Journal of Microbiological Methods 56 (2004) 331 338 www.elsevier.

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Rapid estimation of lipids in oleaginous fungi and yeasts using Nile red fluorescence
K. Kimura *, M. Yamaoka, Y. Kamisaka
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, AIST Tsukuba Central 6, 1-1 Higashi 1-Chome, Tsukuba, Ibaraki, 305-8655 Japan Received 1 September 2003; received in revised form 31 October 2003; accepted 31 October 2003

Abstract A rapid estimation method of the intracellular lipid content in microorganisms using a fluorescent probe, Nile red, was established by optimization of the Nile red staining and data processing. The protocol was designed to be applicable to a wide range of microorganisms and culture conditions. In the optimized procedure, cells diluted with buffer were stained with 0.24 0.47 Ag/ml of Nile red for 5 min, and the fluorescent emission spectra in the wavelength region of 400 to 700 nm excited at 488 nm were acquired before and after the Nile red addition. The fluorescence intensity corresponding to the intracellular lipid amount was determined at the peak of the corrected spectrum. The value showed a linear relation with the lipid content of various oleaginous fungi and yeasts measured by the conventional method. The relative intensities against the unit lipid amounts were almost similar except for one yeast. For the application to mycelia forming various types of pellets, a simple and easy pretreatment of shaking with glass beads for 5 10 min was added to the protocol. The established method was applicable to estimate the lipid content of a wide range of microorganism cultures containing 2 5000 Ag-lipid/ml-broth. D 2003 Elsevier B.V. All rights reserved.
Keywords: Lipids; Oleaginous fungi and yeasts; Nile red fluorescence

1. Introduction Some oleaginous microorganisms accumulate lipids in the cell at more than 50% of the dry cell weight. They have the possibility to be commercial oil producers for food and energy resources (Ratledge, 1989; Ratledge and Wynn, 2002). Since accumulated lipids are localized in the intracellular organelle, the socalled lipid body (Murphy and Vance, 1999), the lipid body formation and maturation are important
* Corresponding author. Tel.: +81-298-861-6666; fax: +81-298861-6171. E-mail address: kykimura@ni.aist.go.jp (K. Kimura). 0167-7012/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2003.10.018

processes for the lipid production in oleaginous microorganisms. How lipid bodies are formed is crucial to the design of microbial oils. We have studied lipid transport pathways into lipid bodies of the oleaginous Mortierella fungus using fluorescent lipid analogues (Kamisaka et al., 1999; Kamisaka and Noda, 2001). Further studies are intended to screen compounds or mutants affecting the lipid body formation. For these screening procedures, a rapid and simple method to determine lipids is indispensable. Conventional methods of lipid determination have many complicated steps, i.e., extraction, purification, concentration, and determination, which are time-consuming. A spectrophotometric method using Sudan

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black B (Thakur et al., 1989) and fluorescence spectrometric methods using Luminor 490PT (Pomoshchnikova et al., 1981) and Nile red (Cooksey et al., 1987; Lee et al., 1998; Cole et al., 1990) were reported to determine the lipid content of yeasts, algae, and ciliates, all of which were dispersed cells without forming large aggregations. For oleaginous fungi, Luminor 490PT (Pomoshchnikova et al., 1983) and Nile blue (Vijayalakshmi et al., 2003) were used as the only examples. Among several dyes, Nile red seems preferable for the intracellular lipid determination but has not been widely used. Although Nile red can stain most lipids, its fluorescence character varies depending on the situation of the lipids. The maximum wavelength of Nile red emission with neutral lipids is shorter than that with polar lipids and the former intensity is higher than the latter (Greenspan and Fowler, 1985). The fluorescence intensity of lipids composed of unsaturated fatty acids is stronger than that of the saturated fatty acids (Fowler et al., 1987). The emission maximum shifts to a shorter wavelength depending on the hydrophobicity of the lipid molecules and their surroundings (Greenspan and Fowler, 1985). Previous studies (Cooksey et al., 1987; Lee et al., 1998; Vijayalakshmi et al., 2003) used special conditions for their microbes so that the fluorescence was measured at the fixed emission wavelength. They were difficult to apply to various microorganisms and their culture conditions without optimization. We have overcome these problems in order to obtain a rapid lipid determination method with a wide range of applicability. In this study, the measurement condition for the lipid determination using fluorescence by Nile red was examined in detail and optimized. The established method can be applicable to various oleaginous microorganisms including mycelia which produce various types of pellets.

