PUBLIC P4
P4 .
RS Medium - Non-Nutrient Medium (NNM) 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.
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Equipment needed: Autoclave
Autoclavable bottles
DURAN screw cap GL45 with 2 hose connections
Plastic tubes
CO2 pump
EHEIM Air pump 400
Autoclavable 0.1 µm and 0.22 µm filters
| Compound | Amount | Unit | Conc. [g/L] | Conc. [mM] | |
|---|---|---|---|---|---|
| NaCl | 26.9000 | g/l | 26.9 | 460.301 | |
| KCl | 0.6000 | g/l | 0.6 | 8.048 | |
| MgCl2 x 6 H2O | 3.0000 | g/l | 3 | 14.756 | |
| MgSO4 x 7 H2O | 6.4000 | g/l | 6.4 | 25.966 | |
| CaCl2 x 2 H2O | 1.2000 | g/l | 1.2 | 8.162 | |
| NaHCO3 | 0.2000 | g/l | 0.2 | 2.381 | |
| H3BO3 | 0.0100 | g/l | 0.01 | 0.162 | |
| SrCl2 x 6 H2O | 0.0300 | g/l | 0.03 | 0.113 | |
| NaF | 0.0004 | g/l | 4.0e-4 | 0.01 | |
|
NaBr
(Stock solution 0.5g/mL) |
2.1000 | μl/l | - | - | |
|
RbCl
(Stock solution 0.01g/mL) |
10.0000 | μl/l | - | - | |
|
LiCl
(Stock solution 0.6g/mL) |
2.0000 | μl/l | - | - | |
|
ZnCl2
(Stock solution 1g/mL) |
5.0000 | μl/l | - | - | |
|
KI
(Stock solution 1g/mL) |
30.0000 | μl/l | - | - | |
|
AlCl3 x 6 H2O
(Stock solution 0.001g/mL) |
0.3000 | μl/l | - | - | |
|
Na2SiO3 x 5 H2O
(Stock solution 0.053g/50mL; prepare by stirring continuously to promote the dissolution of silicon) |
1.0000 | ml/l | - | - | |
|
FeCl3 x 6 H2O
(Stock solution 0.001g/50mL) |
1.0000 | ul/l | - | - | |
|
Na2EDTA x 2 H2O
(Stock solution 0.002g/50mL) |
1.0000 | ul/l | - | - | |
|
MnCl2 x 4 H2O
(Stock solution 0.01g/50mL) |
1.0000 | μl/l | - | - | |
|
ZnSO4 x 7 H2O
(Stock solution 0.001g/50mL) |
1.0000 | ml/l | - | - | |
|
CoCl2 x 6 H2O
(Stock solution 0.0005g/50ml) |
1.0000 | μl/l | - | - | |
|
CuSO4 x 5 H2O
(Stock solution 0.0013g/50mL) |
100.0000 | μl/l | - | - | |
|
NiSO4 x 6 H2O
(Stock solution 0.0005g/50mL) |
100.0000 | ul/l | - | - | |
|
Na2MoO4 x 2 H2O
(Stock solution 0.001g/50mL) |
1.0000 | ml/l | - | - | |
|
H2SeO3
(Stock solution 0.0007g/50mL) |
100.0000 | μl/l | - | - | |
|
Na3VO4
(Stock solution 0.001g/50mL) |
1.0000 | ml/l | - | - | |
|
K2CrO4
(Stock solution 0.0006g/50mL) |
100.0000 | μl/l | - | - | |
|
BaCO3
(Stock solution 1mg/ml. Heavy metal. Filter by using 0.22 µm pore size (do not autoclave). Store in the dark) |
8.0000 | μl/l | - | - | |
|
Cd(NO3)2 x 4 H2O
(Stock solution 1mg/mL. Heavy metal. Store in the dark) |
0.1000 | μl/l | - | - | |
|
PbCl2
(Stock solution 1mg/mL. Heavy metal. Store in the dark) |
0.0200 | μl/l | - | - | |
|
AgNO3
(Stock solution 1mg/mL. Heavy metal. Store in the dark) |
0.0020 | μl/l | - | - | |
|
TiCl3
(Stock solution. Heavy metal. Use Sigma-Aldrich ref. 14010 (10-15% TiCl3 basis) and dilute 1:1000000. Store in the dark) |
1.3000 | μl/l | - | - | |
1
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| 2 Ingredient phase: For the ingredients indicated with "Stock solution", prepare stock solutions in H2O at the specified concentration and autoclave to sterilize (except for the stock solution of BaCO3, which should be filter sterilized with a 0.22 µm pore size). Store stock solutions at room temperature (RT). Heavy metal stock solutions should be stored in the dark at RT. | |||||
| 3 Ingredient phase: In a 2 liter bottle, combine the indicated amount of each solid ingredient or liquid stock solution. | |||||
| 4 Ingredient phase: Fill to a total volume of 1 liter with purified H2O. | |||||
