Diplomonads:Spironucleus Medium
Spironucleus vortens (ATCC 50386) Medium
(modified from ATCC 1141, TY1-S-33)
- This also works for Hexamita inflata
Incubate 12ml cultures in screw cap 16 x 125mm screw-cap falcon tubes (Fisher cat#14-959-42) @25C on slant.
For 500ml:
1. Prepare Basic Medium in an orange screw-cap bottle. In 350 ml dH2O add:
- 10.0g casein digest peptone (BBL97023)
- 5.0g yeast extract (BBL11928)
- 1.0g NaCl
- 5.0g dextrose
- 0.5g L-cysteine HCL (SIGMA 7880)
- 0.01g ascorbic acid
2. pH to 6.8 with NaOH.
3. In a sterile hood add the following to the basic medium; sterile filter:
- 25 ml phosphate buffer (below)
- 0.5 ml ferric ammonium citrate (22.8 mg/ml)
- 15 ml 10X special 107 vitamin mix (below)
- 50 ml fetal bovine serum (GIBCO ok)
- 5 ml pen/strep/fungizone
phosphate buffer stock (250ml)
- 10g K2HPO4
- 6g KH2PO4
Dissolve in 250ml dH2O.
10X special 107 vitamin mix stock for Spironucleus/Monocercomonas:
To 495 ml dH2O add:
- 10 ml SOLN1 (1 mg/ml a-lipoic acid made in ethanol)
- 30 ml SOLN2 (0.4mg/ml vitamin B12)
- 10 ml SOLN3 (Tween 80)
- 0.5 ml SOLN4 (1000x stock = "SOLN4 1000X") or 5 ml "SOLN4 100X"
Stock solutions for making 10x special 107 vitamin mix:
SOLN 1 (1mg/ml a-lipoic acid):
Dissolve 100mg +- a-lipoic acid (d, L-6,8-thioctic acid (SIGMA T1395) in 100ml ethanol. (1mg/ml). Use 10 ml.
SOLN 2 (0.4mg/ml vitamin B12):
Dissolve 40mg vitamin B12 in 100ml dH2O. (0.4mg/ml). Use 30 ml.
SOLN 3 (Tween 80 liquid).
Use 10 ml Tween 80 liquid (undiluted) or dissolve 50g solid in 100ml ethanol and use 10 ml of this solution.
SOLN 4: (1000x vitamins).
1000X: (make 25mg/ml stocks, and add 1ml).
In about 800ml dH2O add
- 1.25g choline chloride
- 10g vitamin B12
- 1ml 25mg/ml a-tocopherol phosphae disodium salt.
- 1ml 25mg/ml d-biotin
- 1ml 25mg/ml calciferol
- 1ml 25mg/ml calcium d+pantohenate
- 1ml 25mg/ml folic acid
- 1ml 12.5mg/ml I-inositol
- 1ml 25mg/ml menadione
- 1ml 62.5mg/ml niacin
- 1ml 62.5mg/ml nicatinamide
- 1ml 12.5mg/ml p-aminobenzoic acid
- 1ml 62.5mg/ml pyridoxal Hcl
- 1ml 62.5mg/ml pyridoxine Hcl
- 1ml 25mg/ml riboflavin
- 1ml 25mg/ml thiamine Hcl
- 1ml 25mg/ml vitamin A (retinol).
Bring volume to 1L for 1000X stock solution.
