Abstract
This is a protocol for precise measurement of chlorophyll a and total carotenoid concentrations in cyanobacteria cells. Cellular chlorophyll concentration is one of the central physiological parameters, routinely followed in many research areas ranging from stress physiology to biotechnology. Carotenoids concentration is often related to cellular stress level; combined pigments assessment provides useful insight into cellular physiological state. The current protocol was established to minimize time and equipment requirements for the routine pigments analysis. It is important to note that this protocol is suitable only for cyanobacteria containing chlorophyll a, and is not designed for species containing other chlorophyll molecules.
Keywords: Pigments, Photosynthesis, Methanol extraction, Spectrophotometry
Materials and Reagents
Equipment
Procedure
Representative data
Figure 2. Representative measurements of chlorophyll a and carotenoids concentrations in cell culture of cyanobacterium Synechocystis sp. PCC 6803. Dense cyanobacteria culture (chlorophyll a: 22 µg/ml, carotenoids: 7 µg/ml) was gradually diluted by half up to pigments concentration 0.1 µg/ml. Each measurement was performed in triplicates; the error bars represent standard deviations.
Notes
Acknowledgments
The protocol was adopted from publication “On the dynamics and constraints of batch culture growth of the cyanobacterium Cyanothece sp. ATCC 51142” (Sinetova et al., 2012). T. Z. and J. C. were supported by the MEYS CR within CzechGlobe Centre, reg. no. CZ.1.05/1.1.00/02.0073 484, the National Sustainability Program I (NPU I), grant number LO1415 and by EC OP project, reg. no. CZ.1.07/2.3.00/20.0256. S. M. A. was supported by grant from Russian Scientific Foundation no. 14-24-00020).
References
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Dear 张捷 ,we have used this protocol exclusively for cyanobacteria. However, methanol can be used for chlorophyll extraction from plants. According to detailed study of Porra et al. (1989) methanol is more efficient solvent for chlorophyll than DMF or 80% acetone.Best regards,Tomas
Dear Allison,May I send you a protocol using ethanol instead methanolContact me at : claude.yepremian@mnhn.fr, I shall send you the pdf.
Dear Alison,I’ve never tried this protocol with diatoms, but based on experience with other algae which is in coincidence with data from thorough study of Ritchie (2006) methanol is a more efficient extractant of chlorophyll than 90% acetone, ethanol should work similarly as methanol. So for spectrophotometric quantitative estimation methanol/ethanol is a better choice. But chlorophylls are less stable in methanol and ethanol and tend to form allomers so for HPLC study it is preferable to use other solvents (acetone, chloroform etc.) and mechanical disruption will be needed in most cases.Best regards,Maria