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Rapid detection of neutral lipid in green microalgae by flow cytometry in combination with Nile red staining—an improved technique

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Abstract

A staining protocol for rapid in situ detection of neutral lipid using flow cytometry in combination with Nile red staining was optimized. Staining efficiency was tested in terms of fluorescence intensity (% grandparent) in varied concentrations of Nile red and dimethyl sulfoxide (DMSO), with variable incubation period, temperature and pH level. The improved method was tested using two microalgae: Chlorella ellipsoidea and Chlorococcum infusionum. Maximum staining efficiency was recorded with a concentration of 5 μg mL−1 Nile red and 40 % DMSO in a 15-min incubation at 40 °C for both taxa (pH 6.5). The forward (FSC) and side scatter (SSC) two-dimensional dot plot showed highly scattered cells containing neutral lipid. The coefficient of variation, standard deviation, mean and median values were determined for quantification of neutral lipid. We also applied this modified method to detect the elevated level of neutral lipid in nitrate (NaNO3)-depleted cells; the efficiency of this technique was justified indicating a prominent 3- to 4-fold increase in neutral lipid in treated cells. Confocal images of stained cells also revealed accumulation of high levels of neutral lipid in treated microalgal cells.

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Acknowledgments

The above study was supported by the New Millennium Indian Technology Leadership Initiative-Council of scientific and Industrial Research (NMITLI-CSIR) and the Department of Science and Technology (DST), New Delhi (India). The authors are thankful to Dr. Sanjaya K. Mallick for his guidance in flow cytometric analysis. The authors are also grateful to the Center for Research in Nanoscience and Nanotechnology (CRNN), University of Calcutta, for instrumental facilities.

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  1. Phycology laboratory, Department of Botany, University of Calcutta, 35, Ballygunge Circular Road, Kolkata, 700019, West Bengal, India

    Gour Gopal Satpati & Ruma Pal

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  1. Gour Gopal Satpati
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  2. Ruma Pal
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Correspondence to Ruma Pal.

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Satpati, G.G., Pal, R. Rapid detection of neutral lipid in green microalgae by flow cytometry in combination with Nile red staining—an improved technique. Ann Microbiol 65, 937–949 (2015). https://doi.org/10.1007/s13213-014-0937-5

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  • DOI: https://doi.org/10.1007/s13213-014-0937-5

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