TITLE: Preparation of defined medium for marine holozoans.
AUTHORS: Sebastian R Najle, Eduard Ocaña-Pallarès, Iñaki Ruiz-Trillo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">In order to study specific metabolic pathways in microorganisms is crucial to have complete control on the components of the growth medium. This protocol describes the components and mode of preparation of a minimum medium (low nutrients medium) of defined composition, suited for marine holozoans (named "modified L1 medium" - mL1). mL1 medium can be used for cultivation of different marine holozoans (i.e. Ichthyosporea and Corallochytrea), allowing to completely control the components (for example: carbon source, nitrogen source, etc). mL1 medium is based on the L1 medium recipe publically available at the National Center for Marine Algae and Microbiota (http://ncma.bigelow.org/algal-recipes).</div></div></div>

[STEPS]
?. [Preparation of stock solutions]
Stock solution A: Trace elements.Dissolve the following components in dH2O to bring a final volume of 1 liter:Na2EDTA • 2H2O ------------------------ 4.36 gFeCl3 • 6H2O ------------------------------ 3.15 gMnCl2 • 4H2O ----------------------------- 178.1 mgZnSO4 • 7H2O ----------------------------- 23 mgCoCl2 • 6H2O ------------------------------ 11.9 mgCuSO4 • 5H2O ----------------------------- 2.5 mgNa2MoO4 • 2H2O ------------------------ 19.9 mgH2SeO3 --------------------------------------- 1.29 mgNiSO4 • 6H2O ------------------------------ 2.63 mgNa3VO4 --------------------------------------- 1.84 mgK2CrO4 ---------------------------------------- 1.94 mgSterilize by filtration through 0.2 µm filter and store at 4 ºC.Stock solution B: Vitamins.Dissolve the following components in dH2O to bring a final volume of 1 liter:thiamine • HCl (vit. B1) -------------------- 200 mgbiotin (vit. H) ----------------------------------- 1 mgcyanocobalamin (vit. B12) -------------- 1 mg Sterilize by autoclaving and store at 4 ºC.Stock solution C: Phosphate.Dissolve 5 mg of NaH2PO4 • H2O in 1 liter of dH2O.Sterilize by filtration through 0.2 µm filter and store at 4 ºC.Na2EDTA • 2H2O ------------------------ 4.36 gFeCl3 • 6H2O ------------------------------ 3.15 gMnCl2 • 4H2O ----------------------------- 178.1 mgZnSO4 • 7H2O ----------------------------- 23 mgCoCl2 • 6H2O ------------------------------ 11.9 mgCuSO4 • 5H2O ----------------------------- 2.5 mgNa2MoO4 • 2H2O ------------------------ 19.9 mgH2SeO3 --------------------------------------- 1.29 mgNiSO4 • 6H2O ------------------------------ 2.63 mgNa3VO4 --------------------------------------- 1.84 mgK2CrO4 ---------------------------------------- 1.94 mgSterilize by filtration through 0.2 µm filter and store at 4 ºC.Stock solution B: Vitamins.Dissolve the following components in dH2O to bring a final volume of 1 liter:thiamine • HCl (vit. B1) -------------------- 200 mgbiotin (vit. H) ----------------------------------- 1 mgcyanocobalamin (vit. B12) -------------- 1 mg Sterilize by autoclaving and store at 4 ºC.Stock solution C: Phosphate.Dissolve 5 mg of NaH2PO4 • H2O in 1 liter of dH2O.Sterilize by filtration through 0.2 µm filter and store at 4 ºC.Stock solution B: Vitamins.Dissolve the following components in dH2O to bring a final volume of 1 liter:thiamine • HCl (vit. B1) -------------------- 200 mgbiotin (vit. H) ----------------------------------- 1 mgcyanocobalamin (vit. B12) -------------- 1 mg Sterilize by autoclaving and store at 4 ºC.Stock solution C: Phosphate.Dissolve 5 mg of NaH2PO4 • H2O in 1 liter of dH2O.Sterilize by filtration through 0.2 µm filter and store at 4 ºC.thiamine • HCl (vit. B1) -------------------- 200 mgbiotin (vit. H) ----------------------------------- 1 mgcyanocobalamin (vit. B12) -------------- 1 mgSterilize by autoclaving and store at 4 ºC.Stock solution C: Phosphate.Dissolve 5 mg of NaH2PO4 • H2O in 1 liter of dH2O.Sterilize by filtration through 0.2 µm filter and store at 4 ºC.Stock solution C: Phosphate.Dissolve 5 mg of NaH2PO4 • H2O in 1 liter of dH2O.Sterilize by filtration through 0.2 µm filter and store at 4 ºC.
It is recommended to prepare primary stock solutions of biotin (100 mg per liter) and cyanocobalamin (1 g per liter), and use, respectively, 10 and 1 ml to prepare 1 liter of stock solution B.
?. [Base mL1 medium preparation (working solution)]
Dissolve 35 g of marine salts (Instant Ocean) and 10 g of glucose in 900 ml of dH2O.Add 1 ml of Stock solution A (trace elements)Add 40 µl of Stock solution B (vitamins)Add 1 ml of Stock solution C (phosphate).Bring to a final volume of 1 liter with dH2O.Sterilize by filtration through 0.2 µm filter and store at 4 ºC.
IMPORTANT: The lack of nitrogen source in this medium is intentional. If the purpose is not to study nitrogen metabolism, it can be supplemented with 75 mg per liter of NaNO3 or NH4NO3.