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Method Article
A simple protocol to determine the neutral lipid content of algal cells using a Nile Red staining procedure is described. This time-saving technique offers an alternative to traditional gravimetric-based lipid quantification protocols. It has been designed for the specific application of monitoring bioprocess performance.
Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal fermentation processes and screening for new oil-rich strains requires a fast and reliable protocol for determination of intracellular lipid content. Current practices rely largely on gravimetric methods to determine oil content, techniques developed decades ago that are time consuming and require large sample volumes. In this paper, Nile Red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of Auxenochlorella protothecoides, a green alga. The method uses ethanol, a relatively mild solvent, to permeabilize the cell membrane before staining and a 96 well micro-plate to increase sample capacity during fluorescence intensity measurements. It has been designed with the specific application of monitoring bioprocess performance. Previously dried samples or live samples from a growing culture can be used in the assay.
Due to their ability to store lipid bodies under certain stress conditions, algae have received a great deal of attention in recent years as a potential renewable fuel source1,2. Neutral lipids can account for over 60% of the cell dry weight under appropriate growth conditions3. Yet the industry does not have a simple, clean, rapid, and reliable standardized protocol to quantitate lipid content of algal cells in order to properly monitor bioprocess performance, analyze cultures, and screen for new strains.
The Bligh-Dyer gravimetric method developed some 50 years ago remains among the most common techniques used today4,5. While this procedure is simple, reliable, and easy to carry out, it is time-consuming, necessitates large sample volumes, and makes use of toxic solvents. It is not practical for analyzing many samples from a fermentation run or screening for new oil-rich strains. Other methods have been developed, but usually require advanced equipment and have not been standardized6.
An alternative that has garnered a great deal of interest is the Nile Red stain. Nile Red, a dye that fluoresces preferentially in non-polar environments, has been used to identify or quantify lipid bodies in various organisms including nematodes7, yeast8, bacteria9, and algae10-19. Initial techniques involving Nile Red were mostly qualitative or semi-quantitative, combining the stain with single-cuvette spectrophotometry or flow cytometry. In addition, some classes of algae such as green algae have thick cell wells that are mostly impermeable to the dye, which limited the range of the technique10.
Recent improvements to the Nile Red staining method have been reported that bypass the initial shortcomings of the protocol10,11. Staining the cells in the presence of a carrier solvent such as DMSO10 or ethanol10,11 linearizes the relationship between oil content and absorbance, allowing for reliable quantitative measurements. The solvent helps permeabilize the cell membrane so that the Nile Red molecules can pass through. In addition, incorporating a spectrophotometer with micro-plate reading capabilities enables high throughput protocols suitable for quantitative analysis.
In this article we detail a simple method for measuring oil content of algal cells by staining cultures with Nile Red in the presence of ethanol, a mild solvent. In order to most accurately account for background noise in the measurements, a standard curve correlating fluorescence intensity to oil content is developed using algal cells of known oil composition. The method is adapted from previously published protocols10,11. By using a 96-well spectrophotometer, one is able to analyze the same amount of samples in an hour that would take days to monitor by gravimetric methods. Furthermore, by calibrating using representative samples of the desired algal species this method produces relatively precise measurements that are directly interpretable. There exist many protocols outlining methods of staining algae with Nile Red optimized for different strains and applications; the protocol presented here was originally developed by de la Hoz Siegler et al.11 for Auxenochlorella protothecoides, Chlorella vulgaris, Scenedesmus dimorphus, and Scenedesmus obliquus, although it is likely suitable for many more species and classes. It has been designed with the specific application of monitoring bioprocess performance and it works equally well for previously dried samples and wet samples from a growing culture.
