Rapid detection of neutral lipid in green microalgae by flow cytometry in combination with Nile red staining—an improved technique
Open accessAbstract
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 (NaNO 3 )-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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In Chloroidium ellipsoideum and Chlorococcum infusionum, total lipid content is maximum in the absence of nitrogen, and increasing nitrate content decreases lipid content.
“It was reported that in Chloroidium ellipsoideum (CUH/Al/MW-189) and Chlorococcum infusionum (CUH/Al/MW-190), the total lipid content was maximum in the absence of nitrogen in the medium, and an increase in nitrate content resulted in a decrease in lipid content [38].”
From Nitrogen and phosphorus stress as a tool to induce lipid production in microalgae (2023) · cites this paper as [38]
The nitrate-stress results report maximum lipid accumulation without nitrate in both studied algae and decreasing accumulation as NaNO₃ increases. However, the collection-and-isolation section identifies voucher CUH/Al/MW-189 as Chlorella ellipsoidea, not Chloroidium ellipsoideum, and the Discussion describes the study’s detection method as measuring neutral lipid only, not total lipid content. No page numbers are available in our copy. (LLM, conf=0.88)
Experimental — accuracy not yet measuredFluorescence-based staining methods face a problem of uniform dye penetration into cells which must be facilitated by different solvents.
“The fluorescence-based staining methods fight a problem of the uniform dye penetration into cells which must be facilitated by different solvents [76][77][78].”
From On-Line Monitoring of Biological Parameters in Microalgal Bioprocesses Using Optical Methods (2022) · cites this paper as [78]
The Discussion (page unavailable) reports that Nile red staining of microalgae has been used with different organic solvents, and the Effect of DMSO concentration section reports stronger staining as DMSO concentration increased. But the Introduction says Nile red can penetrate microalgal cells easily; the paper does not establish a general problem of nonuniform dye penetration or that solvents are necessary to overcome it. (LLM, conf=0.82)
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Nitrogen and phosphorus stress as a tool to induce lipid production in microalgae
Maltsev, Y., Kulikovskiy, M., & Maltseva, S. (2023) · Microbial Cell Factories · doi:10.1186/s12934-023-02244-6 · Cited by 174 · see citing works
“It was reported that in Chloroidium ellipsoideum (CUH/ Al/MW-189) and Chlorococcum infusionum (CUH/Al/ MW-190), the total lipid content was maximum in the absence of nitrogen in the medium, and an increase in nitrate content resulted in a decrease in lipid content [38].”
BackgroundOn-Line Monitoring of Biological Parameters in Microalgal Bioprocesses Using Optical Methods
Havlik, I., Beutel, S., Scheper, T., & Reardon, K. F. (2022) · Energies · doi:10.3390/en15030875 · Cited by 73 · see citing works
Satpati, G. G., Gorain, P. C., & Pal, R. (2016) · Advances in Biology · doi:10.1155/2016/8712470 · Cited by 33 · see citing works
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Visualization and quantification of oil in single microalgal cells
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- Authors
- Satpati, G. G., & Pal, R.
- Affiliations
- University of Calcutta (IN)
- Corresponding author
- Pal, Ruma
- Venue
- Annals of Microbiology
- Published
- 2014-07-22
- Volume
- 65
- Issue
- 2
- Pages
- 937-949
- ISSN
- 0003-4649 (ISSN-L)
- 1590-4261
- 1869-2044
- OpenAlex
- W2037720284
- Cited by
- 54
- References
- 64
- Citations by year
- 2026: 4 · 2025: 7 · 2024: 3 · 2023: 4 · 2022: 6 · 2021: 2 · 2020: 8 · 2019: 5 · 2018: 4 · 2017: 2 · 2016: 7 · 2015: 2
- FWCI
- 1.21
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Annals of Microbiology (publishedVersion)
- Funders
- Department of Science and Technology, Ministry of Science and Technology, India
- Council of Scientific and Industrial Research, India
- University of Calcutta
- Topics
- Physical Sciences › Energy › Renewable Energy, Sustainability and the Environment › Algal biology and biofuel production
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Lipid metabolism and biosynthesis
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Cell Image Analysis Techniques
- Keywords
- Nile Red staining, green microalgae, Chlorella ellipsoidea, dimethyl sulfoxide, staining protocol optimization, nitrate depletion, lipid accumulation, flow cytometry, confocal microscopy
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