Whether you’re working with cyanobacteria, any flavor of E. coli, or any organism for that matter, one thing remains constant: They all have to eat! The most common media we use in lab include Allen and Arnon (AA) for N. punctiforme, Luria Bertani (LB) broth and plates for DH5 E. coli, and the minimal medium M63 for use with BTH101 E. coli. Some cyanobacteria labs use BG11 or other media, but those won’t be listed here. The recipes can be found below:
Allen and Arnon Medium (AA)
(Taken from my lab protocols folder, which was taken from Dr. Meeks at UC Davis)
A) Stock Solutions for A&A media
+Pi Stock Solution
2HPO4 · 3H2O — 28.0 g / 500 mL
or K2HPO4 anhydrous — 21.4g/500ml
Autoclave sterilize
-Pi Stock Solution
Make the following stock solutions and store at +4C. To assemble the -Pi stock, combine 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):
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
Add in order listed. Wait for each to dissolve. Always shake before use. Do not autoclave sterilize. Store at +4oC.
| 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.
Luria Burtani (LB) Broth
Tryptone — 10 g / L
NaCl — 5 g / L
Yeast Extract — 5 g / L
Agar (if plates are desired) — 1.5%, 15 g / L
LB may be supplemented with antibiotics, IPTG, and X-Gal if needed. Typical concentrations include:
Ampicillin — 100 µg / mL
Kanamycin — 30 µg / mL
Streptomycin — 25–50 µg / mL
IPTG — usually 100 µM but this can vary depending on the application, we keep 100 mM stocks in the -20 °C freezer
X-Gal — usually 40 µg / mL from a 20 mg/ mL or 40 mg / mL stock