1. Isolation of Dry Algal Biomass to be Used as Standards for Fluorescence Readings
2. Gravimetric Quantification of Neutral Lipids by Hexane Extraction (Adapted from Bligh and Dyer4)
3. Fluorometric Quantification of Neutral Lipids Using Nile Red (as Reported by de la Hoz Siegler et al.11)
NOTE: Only 10 µl of an algal suspension at 5 g/L is needed for the fluorescence reading. Generally, isolation of dry algal biomass from 1.5 ml of culture broth is more than sufficient. Also, the light intensity of the lamp in the spectrophotometer can degrade over time. It is recommended to include standards in every experiment to ensure that variations in the instrument do not add unnecessary error to the measurements.
4. Fluorescence Microscopy Technique
NOTE: The staining protocol described in section 3 is designed for quantitative analysis, but it can also be useful to provide visual representations of stain-based techniques for educational and illustrative purposes. To produce images of sample fluorescence one requires an optical microscope with traditional transmission and additional epifluorescence illumination sources. Excitation and emission light filters in the 530 nm (green) and 604 nm (red) range, respectively, are needed for the Nile Red stain as well as a microscope mounted camera with associated software. The images shown in this study (Figure 1) were acquired using a bright field microscope equipped with a camera and Monochrome to RGB converter unit. The procedure for producing Nile Red fluorescence images using these tools is outlined below:
Representative algal cells stained with Nile Red dye are depicted in Figure 1. Parts A and B of Figure 1 display images of A. protothecoides grown in excess nitrogen, leading to very low intracellular lipid accumulation. In parts C and D, samples of A. protothecoides grown under nitrogen limitation are shown. Under transmission illumination, the lipid bodies of the cell can be visualized with careful in...
The algae used in the standard curve must be the same species cultivated under the same experimental conditions as those being measured. Significant changes in media composition, cultivation technique, and staining protocol can affect the intensity of the fluorescence reading. Hexane extraction (described in sections 1 and 2) was used to determine the neutral lipid content of samples used in the standard curve. For accurate fluorescence intensity measurements, all samples must be analyzed at the same biomass concentratio...
The authors declare that they have no competing financial interests.
The authors would like to thank the Natural Sciences and Engineering Research Council of Canada for providing financial support for this project.
Name | Company | Catalog Number | Comments |
Dry Weight | |||
25 ml disposable pipettes | Fisher | 13-676-10K | |
Pipette Bulb | Fisher | 13-681-51 | |
40 ml Nalgene Teflon Centrifuge Tubes | Fisher | 05-562-16A | Teflon needed for hexane |
Weigh Dishes (polypropylene) | Fisher | 2-202B | |
1.5 ml micro-centrifuge tubes | Fisher | 05-408-129 | |
Centrifuge | Sorvall | RC6plus | |
Drying Oven (Fisher 625D) | Fisher | 13-254-2 | |
Storage vials | Fisher | 0337-4 | |
Bench-top microcentrifuge (Eppendorf 5415D) | Fisher | 05-40-100 | |
Gravimetric Quantification | |||
Porcelain Mortar (Coorstek) | Fisher | 12-961A | |
Porcelain Pestle (Coorstek) | Fisher | 12-961-5A | |
40 ml Centrifugation tubes (FEP) | Fisher | 05-562-16A | Could also use glass tubes |
Pasteur Glass Pipettes | Fisher | 13-678-20C | |
Aluminum weigh dishes | Fisher | 08-732-101 | |
Hexanes | Fisher | H292-4 | |
Fluorometric quantification of oil content | |||
Fluorescence multi-well plate reader | Thermo Lab Systems | Fluoroskan Ascent | |
Fluorescence reader software | Thermo Lab Systems | Ascent Software 2.6 | |
COSTAR 96 well plate with round bottom | Fisher | 06-443-2 | |
Nile Red | Sigma | N3013-100MG | |
Ethanol (Alcohol reagent grade) | Fisher | AC65109-0020 | |
Imaging Fluorescent cells | |||
Leica DMRXA2 (or equivalent) microscope | Leica | DMRXA2 | |
Microscope slides | Fisher | 12-550-15 | |
Microscope cover slips | Fisher | 12-541B | |
Camera | Qimaging | Retiga Ex | |
Imaging software | Qimaging | QCapture v.1.1.8 |
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