RS Medium - IBEMicrobeHub/culturing GitHub Wiki

Richter-Sigona (RS) Medium

The RS medium is a fully defined medium for growing marine diverse protists, able to mimic the oligotrophic conditions present in the open ocean. It has:

  • pH= ~7
  • salinity=3,3%

And it includes two parts:

  1. Non Nutrient Medium (NNM)
  2. Nutrient Medium (NM).

1) NON NUTRIENT MEDIUM (NNM)

INGREDIENTS:

Major elements mmol/Kg g/L
NaCl 461 26,9
KCl 8 0,6
MgCl2*6H2O 14,8 3
MgSO4*7H2O 26,1 6,4
CaCl2*2H2O 8,5 1,2
NaHCO3 2,4 0,2
Minor Elements mmol/Kg g/L
NaBr 0,01 0,001
H3BO3 0,1 0,01
SrCl2*6H2O 0,1 0,03
NaF 0,01 0,0004
RbCl 0,001 0,0001
LiCl*H2O 0,02 0,001
ZnCl2 0,00004 0,00001
KI 0,0002 0,00003
Minor Elements (others) mmol/Kg g/L
AlCl3*6H2O 0,000001 0,0000003
Na2SiO3*5H2O 0,005 0,001
Trace Elements mmol/Kg g/L
FeCl3*6H2O 0,0000001 0,00000003
Na2EDTA*2H2O 0,0000001 0,00000004
MnCl2*4H2O 0,000001 0,0000002
ZnSO4*7H2O 0,0001 0,00002
CoCl2*6H2O 0,00000004 0,00000001
CuSO4*5H2O 0,00001 0,000003
NiSO4*6H2O 0,000004 0,000001
Na2MoO4*2H2O 0,0001 0,00002
H2SeO3 0,00001 0,000001
Na3VO4 0,00004 0,00002
K2CrO4 0,000003 0,000001
Heavy Metals mmol/Kg g/L
BaCO3 0,00004 0,00001
Cd(NO3)2*4H2O 0,0000001 0,0000001
PbCl2 0,0000001 0,00000002
AgNO3 0,00000001 0,000000002
TiCl3 0,00000001 0,000000002

RS medium NNM

A) Ingredient phase:

  1. 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.

  1. In a 2 liter bottle, combine the indicated amount of each solid ingredient or liquid stock solution.
  2. Fill to a total volume of 1 liter with purified H2O.

B) Sparging apparatus assembly phase:

(Optional): We have found that directly autoclaving the sparging apparatus materials after assembly (step 10 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.

  1. Replace the normal cap of the 2 liter bottle with a GL45 screw cap (which has two pass-through connections for flexible plastic tubes).
  2. 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.
  3. 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.
  4. 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.
  5. 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.
  6. 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.
  7. Autoclave the 2 liter bottle, containing 1 liter of liquid, with the attached sparging apparatus. Let sit until cool.

C) Sparge CO2 and air phase:

  1. Connect the long plastic inflow tube to a source of CO2 and sparge for 4h.
  2. Disconnect the tube from the CO2 source and connect to an air pump. Sparge for 12-24h to remove excess CO2.
  3. Disconnect from the air pump. Working in sterile conditions, replace the sparging apparatus with a sterile non-vented cap. Store at RT.

(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.

Acknowledgements: We would like to thank Ben Temperton (University of Exteter) for fundamental advice and ideas in the creation of RS medium.

2) NUTRIENT MEDIUM (NM)

INGREDIENTS:

Si&Fe+EDTA (Fridge) mmol/Kg g/L SINGLE STOCKS [g (x400) in 50 mL NNM] INTERMEDIATE STOCKS (50mL)
Na2SiO3*5H2O 0,106 0,02 0,45 g 2,27 mL
FeCl3*6H2O 0,0117 0,003 0,06 g 2,27 mL
Na2EDTA*2H2O 0,0117 0,004 0,09 g 2,27 mL
NO3-PO4 (Room TºC) mmol/Kg g/L SINGLE STOCKS [g (x400) in 50 mL NNM] INTERMEDIATE STOCKS (50mL)
NaNO3 0,88 0,08 1,50 g 2,27 mL
NaH2PO4*H2O 0,36 0,1 1 g 2,27 mL
K2HPO4 0,57 0,1 2 g 2,27 mL
NH4Cl 0,05 0,003 0,05 g 2,27 mL
Vitamins (Freezer) mmol/Kg g/L INTERMEDIATE STOCKS (µL)
Thiamine HCl (B1) 0,01 0,002 4,5
Biotin (B8) 0,000004 0,000001 4,5
Cyanocobalamin (B12) 0,000001 0,000001 4,5
Folic acid (Folate) (B9) 0,000004 0,000002 4,5
Pyridoxine (B6) 0,0005 0,0001 4,5
Riboflavin (B2) 0,0004 0,0002 4,5
Niacin (B3) 0,001 0,0001 4,5
D-pantothenate acid hemicalcium salt (B5) 0,0004 0,0002 4,5
Myo-Inositol (B7) 0,01 0,001 4,5
4-Aminobenzoic acid 0,0001 0,000008 4,5
Amino acids with L-glutamine (Fridge) mmol/Kg g/L INTERMEDIATE STOCKS
L-Alanine 0,39 0,03 2,25 mL (50X) & 2,27 mL (100X)
Arginine 0,16 0,03 " "
Cysteine HCl 0,22 0,03 " "
Glycine 0,47 0,03 " "
Histidine 0,22 0,03 " "
L-Isoleucine 0,27 0,03 " "
L-Leucine 0,27 0,03 " "
Lysine HCl 0,24 0,04 " "
L-Methionine 0,23 0,03 " "
L-Phenylalanine 0,21 0,03 " "
L-Proline 0,30 0,04 " "
L-Threonine 0,29 0,04 " "
L-Tryptophan 0,22 0,05 " "
L-Tyrosine 0,19 0,04 " "
L-Valine 0,30 0,03 " "
L-Asparagine *H2O 0,34 0,04 " "
Aspartate 0,27 0,04 " "
Glutamate 0,24 0,04 " "
Serine 0,33 0,03 " "
L-Glutamine (Freezer) 0,24 0,03 2,27 mL