Japan) and maintained on YM agar (glucose 10 g/l, peptone 5 g/l, yeast extract 3 g/l, malt extract 3 g/l, agar 20 g/l). All the microorganisms were precultured in YM broth (glucose 10 g/l, peptone 5 g/l, yeast extract 3 g/l, malt extract 3 g/l) and cultured for observation of the lipid accumulation in medium A (glucose 30 g/l, yeast extract 1.5 g/l, NH4Cl 0.5 g/l, KH2PO4 7.0 g/l, Na 2 HPO 4 12H 2 O 5.0 g/l, MgSO 4 7H 2 O 1.5 g/l, FeCl 3 6H 2 O 0.08 g/l, ZnSO 4 7H 2 O 0.01 g/l, CaCl 2 2H 2 O 0.1 g/l, MnSO 4 5H 2 O 0.1 mg/l, CuSO45H2O 0.1 mg/l, Co(NO3)26H2O 0.1 mg/l; pH 5.5 (Suutari et al., 1993)). For cultivation of the yeasts, 1 ml of the 2-day preculture in YM broth was inoculated to 100 ml of medium A in a 500 ml Erlenmeyer flask with three baffles. For cultivation of the Mortierella fungi, a 2-day preculture in YM broth was pretreated by shaking with the same volume of about 3mm diameter glass beads for 5 min to provide a homogeneous suspension for inoculation. Ten milliliters of medium A broth with a 2% inoculation was pipetted into a 50-ml plastic conical tube with a ventilation plug. The entire broth in a tube was harvested at once for the sampling. Flasks and tubes were shaken in a rotary shaker at 120 rpm at 27 jC. Lipid accumulation profiles of oleaginous yeasts and fungi were demonstrated as average data of two batches. 2.2. Nile red staining and fluorescence spectrometry Nile red (9-diethylamino-5H-benzo[a]phenoxa]phenoxazine-5-one) obtained from the Aldrich Chemical (Milwaukee, USA) was dissolved in 0.1 mg/ml with acetone. Fluorescence spectra were measured with the spectrofluorometer FP-750 with PC control equipment (JASCO, Tokyo, Japan). The acquiring and processing of the data were done using the PC software (JASCO). In order to eliminate the effect of autofluorescence of the cells and scattering of the cell suspension, the fluorescence spectra were obtained as follows. A 100-Al aliquot of the culture broth of was mixed with 2 ml of 10 mM potassium phosphate buffer with 0.15M KCl (pH 7.0; PBS) in a 10-mm acryl cuvette. The spectrum in a wavelength region of 400 to 700 nm for the cell suspension without Nile red was recorded. The 10-Al Nile red solution was then added and mixed well. Five minutes later, the spectrum in the same wavelength region was recorded again. The cell suspension in a cuvette was mixed well by an upside-

2. Materials and methods 2.1. Organism and culture Oleaginous yeasts (Lipomyces starkeyi IFO-10381, Rhodosporidium toruloides IFO-0559, Cryptococcus curvatus IFO-1159) and fungi (Mortierella isabellina IFO-7884, Mortierella nana IFO-8794, Mortierella ramanniana var. angulispora IFO-8187) were obtained from the Institute for Fermentation (Osaka,

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down inversion just before measurement in order to avoid cell sedimentation. Spectra were corrected by subtracting the spectra before and after the Nile red addition using the PC software. The fluorescence intensity corresponding to the lipid amount was determined at the peak of the corrected spectrum (see Fig. 1). 2.3. Time course of fluorescence development and fading Spectra of the cell suspension were recorded at 1, 5, 10, 15, and 20 min after the Nile red addition in various amounts (2 100 Al). To avoid cell sedimentation, the cell suspension was well mixed just before every data acquisition. To minimize emission fading, irradiation of the cell suspension was done only during the data acquisition.

