Oct 28, 2021

Public workspaceRecipe for standard BG-11 media

 Forked from Recipe for standard BG-11 media
DOI
https://dx.doi.org/10.17504/protocols.io.bzjup4nw
  • Anna Behle1,
  • Alice Pawlowski1
  • 1Institute for Synthetic Microbiology
  • Axmann Lab
Alice Pawlowski
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DOI: https://dx.doi.org/10.17504/protocols.io.bzjup4nw
Protocol Citation: Anna Behle, Alice Pawlowski 2021. Recipe for standard BG-11 media. protocols.io https://dx.doi.org/10.17504/protocols.io.bzjup4nw
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: October 28, 2021
Last Modified: October 28, 2021
Protocol Integer ID: 54612
Keywords: cyanobacteria, Synechocystis, Synechococcus, culture, media, medium, BG11, culturing freshwater cyanobacteria, freshwater cyanobacteria, suitable for marine cyanobacteria, marine cyanobacteria, properties of pure culture, microbiology, synechocystis sp
Abstract
Stanier RY, Deruelles J, Rippka R, Herdman M, Waterbury JB: Generic Assignments, Strain Histories and Properties of Pure Cultures of Cyanobacteria. Microbiology 1979, 111:1–61.
Recipes for standard and alternative BG11 for culturing freshwater cyanobacteria, such as Synechocystis sp. PCC 6803, as described.
Media is usually not suitable for marine cyanobacteria.
Final Concentration of Medium.
CaCl2*2 H2O 0.036 g/L
Citric acid 0.006 g/L
NaNO3 1.4958 g/L
MgSO4* 7 H2O 0.0749 g/L
0.25M Na2EDTA (pH 8) 0.0056 mL/L
Na2CO3 20 µg/ml
Fe(III) Ammonium citrate 6 µg/ml
K2HPO4 * 3H2O 30 µg/ml
TES Buffer (pH 8) 10 mM
H3BO3 2.86 mg/L
MnCl2 * 4 H2O 1.81 mg/L
ZnSO4 * 7 H2O 0.222 mg/L
Na2MoO4 * 2 H2O 0.390 mg/L
Co(NO3)2 *6 H2O 0.049 mg/L
(CuSO4 * 5 H2O 0.079 mg/L if required)
Guidelines
Always work under sterile conditions when handling sterile media or stocks. Work under the clean bench.
Troubleshooting
Safety warnings
Wear gloves when preparing stocks!
Heavy metals are toxic for the environment and need to be discarded accordingly.
Before start
For plates:
Thaw antibiotic stocks before pouring plates.
100 x BG11 stock:
  • CaCl2 2H2O (3.6 g · L-1)
  • Citric acid (0.6 g · L-1)
  • NaNO3 (149.58 g · L-1)
  • MgSO4 · 7 H2O (7.49 g · L-1)
  • 0.25 M Na2-EDTA, pH 8.0 (0.56 ml · L-1)

For BG11-N prepare a 50 X stock solution without NaNO3:

  • CaCl2 x 2 H2O: 1.8 g/L
  • Citric acid: 0.3 g/L
  • MgSO4 x 7 H2O: 3.75 g/L
  • 0.25 M Na2-EDTA, pH8.0: 0.28 ml/L
Supplemental stocks for standard media:
  • 1000x Na2CO3: 20 mg mL-1
  • 100x TES-buffer, pH 8.0 (1M), adjust with KOH
  • 1000x K2HPO4 x 3 H2O: 30 mg · mL-1
  • 1000x Fe(III) ammonium citrate (6 mg ·m L-1)
  • 5000x CuSO4 5 H2O (395 ng · mL-1) (sterilize using a filter)
Trace metal mix:
1000x concentration:
  • H3BO3 (2.86 g · L-1)
  • MnCl2 · 4 H2O (1.81 g · L-1)
  • ZnSO4 · 7 H2O (0.222 g · L-1)
  • Na2MoO4 · 2 H2O (0.390 g · L-1)
  • Co(NO3)2 · 6 H2O (0.049 g · L-1)
For BG11 lacking certain metals (e.g. for working with metal inducible promoters PpetE, PcoaT, PziaA etc., trace metal mix can be prepared lacking these chemicals and used instead of standard trace metal mix.
Standard 1x BG11
Fill 1 L bottle with 500 mL ultra pure water. Add stock solutions as shown below.
Stock solutionVolume
100x BG11 Stock10 mL
1000x Na2CO31 mL
1000x K2HPO4 x 3 H2O1 mL
100x TES-buffer10 mL
1000x Trace Metal Mix1 mL
Add ultra pure water to 1 L.
Autoclave.
After autoclaving, add 1 mL 1000x Fe(III) ammonium citrate.
Optional: After autoclaving, add 200 µL 5000x CuSO4
Standard 1x BG11 -N
Fill 1 L bottle with 500 mL ultra pure water. Add stock solutions as shown below.
AB
Stock solutionVolume
50x BG11 Stock -N20 mL
1000x Na2CO31 mL
1000x K2HPO4 x 3 H2O1 mL
100x TES-buffer10 mL
1000x Trace Metal Mix1 mL
Add ultra pure water to 1 L.
Autoclave.
After autoclaving, add 1 mL sterile 1000x Fe(III) ammonium citrate.
Optional: After autoclaving, add 200 µL sterile 5000x CuSO4
Standard 2x BG11 for agar plates
Fill 500 mL bottle with 250 mL ultra pure water. Add stock solutions as shown below.
Stock solutionVolume
100x BG11 Stock -N10 mL
1000x Na2CO31 mL
1000x K2HPO4 x 3 H2O1 mL
100x TES-buffer, pH = 8.010 mL
1000x Trace Metal Mix1 mL
Add ultra pure water to 500 mL.
Autoclave.
After autoclaving, add 1 mL sterile 1000x Fe(III) ammonium citrate.
Optional: After autoclaving, add 200 µL sterile 5000x CuSO4
BG11 plates
Prepare 1.5 % agar: Weigh 4.5 g Bacto Agar. Fill up to 300 mL. Autoclave.
Microwave agar until liquid. Let cool.
In a 50 mL Falcon, add 1 vol 2x BG11 and 1 vol liquid 1.5 % agar. (Note: Usually, one plate requires 30-40 mL total volume.)
When mixture is hand warm, add appropriate antibiotics, if required. Quickly pour plate, avoiding air bubbles.