Diplomonads:Spironucleus Medium

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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.

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