A) Stock Solutions for A&A media
+Pi Stock Solution
2HPO4 · 3H2O |
28.0g/500ml |
or K2HPO4 anhydrous |
21.4g/500ml |
autoclave sterilize |
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-Pi Stock Solution
Make the following stock solutions and store at +4C. To assemble the -Pi stock, combined them
1:1:1:1 (and store at 4oC).
1) MgSO4 stock: MgSO4·7H2O |
20g/500ml,autoclave sterilize |
2) CaCl2 stock: CaCl2·2H2O |
6g/500ml, autoclave sterilize |
3) NaCl stock: NaCl |
20g/500ml, autoclave sterilize |
4) Microelement stock ( shake before each use): |
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Add in order listed. Wait for each to dissolve. Always shake before use. Do not autoclave sterilize.
Store at +4oC.
1) Double distilled water |
1090ml |
2) A&A Fe-EDTA solution (see below) |
160ml |
3) MnCl2·4H2O |
360mg |
4) MoO3 (molybdic acid) 85% purity |
36.0mg |
or Na2Mo4·2H2O (99% purity) |
61.1mg |
5) ZnSO4·7H2O |
44.0mg |
6) CuSO4·5H2O |
15.8mg |
7) H3BO3 (boric acid) |
572mg |
8) NH4VO3 (NH4+ metavanadate) |
4.6mg |
9) CoCl2·6H2O |
8.0mg |
A&A Fe-EDTA solution
1) Dissolve 5.2g KOH pellets in 186ml double distilled water. Add 20.4g Na2EDTA.2H2O
( Na2 ethylene dinitro tetra acetate 2H2O).
2) Dissolve 13.7g FeSO4.7H2O in 364ml double distilled water [or use Fe2(SO4)3.nH2O;
Ferric sulfate, n-hydrate]. This solution is cloudy, but will clear when mixed with #1.
3) Mix solutions 1 and 2.
4) Bubble Millipore filtered air ( 0.45mm) through the combined solution until the color changes
(darkens to brownish-orange). May take 4 minutes or 4 hours. Typically turns brown to burgandy
in about 2.5 hours.
5) Makes ~570ml FeEDTA soultion.
6) Store at +4oC
B) Prepartation of A&A for plates
Liters |
-Pi stock |
+Pi stock |
0.5 |
12.5ml |
3.13ml |
1.0 |
25ml |
6.25ml |
2.0 |
50ml |
12.5ml |
3.0 |
75ml |
18.8ml |
4.0 |
100ml |
25.0ml |
C) Preparation of A&A/4 for liquid media
Liters |
-Pi stock |
+Pi stock |
0.5 |
3.15ml |
1.55ml |
1.0 |
6.30ml |
3.10ml |
2.0 |
12.6ml |
6.20ml |
3.0 |
18.9ml |
9.30ml |
4.0 |
25.2ml |
12.4ml |
5.0 |
31.5ml |
15.5ml |
1. Shake the -Pi stock before measuring out the amount required
2. Add -Pi and +Pi stocks to double distilled water ( if they are mixed without water insoluble
precipitates will form.
3. Check pH before autoclaving; it should be 7.8. If it is less, the +Pi stock should be examined
and remade if needed.
4. For plates or slants, add 1.0% (10g/liter) noble agar.
5. Unless they are plain A&A plates, add a stir bar before autoclaving media containing agar to
facilitate mixing of later additions prior to pouring plates.
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