Jul 01, 2026
Allen and Arnon Medium diluted 4-fold (AA/4)
- 1UCCS
- Risser Lab (N. punctiforme)
Protocol Citation: Douglas Risser 2026. Allen and Arnon Medium diluted 4-fold (AA/4). protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3mrnql25/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 22, 2026
Last Modified: July 01, 2026
Protocol Integer ID: 317792
Abstract
This recipe describes Allen and Arnon medium diluted by fourfold (AA/4), adapted from Allen and Arnon (1955). AA/4 is routinely used for cultivation of Nostoc punctiforme in liquid or solid media.
Materials
- 2HPO₄ • 3H₂O
- K₂HPO₄ anhydrous
- MgSO₄•7H₂O
- CaCl₂•2H₂O
- NaCl
- Double distilled water
- MnCl₂•4H₂O: 360mg
- MoO₃ (molybdic acid) 85% purity or Na₂MoO₄•2H₂O (99% purity)
- ZnSO₄•7H₂O
- H₃BO₃ (boric acid)
- NH₄VO₃ (NH₄ metavanadate)
- CoCl₂•6H₂O
- KOH pellets
- Na₂EDTA•2H₂O (Na₂ ethylene dinitro tetra acetate 2H₂O).
- FeSO₄•7H₂O [or use Fe₂(SO₄)₃•nH₂O; Ferric sulfate, n-hydrate]
+Pi Stock Solution
Prepare the +Pi Stock Solution by dissolving 28.0g of 2HPO₄ • 3H₂O or 21.4g of K₂HPO₄ anhydrous in 500ml of water. Autoclave sterilize the solution.
-Pi Stock Solution
To prepare the -Pi Stock Solution, make the following stock solutions and store at +4°C. Combine them in a 1:1:1:1 ratio and store at 4°C.
1) MgSO₄ stock: Dissolve 20g of MgSO₄•7H₂O in 500ml of water and autoclave sterilize.
2) CaCl₂ stock: Dissolve 6g of CaCl₂•2H₂O in 500ml of water and autoclave sterilize.
3) NaCl stock: Dissolve 20g of NaCl in 500ml of water and autoclave sterilize.
4) Microelement stock (shake before each use) - See below for details
Microelement Stock Solution
Add the following in order, waiting for each to dissolve. Always shake before use. Do not autoclave sterilize. Store at +4°C.
Double distilled water: 1090ml
AA/4 Fe-EDTA solution: 160ml (see below)
MnCl₂•4H₂O: 360mg
MoO₃ (molybdic acid) 85% purity: 36.0mg or Na₂MoO₄•2H₂O (99% purity): 61.1mg
ZnSO₄•7H₂O: 44.0mg
CuSO₄•5H₂O: 15.8mg
H₃BO₃ (boric acid): 572mg
NH₄VO₃ (NH₄ metavanadate): 4.6mg
CoCl₂•6H₂O: 8.0mg
AA/4 Fe-EDTA solution (For microelements stock solution)
Dissolve 5.2g KOH pellets in 186ml double distilled water. Add 20.4g Na₂EDTA•2H₂O (Na₂ ethylene dinitro tetra acetate 2H₂O).
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 solution 1.
Mix solutions 1 and 2.
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 burgundy in about 2.5 hours.
Makes ~570ml FeEDTA solution.
Store at +4°C.
Preparation of AA/4 media
Refer to the table for the volumes of -Pi and +Pi stock solutions needed for different volumes of 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
Shake the -Pi stock before measuring out the amount required.
Add -Pi and +Pi stocks to double distilled water (if they are mixed without water, insoluble precipitates will form).
Check pH before autoclaving; it should be 7.8. If it is less, the +Pi stock should be examined and remade if needed.
Autoclave to sterilize
For plates or slants, add 1.0% (10g/liter) noble agar before autoclaving.
Unless they are plain AA/4 plates, add a stir bar before autoclaving media containing agar to facilitate mixing of later additions prior to pouring plates.
To supplement medium with a source of fixed nitrogen, prepare a stock solution of 250 mM NH4Cl, 500 mM MOPS, adjust pH to 7.8 with NaOH, filter sterilize and store at 4°C. Add to media after autoclaving to final concentration of 2.5 mM NH4Cl, 5 mM MOPS.
Protocol references
M. B. Allen, Daniel I. Arnon, Studies on Nitrogen-Fixing Blue-Green
Algae. I. Growth and Nitrogen Fixation by Anabaena Cylindrica Lemm, Plant Physiology, Volume 30, Issue 4, July 1955, Pages 366–372, https://doi.org/10.1104/pp.30.4.366