There are 2 stocks (50X & 100X) in the fridge with all the amino acids, less L-Glutamine. Add 2,25 mL of the 50X stock, and then, 2,27 mL of 100X stock. At the end, add 2,27 mL of L-glutamine.

High Nutrient mmol/Kg g/L SINGLE STOCKS [g (x400) in 50 mL NNM] INTERMEDIATE STOCKS
Glycerol 0,50 0,05 1,26 g/mL Sigma 45 µL
Sodium pyruvate 0,90 0,10 1,98 g 2,3 mL
D-glucose 0,55 0,10 1,98 g 2,3 mL
N-acetylD-glucosamine 0,36 0,08 1,59 g 2,3 mL
D-Ribose 0,67 0,10 2 g 2,3 mL
Sodium succinate 0,84 0,14 2,72 g 2,3 mL
y-aminobutyric acid 0,75 0,08 1,55 g 2,3 mL
Sodium acetate 0,75 0,06 1,23 g 2,3 mL
Decanoic acid 0,002 0,0003 0,007 g 2,3 mL
L-Arabinose 0,44 0,07 1,32 g 2,3 mL
L-Rhamnose 0,55 0,09 1,81 g 2,3 mL
Fucose 1,10 0,18 3,61 g 2,3 mL
L-Mannose 1,10 0,20 3,96 g 2,3 mL
Galactose 0,55 0,10 1,98 g 2,3 mL
Xylan (D-xylose) 0,002 0,40 8 g 2,3 mL
Sodium alginate 0,022 0,0047 0,09 g 2,3 mL
Methyl-cellulose 0,08 0,05 1,02 g 2,3 mL
Pectin 0,52 0,10 2 g 2,3 mL
Guanosine 0,006 0,002 0,03 g 2,3 mL
Uracil 0,07 0,008 0,16 g 2,3 mL

Guanosine is on the freezer.

RS medium NM

A) Prepare the intermediate stocks from the single stocks:

  1. Si & Fe+EDTA:
  • Weigh each ingredients and put them in a 50 mL falcon.

The nexts steps must be done under the chemical hood.

  • Fill the falcon with NNM (not sterilized) till 50 mL.
  • Filter thought 0,22 µm and place it into a 100 mL bottle.
  1. NO3-PO4:
  • Weigh each ingredients and put them in a 50 mL falcon.

The nexts steps must be done under the chemical hood.

  • Fill the falcon with NNM (not sterilized) till 50 mL.
  • Filter thought 0,22 µm and place it into a 100 mL bottle.
  1. Vitamins:

The vitamins stocks are in the freezer.

Under the chemical hood.

  • Add the amounts of each ingredients in a 50 mL falcon.
  • Fill the falcon with NNM (not sterilized) till 50 mL.
  • Filter thought 0,22 µm and place it into a 100 mL bottle.
  1. Amino acids:

The 2 amino acids stocks are in the fridge: 50X and 100X.

Under the chemical hood.

  • Add 2,25 mL of 50X amino acid stock in a 50 mL falcon.
  • Add 2,27 mL of 100X amino acid stock in the previous falcon.
  • Add 2,27 mL of L-glutamine from the freezer in the falcon.
  • Fill the falcon with NNM (not sterilized) till 50 mL.
  • Filter thought 0,22 µm and place it into a 100 mL bottle.
  1. High nutrient:

B) Prepare the Intermediate stocks:

  1. Si & Fe+EDTA:

  2. NO3-PO4:

  3. Vitamins:

  4. Amino acids:

  5. High nutrient:

C) Prepare the final solution NM: