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| - | AF-6 Medium | + | ==AF-6 Medium Notes== |
| | + | Use this when you culture most all of the phototrophic protists. It is a defined media, meaning that all of its components are known in exact quantity. |
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| - | 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).
| + | ==View the Recipe== |
| | + | *Click on the link below: |
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| - | 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.
| + | http://editthis.info/images/protists/3/33/AF-6.doc |
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| - | 2. Add 10mL of each 100X Nutrient Stock Solutions to the cylinder (using different sterile volumetric pipets for each solution).
| + | ==See Also== |
| - | | + | *[[Recipes]] |
| - | Nutrient Stocks AF-6 Medium 100X Stock solutions
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| - | #1 NaNO3 14.00 mg 1.40 g/100mL
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| - | #2 NH4NO3 2.20 mg 0.22 g/100mL
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| - | #3 MgSO4 x 7H20 3.00 mg 0.30 g/100mL
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| - | #4 KH2PO4 1.00 mg 0.10 g/100mL
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| - | #5 CaCl2 x H20 1.00 mg 0.10 g/100mL
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| - | #6 K2HPO4 0.50 mg 0.05 g/100mL
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| - | #7 Fe-citrate 0.20 mg 0.02 g/100mL
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| - | #8 Citric acid 0.20 mg 0.02 g/100mL
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| - | #9 MES buffer* 40.00 mg 4.00 g/100mL
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| - | *MES = 2-morpholinoethanesulfonic acid monohydrate 98% from VWR (cat.# AAA16104-22)
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| - | _____________________________________________________________________________
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| - | 3. Add 5mL of the P IV Trace Metal Working Solution.
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| - | 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.
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| - | P IV Trace Metals Working Solution 100X Stock Solution
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| - | FeCl3 x 6H20 19.60 mg 1.960 g/100mL
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| - | MnCl2 x 4H20 3.60 mg 0.360 g/100mL
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| - | ZnSO4 x 7H20 2.20 mg 0.220 g/100mL
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| - | CoCl x H20 0.40 mg 0.040 g/100mL
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| - | Na2MoO4 x 2H20 0.25 mg 0.025 g/100mL
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| - | Na2EDTA x 2H20 100.00 mg 0.100 g crystals directly
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| - | _____________________________________________________________________________
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| - | 4. Add 10uL of each of the Vitamin Stocks.
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| - | Vitamin Stocks AF-6 Medium 100,000X Stock Solutions
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| - | Biotin (vitamin H or B7) 0.2 ug 0.02 g/100mL†
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| - | Thiamine HCl (vitamin B1) 1.0 ug 0.10 g/ 10mL
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| - | Vitamin B6 (pyridoxine) 0.1 ug 0.01 g/ 10mL
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| - | Vitamin B12 (cyanocobalamin) 0.1 ug 0.01 g/ 10mL‡
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| - | † Instead of 10uL of 0.02g/100mL stock solution, can add 20uL of 0.1g/L stock solution.
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| - | ‡ Instead of 10uL of 0.01g/ 10mL stock solution, can add 10uL of 1.0g/L stock solution.
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| - | _____________________________________________________________________________
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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.
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| - | 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).
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| - | 7. Bring final volume up to 1000mL with distilled water.
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| - | 8. Aliquot media into media bottles and autoclave and then allow to cool before using.
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Use this when you culture most all of the phototrophic protists. It is a defined media, meaning that all of its components are known in exact quantity.