2.4. Microscopy Microscopic photographs were taken with a Nikon E600 microscope (Nikon, Tokyo, Japan) equipped with a color CCD digital camera (DP12, Olympus, Tokyo, Japan) using a 450 490-nm excitation filter, a 505-nm diachronic mirror and a 520-nm barrier filter with 40 or 60 objective lens. The obtained digital color pictures were corrected to grey scale figures by PC software. 2.5. Lipid analysis The total lipid concentration was determined by gas chromatographic analysis of the total fatty acids directly transmethylesterified from dried cells (Kumon et al., 2002). One milliliter of 10% methanolic HCl and 0.5 ml methylene chloride were added to the dried cells and kept at 60 jC for 3 h for the direct methylesterification. The reaction was stopped by the addition of 2 ml saturated NaCl solution and 1 ml hexane. The resultant methyl esters recovered in the hexane layer were then applied to a gas chromatograph (GC-17-A; Shimadzu, Kyoto, Japan) equipped with a TC-70 capillary column (30 m 0.25 mm i.d., GL Science, Tokyo, Japan) under temperature programming (180 220 jC at 4 jC/min increments). Peanut oil (Nacalai Tesque, Kyoto, Japan) was transmethylesterified and used as the reference material. 2.6. Other Analytical Methods The glucose concentration was determined using Glucose CII Test Wako (Wako, Osaka, Japan). Cell growth was based on the dry cell weight. Cells in the 1 3-ml culture broth were collected after washing with the same volume of water by centrifugation, and weighed after being dried at 105 jC overnight.

Fig. 1. Emission spectra of Nile red-stained cell suspension. Culture broth (100 Al) of L. starkeyi in log phase was mixed with 2 ml of PBS. Before (: : :) and after (- - -) the addition of 10 Al Nile red solution, emission spectra of the cell suspension were recorded with excitation wavelength at 488 nm (A) and 552 nm (B). The corrected spectra () were derived by subtracting the spectra before and after the Nile red addition. The fluorescence intensity ( X ) was derived at the peak of the corrected spectrum. The emission spectra were recorded at 5 min after the Nile red addition.

3. Results and discussion 3.1. Optimization of Nile red staining and fluorescence data collection To develop a widely applicable method, optimization of the Nile red staining for fluorescence measurement of intracellular lipids was studied. Lipid

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detection by Nile red has been commonly measured with excitation at 480 490 and at 510 560 nm. The former target is neutral lipids to fluoresce and the latter target is polar lipids (Greenspan et al., 1985). Emission spectra of typical excitation wavelengths at 488 and at 552 nm are compared in Fig. 1. Both emission spectra, corrected as described in the Materials and methods, showed similar peaks at 565 575 nm, but had different peak fluorescence intensities. Fluorescence with 488-nm excitation showed a higher intensity than with 522-nm excitation. In addition, the spectrum with the 488-nm excitation was much easier and more reproducible to separate the effect of scattering by excitation than the spectrum with 552-nm excitation. In all the tested microorganisms, the peak wavelengths of emission varied between 565 and 585 nm. Therefore, we chose the excitation wavelength of 488 nm and observed the emission of 565 585 nm as lipids fluorescence. The fluorescence of Nile red by itself without lipids existed at around 600 605nm with a slight peak, but was negligible. Nile red is nearly insoluble in water and its fluorescence immediately quenches in aqueous solution (Sackett and Wolff, 1987). Based on these results, removal of excess Nile red from the suspension after the staining is not necessary. Fig. 2a shows the time course of the fluorescence of the Lipomyces cell suspension with different Nile red concentrations. Upon the microscopic observation, fluorescence of the Nile red stained cells rapidly faded in the first few seconds of irradiation. For the spectrometric measurement, fluorescence of the cell suspension did not fade so rapidly. At every Nile red concentration, full-staining was achieved between 1 and 5 min. After 5 min, fluorescence fading was observed especially at high Nile red concentrations above 2.3 Ag/ml. Therefore, we chose 5 min as the most efficient staining period. The fluorescence intensity increased with an increase in the Nile red concentration up to 2.3 Ag/ml, but not linearly with the Nile red concentration. This means that an increase in the Nile red concentration can amplify the fluorescence intensity, but its amplification will not linearly increase with the Nile red concentration. Fig. 2b shows the fluorescence with various amount of culture broth at 5 min after the Nile red addition. The fluorescence intensity increased with an increase in culture broth amount; the fluorescence

