DRL Lab 1-4

Artificial Pore Water Medium (APM) Ferric Citrate Marine Media

1 liter

Use iron contaminated beaker, stir bar, canulas, and disposable pipettes

Protect media from light

  1. Put approximately 150ml/L of milli-Q H2O in a small beaker and begin heating on a hot plate.
  2. Put approximately 600ml/L of milli-Q H2O in a large beaker and set it aside.
  3. Weigh out 13.7 g Ferric Citrate (FC)
  4. When the water on the hotplate is steaming add the FC.
  5. Wait for the FC to dissolve fully.
  6. Remove the FC from the heat and pour it into large beaker of water.
  7. Place the beaker on an un-heated stir plate.
  8. Adjust pH of the solution to 6.0-6.5 with 10N NaOH
  9. Add 7.0ml/L of 10N NaOH all at once.
  10. When the pH approaches 5.0 use a lower normality base such as 5N or even 2N.
  11. Once the solution is at a sufficient pH and at room temperature add the rest of the ingredients for 1 liter.

Ingredients / 1 L
NaCl / 20 g
KCl / 0.67 g
NaHCO3 / 2.5 g
DL vitamins / 10 ml
DL minerals / 10 ml
RST minerals / 20 ml
Complete volume with Milli-Q H2O to / 1000 ml
  1. Complete volume with milli-Q water in a graduated cylinder
  2. Aliquot 10ml per pressure tube or 100ml per 156ml serum bottle
  3. Bubble out media with 80:20 of N2:CO2
  4. 10ml in Pressure Tubes:

·  6 minutes open with canulas in liquid

·  6 minutes with stopper on and canulas in liquid

·  3 minutes with stopper on and canulas in headspace above liquid

  1. 100mL in 156ml Serum Bottles:

·  30 minutes open with canulas in liquid

·  15 minutes with stopper on and canulas in headspace above liquid

  1. 25mL in 60ml Serum Bottles:

·  15 minutes open with canulas in liquid

·  15 with stopper on and canulas in liquid

·  10 minutes with stopper on and canulas in headspace above liquid

  1. 800mL in 1L Pyrex Bottles:

·  at least 1 hour open with diffuser (not canula) in liquid

·  at least 30 minutes with stopper on and canulas in headspace above liquid

  1. Crimp to close tightly
  2. Autoclave on a fast/wrapped cycle for 30 minutes
  3. Do not expose this media to direct sunlight
  4. Add aseptically and anaerobically after autoclaving!!
  5. Salt Stock 50 ml 25 ml

DL Vitamin Solution

1 Liter

Ingredient / 1 L
Milli-Q H2O / 800 ml
In Fridge
Biotin / 0.002 g
Pantothenic Acid / 0.005 g
B-12 / 0.0001 g
p-aminobenzoic acid / 0.005 g
Vitamin Box
Thioctic Acid (alpha lipoic) / 0.005 g
Nicotinic Acid / 0.005 g
Thiamine / 0.005 g
Riboflavin / 0.005 g
Pyridoxine HCl / 0.01 g
Folic Acid / 0.002 g
Complete volume with Milli-Q H2O to / 1000 ml

Use iron-free beaker and stir bar

Protect solution from light

  1. Put 800 ml milli-Q water into a beaker with a stir bar
  2. Add all ingredients
  3. Allow to mix and dissolve completely
  4. Complete volume with milli-Q water in a graduated cylinder
  5. Store at 4oC in a dark bottle

DL Mineral Mix

1 Liter

Ingredient / 1 L
Milli-Q H2O / 800 ml
NTA Trisodium Salt (Free acid) / 1.5 g
MgSO4 / 3.0 g
MnSO4 * H2O / 0.5 g
NaCl / 1 g
FeSO4 * 7 H2O / 0.1 g
CaCl2 * 2 H2O / 0.1 g
CoCl2* 6 H2O / 0.1 g
ZnCl2 / 0.13 g
CuSO4* 5 H2O / 0.01 g
AlK(SO4)2 * 12 H2O / 0.01 g
H3BO3 / 0.01 g
Na2MoO4 * 2 H2O / 0.025 g
NiCl2 * 6 H2O / 0.024 g
Na2WO4 * 2 H2O / 0.025 g
Complete volume with Milli-Q H2O to / 1000 ml

Use iron-free beaker and stir bar

  1. Put 800 ml milli-Q water into a beaker with a stir bar
  2. Add all ingredients
  3. Allow to mix and dissolve completely
  4. Complete volume with milli-Q water in a graduated cylinder
  5. Store at 4oC

50X RST Minerals Stock Solution for APM Marine Media

100 ml

Ingredient / 100 mL
Milli-Q H2O / 80 ml
NH4Cl / 5.0 g
KCl / 0.5 g
KH2PO4 / 0.5 g
MgSO4 * 7 H2O / 1.0 g
CaCl2 * 2 H2O / 0.1 g
Complete volume with Milli-Q H2O to / 100 ml

Use iron-free beaker and stir bar

2.  Put 800 ml milli-Q water into a beaker with a stir bar

3.  Add all ingredients

4.  Allow to mix and dissolve completely

5.  Complete volume with milli-Q water in a graduated cylinder

Salt Stock Solution for APM Marine Media

100 ml

Ingredient / 100 mL
Milli-Q H2O / 80 ml
MgCl2 * 6 H2O / 21.2 g
CaCl2 * 2 H2O / 3.04 g
Complete volume with Milli-Q H2O to / 100 ml

Use iron-free beaker and stir bar

6.  Put 800 ml milli-Q water into a beaker with a stir bar

7.  Add all ingredients

8.  Allow to mix and dissolve completely

9.  Complete volume with milli-Q water in a graduated cylinder