BG11 Medium
BG-11 Medium (modified by J.Acreman)
[edit]Reference: Rippka, R., J. Deruelles, J. Waterbury, M. Herdman and R. Stanier. 1979. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol. 111: 1-61.
This medium is used for successfully for most cyanobacteria. Vitamin B12 may be added for those species that require it. Use f/2 vitamin solution.
| Stock | Stock solution | mL/Litre |
|---|---|---|
| 1. NaNO3 (omitted for heterocystous species) |
150 g/L | 10 mL |
| 2. K2HPO4•3H2O or *K2HPO4 | 40 g/L or *30 g/L | 1 mL |
| 3. MgSO4•7H2O | 75 g/L | 1 mL |
| 4. CaCl2•2H2O | 36 g/L | 1 mL |
| 5. Citric Acid | 6 g/L | 1 mL |
| combined with Ferric Citrate | 6 g/L | |
| 6. Na2EDTA•2H2O | 1 g/L | 1 mL |
| 7. Na2CO3 | 20 g/L | 1 mL |
| 8. Trace Metal solution | See below | 1 mL |
| 9. F/2 vitamins | See below | 1 mL |
Adjusting the pH of the medium to approximately 7.5 will avoid heavy precipitation. (Initial pH is approximately 8.5.) When making solid media, you can add agar directly to medium. Omit NaNO3 for media used to culture heterocystous cyanobacteria e.g. Nostoc, Anabaena in order to maintain their ability to continue to produce the heterocysts.
Option: 0.5 g/L of HEPES buffer can be added to the final medium as a buffer. FeCl3 and EDTA added in a 1:1 ratio may be substituted.
Trace metal stock solution
[edit]| Substance | g/Litre |
|---|---|
| 1. H3BO3 | 2.86 g |
| 2. MnCl2•4H2O | 1.81 g |
| 3. ZnSO4•7H2O | 0.222 g |
| 4. Na2MoO4•2H2O | 0.390 g |
| 5. CuSO4•5H2O | 0.079 g |
| 6. Co(NO3)2•6H2O | 0.0494 g |
Add each substance in the order that they appear here and ensure that each is visually dissolved prior to adding the next on the list. After all substances have been added the stock solution should be topped up with distilled or MilliQ water to 1000 mL.
<br/>Medium<br/>ATCC® Medium 616: Medium BG11 for bluegreen algae<br/>Growth Conditions<br/>Temperature: 26°C<br/>Atmosphere: Aerobic under light intensity of 2,000~3,000 LUX
Propagation Procedure<br/>Incubate test tube cultures under above conditions upon receipt. DO NOT STORE IN A REFRIGERATOR. It is<br/>helpful to incubate test tubes in a slanted position to increase gas exchange in broth and to enhance<br/>exposure to light. Transfer culture to fresh media within one week of arrival, as follows:<br/>1. Withdraw 0.6 mL from the base of a broth culture where cells are concentrated, or harvest cells from a slant culture with 0.6 mL of #616 broth.<br/>2. Using this aliquot, inoculate one broth and one slant tube with 0.2 and 0.4 mL respectively.<br/>3. Incubate tubes at 26°C under 2000~3000 LUX light. Good growth, indicated by increased pigmentation in the broth or on the slant, should occur after one to two<br/>weeks of incubation. Examine cells microscopically to assure that they are intact and healthy. At this time,<br/>additional test tubes or flasks can be inoculated. A 5% inoculum is recommended (i.e. 5 mL of culture to 100<br/>mL fresh medium).<br/>To minimize change in a culture, it is recommended that a frozen seed stock be established from early<br/>passage cells. This may be accomplished by propagating the strain under ideal conditions, utilizing<br/>recommended medium, temperature and light. Prepare a concentrated cell suspension, after good growth is<br/>achieved. If grown in broth, pellet the cells by centrifugation. Decant the supernatant and resuspend the<br/>pellet in fresh #616 broth using 1/10 or less of the original volume. For slant cultures, wash cells off the agar<br/>surface with a minimal amount of #616 broth so that a concentrated cell suspension is attained. Add 50%<br/>DMSO solution to the concentrated cell suspension so that the final concentration of DMSO in the suspension<br/>is 5%. Dispense small aliquots (0.5 to 1 mL) of the suspension into small sterile vials. Store the vials at 50°<br/>C or below.<br/>When needed, remove vials from storage, thaw contents in a 37°C water bath and inoculate into<br/>recommended medium. A minimum of 0.2 mL of the thawed stock should be used to inoculate 5 mL of broth or<br/>1 agar slant. Cultures may require up to a month to recover from being frozen. Once grown, however,<br/>transfers will grow in a week.
Cyanothece 51142 were grown under 8 different conditions in 250-ml flasks on a shaker at 125 rpm at 30°C: in continuous light or 12 h light/12 h dark (50 μmol photons m−2 s−1), in BG11 medium (for Cyanothece 7822) with or without NaNO3 or in ASP2 medium (for Cyanothece 51142) with or without NaNO3, and with or without glycerol. Cultures were grown to mid-log phase, and 200 ml cells was harvested after 3 days at 4 h into either the light period (L4) or the dark period (D4), frozen immediately in liquid nitrogen, stored at −80°C, and later used for protein extraction (see below). Some of the cells cultivated in BG11-NF or ASP-NF medium (without NaNO3) were inoculated into a 6-liter bioreactor (Bioflo 3000; New Brunswick Scientific) and grown in 12 h light/12 h dark (100 μmol photons m−2 s−1 at 30°C) (20, 28). Measurements of nitrogenase and H2 have been highly reproducible in this bioreactor over many years, and samples were taken from the bioreactor at various times through the diurnal cycle and used for the following measurements.