Fig. 2. Fluorescence of Lipomyces cell broth stained with Nile red with excitation at 488 nm. Into the suspension of 2 ml PBS and culture broth of L. starkeyi in stationary phase, 0.1 mg/ml Nile red solution in acetone was added. (a) Time course of staining of 100 Al culture broth with Nile red in various concentrations. Spectra were recorded at time intervals and the fluorescence with time was shown as intensity of the corrected spectrum peak. (b) Fluorescence with various amount of culture broth of Lipomyces at 5 min. Final Nile red concentration in the suspension was as follows: o, Nile red 0.095 Ag/ml; 4, 0.24 Ag/ml; 5, 0.47 Ag/ml; ., 0.94 Ag/ml; E, 2.3 Ag/ml; n, 4.5 Ag/ml. Values are average of two determinations. (c) Variations of emissions spectra of the same culture broth of Lipomyces in early stationary phase at 5 min after 10 Al Nile red addition. Average fluorescence intensity at the peak wavelength (569 nm) was 120.1 unit with S.D. of 2.6 unit for n = 12.

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intensity of suspension with more than 100 Al culture broth did not linearly increase with lipid amount in cell suspension. Usually we used 100 Al culture broth throughout the one-batch culture for the relative comparison. The minimum lipid amount by this protocol was 20 Ag in a 2.1-ml final suspension at 0.095 2.3 Ag/ ml Nile red for Lipomyces cells (data not shown). Sensitivity for the low lipid concentration was not enhanced by an increase in the Nile red concentration. The fluorescence fading became remarkable for high Nile red concentrations above 2.3 Ag/ml with time. For the various lipid concentrations tested, the Nile red concentration of 0.24 0.47 Ag/ml showed reproducible fluorescence since higher Nile red concentrations could produce higher experimental errors.

As a result, the rapid estimation method of intracellular lipids was optimized as follows. Nile red (0.1 mg/ml, 10 Al) was added to the cell suspension, the mixture of 50 100 Al culture broth and 2 ml PBS, and held for 5 min after mixing well. Spectra in the emission wavelength 400 700 nm with excitation at 488 nm were recorded before the Nile red addition and 5 min after the addition. The corrected fluorescence spectrum was derived by subtracting the spectra before and after the Nile red addition. The fluorescence intensity at the peak of the corrected spectrum corresponded to the intracellular lipid amount. With the established method described above, the emission spectra of the culture broth was analyzed repeatedly in Fig. 2c; it shows the good reproducibility.

Fig. 3. Time course of lipid accumulation of oleaginous fungi. (a) L. starkeyi IFO-10381; (b) R. toruloides IFO-0559; (c) C. curvatus IFO-1159; (d) M. rammanniana var. angulispora IFO-8187; (e) M. isabellina IFO-7884; (f) M. nana IFO-8794. n , Glucose concentration in the culture broth; E , dry cell weight (DCW); . , lipid concentration; x , fluorescence intensity. Lipid concentration and fluorescence intensity were the calculated values originally contained in 1 ml of culture broth. Values are average of two determinations.

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3.2. Rapid estimation of lipid concentration in the culture of various oleaginous fungi We then investigated whether the established method was applicable to various oleaginous yeasts and fungi having different cell and lipid body morphologies. The time courses of the cultivation with lipid accumulation of yeasts and fungi are shown in Fig. 3. The lipid concentration of the culture broth was determined as the total fatty acids in 1 ml culture broth, as described in the Materials and methods (Fig. 3a c). In oleaginous yeasts, the total lipid concentration and fluorescence intensity increased with an

increase in the dry cell weight, following the glucose consumption from the culture broth. Lipid bodies, in which lipids accumulate in oleaginous microorganisms, have different shapes and development depending on the species and culture conditions. Fig. 4 is microscopic photographs of the tested microorganisms at the beginning of the lipid accumulation. L. starkeyi (Fig. 4a) produced one or two large lipid bodies in the spherical cells, as previously shown (Naganuma et al., 1986). Small lipid bodies with less than a 0.5-Am diameter in the cell with a 2 3-Am diameter at the beginning of the culture increased in size to more than 5 Am after a 100-h cultivation. The

