macrostomum work: different media

Media are very important for culturing both the algae and the worm cultures in our lab. Please download and read the general MacLab guidelines thoroughly and make sure that they are followed when attempting to culture Macrostomum and Nitzschia.

Here is a list of the different media we use for maintenance and work with Macrostomum.
When available, we provide the Sigma number between brackets. dH₂O = deionized H₂O

solution	recipe (adjust your quantities)		used for

ASW	for 1 liter: 32 gr HW Sea Salt 1 L dH₂O	Dissolve the sea salt in the distilled water. It helps to shake it vigorously with little distilled water and then add up to the one liter. Check that the salinity is 32 per mil, and add more salt or water if necessary: the weight of the salt changes after the container has been open because it absorbs humidity from the air. Therefore, keep your salt dry! Store in air-tight flask, and use within two-three weeks. Discard if there are changes in smell, transparency, or if a precipitate occurs. NOTE that when working with other Macrostomum species, the salinity may change. Change the amount of salt accordingly (e.g. for a 19 per mil salinity, add 19 gr of salt to 1000 ml of distilled water).	worm washing iso-female lines f/2

f/2 pdf	for 2 litres: 2 L ASW 1 mL Stock I 1 mL Stock II 1 mL Stock III 1 mL Stock IV 0.5 mL Vitamins	(aka Guilliard's medium) Prepare the Artificial Sea Water in an autoclavable bottle. Add the different stock solutions in order, shaking well after each addition. You can also add the Vitamins after autoclaving, by sterile-filtering (Thiamine is heat-unstable, but will denature even at room temperature after a few days). Autoclave for at least 48 minutes at 120 degrees Celsius. Note that a white precipitate occurs during autoclaving. Check the salinity (it may change during autoclaving) and correct if necessary. Before use, filter to a clean flask. Keep well covered (use an air-tight flask or cover with parafilm). Use within three weeks after filtering, and discard if there are changes in smell, transparency, or if a new precipitate occurs.	algae culturing

Stock I	NaNO₃ 150 g/L for 100 mL: 15 g NaNO₃(S5506) 100 ml dH₂O	Using an autoclavable bottle, dissolve the salt in the distilled water. Autoclave for at least 20 minutes at 120°C (248°F). Store cool and dark, and use within six months. Discard if there are changes in transparency, colour, or if a precipitate occurs. ATTENTION: TOXIC.	f2

Stock II	NaH₂PO4H₂O 10 g/L for 100* mL: 1 g NaH₂PO4*H₂O (S9638) 100 ml dH₂O	Using an autoclavable bottle, dissolve the salt in the distilled water. Autoclave for at least 20 minutes at 120°C (248°F). Store cool and dark, and use within six months. Discard if there are changes in transparency, colour, or if a precipitate occurs. ATTENTION: TOXIC.	f2

Stock III	Na₂SiO₃9H₂O 30 g/L for 100* mL: 3 g Na₂SiO₃*9H₂O (S4392) 100 mL dH₂O Note that this is half the concentration than in Andersen & al. Double if original recipe wanted.	Using an autoclavable bottle, dissolve the salt in the distilled water. Autoclave for at least 20 minutes at 120°C (248°F). A precipitate will likely occur. Store cool and dark, and use within six months. Discard if there are changes in transparency, colour, or if a new precipitate occurs. ATTENTION: TOXIC.	f2

Stock IV:	Trace metals for 100 mL: 0.88 g Na₂EDTA (E1644) 0.63 g FeCl₃6H₂O (F2877) 0.2 mL of each 1° stock 99 ml dH₂O	Into 80 mL of dH₂O, dissolve the EDTA entirely, then add the other components. Bring the final volume to 100 mL with dH₂O. Autoclave for at least 20 minutes at 120°C (248°F). Store cool and dark, and use within six months. Discard if there are changes in transparency, colour, or if a precipitate occurs. Primary stocks are prepared in 20 mL and stored at room temperature. ATTENTION: TOXIC.	f2

Trace metals: 1° stocks	for 20 mL each: 3.58 g MnCl₂4H₂O (M3634) 0.44 g ZnSO₄7H₂O (Z0251) 0.20 g CoCl₂6H₂O (C2644) 0.20 g CuSO₄5H₂O (C7631) 0.12g NaMoO₄*2H₂O (M1003)	Prepare each primary stock separatedly . Add the corresponding quantities of metals to 20 mL of deionized H₂O for each stock. Shake until dissolved. Store cool and dark, and use within six months. Discard if there are changes in transparency, colour, or if a precipitate occurs. ATTENTION: TOXIC.	f2

Vitamins	for 100 mL: 0.04 g ThiamineHCl (T3902) 1 mL* Biotin 1° Stock 0.2 mL B12 1° Stock dH₂O	Dissolve the Thiamine in about 80 mL of distilled H₂O. Add the vitamin primary stocks. Bring the final volume to 100 mL with dH₂O. Distribute in 0.5 mL aliquotes and deep-freeze. NOTE that the quantity from each 1° stock differ. ATTENTION: TOXIC.	f2

Biotin 1° stock	for 100 mL: 0.02 g Biotin (B4639) 100 mL dH₂O	Dissolve the Biotin in the distilled water. It will require some stirring. You may want to use a magnetic stirrer ATTENTION: TOXIC.	f2

B12 1° stock	for 20 mL: 0.02 g B12, (V6629) 20 mL dH₂O	Dissolve the vitamin B12 in the distilled water. ATTENTION: TOXIC.	f2

MgCl₂	for 1 liter: 71.4 gr MgCl (63064)2 1 L dH₂O	Dissolve the Magnesium Chloride in the distilled water. Store in air-tight flask for no longer than two months. Discard if there are changes in smell, transparency, or if a precipitate occurs. To anaesthetise worms living in full-strength sea water, mix MgCl2 with f2 in a ratio from 1:1 to 5:3 (do some trials to find the best results). Place worm in the anaesthetic for about 10 minutes (also do some trials for the time). The effects go away when substituting the solution for f2. NOTE that the optimal amount of MgCl2 will strongly depend on the salinity of your cultures. Species from lower salinities will require less MgCl2.	anaesthetic

agar	for 100 ml: 100 ml f/2 0.9 gr soft agar (A1296)	Dissolve the agar in the f/2, either using a microwave oven, or over inductive heat (a burner or a plate). If using an oven: Use a microwave-safe container and cover. Stirr frequently, and remove any time that it starts bubbling. Check that there are no particles left, heating again and again until the agar is completely dissolved. If using induction: Use a heat-proof flask, and gloves or a heat-proof flask-holder. Stirr continuously over the heat to prevent burning of the agar, and remove from heat any time it starts bubbling. Check that there are no particles left, heating again and again until the agar is completely dissolved.

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