with NNM. Filter through a 0.22 µm pore size by using the vacuum filter (not by syringe) because the solution is viscous. Store at 4C. | |||||
| 10 Final phase: Combine 4 ml of Mix solution 1 (Si and Fe+EDTA), 30 ml from Mix solution 2 (NO3-PO4), 10.7 ml from Mix solution 3 (Vitamins), 10.9 ml of Mix solution 4 (Amino Acids) and 10.7 ml of Mix solution 5 (Carbon sources). Fill to 150 ml with NNM. Filter through a 0.22 µm pore size by using a vacuum filter. Store Nutrient Medium (NM) component at 4C. | |||||
| 11 Acknowledgements: We would like to thank Ben Temperton (University of Exteter) for fundamental advice and ideas in the creation of RS medium. | |||||
Metadata
| Creator: | Cristiana Sigona |
| Author(s): | Cristiana Sigona, Daniel J. Richter |
| Published: | 30.06.23 |
| Last modified: | 30.06.23 |
| Source: | MediaDive: public |
| Medium type: | Complex medium |
| Final pH: | 7 |
| Equipment needed: | Autoclave, Autoclavable bottles, 0.22 µm vaccum filters, 0.22 µm syringe filters, 50 ml conical tubes |
|
Description:
The RS medium is a fully defined medium designed for growing diverse marine protists. RS medium presents a natural buffer system with a pH of ~7 and a salinity of 3,3%. It includes two components: the Non-Nutrient Medium (NNM) and the Nutrient Medium (NM). Here we show the preparation of the NM. |
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