| 5 Sparging apparatus assembly phase (optional): We have found that directly autoclaving the sparging apparatus materials after assembly (step 12 below) can result in small particles from the materials entering the liquid. These particles mimic the appearance of precipitates that might result from autoclaving the liquid, making it difficult to distinguish between particles from the materials (which could be filtered out) versus precipitates (which are to be avoided). To prevent the accumulation of these particles, we autoclave all sparging apparatus materials (and let them dry and cool) before beginning assembly in the next step. | |||||
| 6 Sparging apparatus assembly phase: Replace the normal cap of the 2 liter bottle with a GL45 screw cap (which has two pass-through connections for flexible plastic tubes). | |||||
| 7 Sparging apparatus assembly phase: Attach a plastic tube to one of the connections on the inside of the cap. The tube should be long enough such that one end reaches inside the liquid in the bottle. This will be the tube through which CO2 and air will flow into the liquid. | |||||
| 8 Sparging apparatus assembly phase: Still working on the inflow of CO2/air, attach a short piece of plastic tubing to the corresponding connector on the top of the cap, and insert an 0.1 µm autoclavable filter into the tube. | |||||
| 9 Sparging apparatus assembly phase: Still working on the inflow of CO2/air, attach another small piece of plastic tubing to the open end of the 0.1 µm filter from the previous step. Attach an autoclavable filter with 0.2 µm pore size to this tube. | |||||
| 10 Sparging apparatus assembly phase: Still working on the inflow of CO2/air, attach a long piece of plastic tubing to the open end of the 0.2 µm filter from the previous step. This tube will attach to the supply of CO2 or air in the sparging step. | |||||
| 11 Sparging apparatus assembly phase: Changing to work on the outflow of excess CO2/air, attach a small piece of plastic tubing to the connection on the top of the cap. Attach an autoclavable filter with 0.2 µm pore size to this tube. | |||||
| 12 Autoclave the 2 liter bottle, containing 1 liter of liquid, with the attached sparging apparatus. Let sit until cool. | |||||
| 13 Sparge CO2 and air phase: Connect the long plastic inflow tube to a source of CO2 and sparge for 4h. | |||||
| 14 Sparge CO2 and air phase: Disconnect the tube from the CO2 source and connect to an air pump. Sparge for 12-24h to remove excess CO2. | |||||
| 15 Sparge CO2 and air phase: Disconnect from the air pump. Working in sterile conditions, replace the sparging apparatus with a sterile non-vented cap. Store at RT. | |||||
| 16 (Optional) Create agar plates with RS medium: for cultures that grow on solid media. Add desired percentage of agar (for example, 1.2%) to Non-Nutrient Medium (NNM) component from the previous step, autoclave again to sterilize and melt the agar, let cool for several minutes, add desired concentration of Nutrient Medium (NM) component (see corresponding recipe on MediaDive), mix and pour into plates. | |||||
| 17 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, DURAN screw cap GL45 with 2 hose connections, Plastic tubes, CO2 pump, EHEIM Air pump 400, Autoclavable 0.1 µm and 0.22 µm filters |
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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 NNM. |
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