AF-6 Medium

From Protists

Note: All nutrient stock solutions are stored at 4˚C in the walk-in and the trace metal and vitamin stocks are in the -20˚C (thaw these completely in warm water bath with parafilm around caps or at room temperature…invert several times before using).

1. Begin with 600mL of distilled water in the plastic 1L graduated cylinder. Continue the rest of the process in the sterile environment of the transfer hood.
2. Add 10mL of each 100X Nutrient Stock Solutions to the cylinder (using different sterile volumetric pipets for each solution).

Nutrient Stocks AF-6 Medium 100X Stock solutions

1. 1 NaNO3 14.00 mg 1.40 g/100mL
2. 2 NH4NO3 2.20 mg 0.22 g/100mL
3. 3 MgSO4 x 7H20 3.00 mg 0.30 g/100mL
4. 4 KH2PO4 1.00 mg 0.10 g/100mL
5. 5 CaCl2 x H20 1.00 mg 0.10 g/100mL
6. 6 K2HPO4 0.50 mg 0.05 g/100mL
7. 7 Fe-citrate 0.20 mg 0.02 g/100mL
8. 8 Citric acid 0.20 mg 0.02 g/100mL
9. 9 MES buffer* 40.00 mg 4.00 g/100mL

• MES = 2-morpholinoethanesulfonic acid monohydrate 98% from VWR (cat.# AAA16104-22)

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Add 5mL of the P IV Trace Metal Working Solution

P IV Trace Metal Working Solution: Start with 85mL of distilled water. Add 0.1 g of Na2EDTA x 2H2O, stirring continuously. Add 1mL of each of the FIVE trace metal 100X stock solutions. Bring final volume to 100mL with distilled water.

P IV Trace Metals Working Solution 100X Stock Solution FeCl3 x 6H20 19.60 mg 1.960 g/100mL MnCl2 x 4H20 3.60 mg 0.360 g/100mL ZnSO4 x 7H20 2.20 mg 0.220 g/100mL CoCl x H20 0.40 mg 0.040 g/100mL Na2MoO4 x 2H20 0.25 mg 0.025 g/100mL Na2EDTA x 2H20 100.00 mg 0.100 g crystals directly

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4. Add 10uL of each of the Vitamin Stocks.

Vitamin Stocks AF-6 Medium 100,000X Stock Solutions Biotin (vitamin H or B7) 0.2 ug 0.02 g/100mL† Thiamine HCl (vitamin B1) 1.0 ug 0.10 g/ 10mL Vitamin B6 (pyridoxine) 0.1 ug 0.01 g/ 10mL Vitamin B12 (cyanocobalamin) 0.1 ug 0.01 g/ 10mL‡

† Instead of 10uL of 0.02g/100mL stock solution, can add 20uL of 0.1g/L stock solution. ‡ Instead of 10uL of 0.01g/ 10mL stock solution, can add 10uL of 1.0g/L stock solution.

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5. Add 7.5mL of undiluted Carolina Biological Supply Soil Water Supernatant for each liter of AF-6. If diluted to the specified 50mL of Soil Water Supernatant into 1L of distilled water, then add 150mL to the AF-6 cylinder mix instead.

6. The solution will be acidic, adjust pH to 6.6 with 1N NaOH (=1M; found in chemical cabinet, use NaOH hydroxide pellets to make more solution if necessary). Use pH paper or pH meter. If overshot, then add a drop or two of concentrated HCl (acid storage under fume hood).

7. Bring final volume up to 1000mL with distilled water.

8. Aliquot media into media bottles and autoclave and then allow to cool before using.