A) Stock Solutions for A&A media

+Pi Stock Solution

-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).
        

Add in order listed.  Wait for each to dissolve.  Always shake before use.  Do not autoclave sterilize.
Store at +4oC.

           
     
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

C) Preparation of A&A/4 for liquid media

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.