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Spot Assays for Viability Analysis of Cyanobacteria

蓝藻细菌活性分析的斑点试验

AD Anja Katharina Dörrich
AW Annegret Wilde

发布: 2015年09月05日第5卷第17期 DOI: 10.21769/BioProtoc.1574 浏览次数: 12141

评审: Maria SinetovaElizabeth LibbyAmit Dey

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Cover of Microbiology, featuring study using the protocol.

Nov 2014

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Abstract

Cyanobacteria are prokaryotic organisms performing oxygenic photosynthesis. The cyanobacterium Synechocystis sp. PCC 6803 is a model organism for the study of photosynthesis, gene regulation and biotechnological applications because it is easy to manipulate genetically. Moreover, this cyanobacterium can grow photoautotrophically as well as chemoheterotrophically in the dark utilizing glucose. Microbiologists often use optical density measured with a spectrophotometer for the comparison of growth performance of different strains in liquid cultures. Because Synechocystis sp. PCC 6803 (especially motile strains) tend to form aggregates under stress conditions this method might be not suitable for evaluation of different strains under different growth conditions. In addition, many labs are not well equipped with standardized photobioreactors and illumination facilities to ensure reproducibility of growth curves. Here, we describe a highly reproducible spot assay for viability analysis of Cyanobacterial strains.

Keywords: Cyanobacteria (蓝藻) search Synechocystis 6803 (集胞藻6803) search Spot assay (斑点法) search Growth assay (生长测定) search

Materials and Reagents

  1. Synechocystis sp. PCC 6803 strain (wild type obtained from S. Shestakov, Moscow State University, Russia)
  2. CaCl2.2H2O (Carl Roth, catalog number: 5239.1 )
  3. Citric acid (Carl Roth, catalog number: X863.2 )
  4. NaNO3 (Carl Roth, catalog number: A136.2 )
  5. MgSO4.7H2O (Carl Roth, catalog number: P027.1 )
  6. Na2-EDTA (Carl Roth, catalog number: 8043.2 )
  7. H3BO3 (Carl Roth, catalog number: 6943.1 )
  8. MnCl2.4H2O (Carl Roth, catalog number: T881.3 )
  9. ZnSO4.7H2O (Carl Roth, catalog number: T884.1 )
  10. Na2MoO4.2H2O (Carl Roth, catalog number: 0274.1 )
  11. Co(NO3)2.6H2O (Carl Roth, catalog number: HN16.1 )
  12. CuSO4.5H2O (Carl Roth, catalog number: P024.1 )
  13. Na2CO3 (Carl Roth, catalog number: P028.2 )
  14. Ammonium iron (III) citrate (Carl Roth, catalog number: P027.1)
  15. K2HPO4.3H2O (Merck KGaA, Calbiochem®, catalog number: 105099 )
  16. 2-[(2-Hydroxy-1, 1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid (TES) (Carl Roth, catalog number: 9137.3 )
  17. Bacto Agar (BD Bioscience, catalog number: 214010 )
  18. Syringes (50 ml volume)
  19. Sterile syringe filters (Sarstedt Filtropur S 0.2 µm, catalog number: 83.1826.001 and 0.45 µm, catalog number: 83.1826 )
  20. Sterile 96-well microliter plates (i.e. VWR International, catalog number: 734-2328 )
  21. 100x BG11 medium (see Recipes)
  22. Trace metal mix (see Recipes)
  23. Stock Solutions for BG-11 medium (see Recipes)
  24. 1x BG11 (2x BG11) (see Recipes)
  25. BG11 agar (see Recipes)
  26. Na2S2O3 solution (see Recipes)
  27. 0.25 M Na2-EDTA (see Recipes)

Equipment

  1. Wide neck Erlenmeyer flasks with 100 ml volume (Carl Roth, catalog number: C146.1 )
  2. Light source (18 W/840) (Philips, model: MASTER TL-D Super 80 )
  3. Laboratory shaker (one-dimensional orbital motion) (Heidolph Instruments GmbH, catalog number: 036130180 )
  4. UV/Vis spectrophotometer (Shimadzu, model: UV-2401PC )
  5. Multichannel pipettes (8-channel pipette, 0.5-10 µl and 10-100 µl) (Starlab ErgoOne®, catalog number: S7108-0510 and S7108-1100 )
  6. Petri dishes with square shape (120 x 120 x 17 mm) (Greiner Bio-One GmbH, catalog number: 688102 )
  7. Transmitted light scanner with adjustable cover (i.e. Epson perfection v700 photo, a good camera can be used as an alternative)
  8. Laminar flow hood (i.e. Heraeus HeraSafe HS)
  9. LI-190R Quantum Sensor

Procedure

English
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文章信息

版权信息

© 2015 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Dörrich, A. K. and Wilde, A. (2015). Spot Assays for Viability Analysis of Cyanobacteria. Bio-protocol 5(17): e1574. DOI: 10.21769/BioProtoc.1574.

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分类

微生物学 > 微生物细胞生物学 > 细胞活力

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