Fig. 4. Microscopic photographs of differential interference contast ( 1) and Nile red fluorescence ( 2) of oleaginous fungi at the beginning of lipid accumulation. (a) L. starkeyi IFO-10381; (b) R. toruloides IFO-0559; (c) C. curvatus IFO-1159; (d) M. rammanniana var. angulispora IFO-8187; (e) M. isabellina IFO-7884; (f) M. nana IFO-8794. In the fluorescence photograph, the lipid bodies which are fluoresced by Nile red are reflected as white circlets in the field identical to the differential interference contrast photograph. Bar indicates 10 Am.

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cell expanded in diameter followed by growth of the lipid body. The largest lipid body reaching to 10 Am in diameter was produced in cells with more than a 12Am diameter. R. toruloides (Fig. 4b) produced two to four lipid bodies with 1 3-Am diameter in the ellipsoidal cells. Small cells with small lipid bodies of less than 0.5-Am diameter became bigger cells with two to three lipid bodies up to 2 3 Am and some smaller ones around 1 Am. C. curvatus (Fig. 4c) had initially many small lipid bodies with less than 0.5 Am in long rod-shaped cells. After a 140-h cultivation, about half of the cells transformed into the enlarged spherical or ellipsoidal cells contained one or two 2 3-Am lipid bodies and many smaller ones of less than 1 Am. Fig. 3d f shows the time course of the cultivation of mycelia which made various types of pellets depending on the species and its cultivation condition. For the application of our protocol to mycelial grown fungi, a pretreatment was required. Into the conical tube culture, the same volume of glass beads (3-mm diameter) was added to break down the pellets by shaking for 10 min. A nearly homogeneous suspension was used for the fluorescence measurement. The fluorescence intensity increased with an increase in the cell biomass and lipid concentration. Mortierella rammanniana var. angulispora (Fig. 4d) formed an irregular soft pellet mixture, from small particles of less than 0.5 mm to flattened pellets like torn-off clay with long side of more than 5 mm. In the hyphae lipid bodies with less than a 0.5-Am diameter, initially enlarged up to 3-Am diameter, but did not grow thereafter and increased in number after a 50-h cultivation. M. isabellina (Fig. 4e) formed small soft particulate pellets like a wet powder of less than 1mm diameter having lipid bodies with up to 1-Am diameter. M. nana (Fig. 4f) formed irregular pellets of soft particles with a 0.5 3-mm diameter. In the hyphae, small lipid bodies with less than 0.5-Am diameters at the beginning of the culture enlarged to 2 3-Am diameters and increased in number. In all the Mortierella fungi tested, the inside of the hyphae became filled with lipid bodies as the lipid accumulation proceeded. In all the fungi tested, the change in fluorescence intensity well corresponded to the change in the lipid concentration during the cultures. In Fig. 5, the fluorescence intensity is plotted versus the lipid concentration based on the data in Fig. 3. Linear relationships between the lipid concen-

tration and fluorescence intensity were obtained up to a 2.5 mg/ml lipid concentration among the various microorganisms which had different types of cells and lipid bodies in size, shape, and number. The relation coefficients (r), mentioned in the legend of the figure, were high irrespective of the variation of cells and lipid bodies throughout the culture. The slopes of the linear lines were not identical among species but similar among them except for R. toruloides. Similar slopes with high relation coefficients were also obtained in other Mortierella fungi such as Mortierella alpine (data not shown). On the other hand, R. toruloides was the exception which showed a lower slope than the other species.

Fig. 5. Relationship between lipid concentration and fluorescence in oleaginous yeast (a) and fungi (b) throughout the cultivation. . , L. starkeyi IFO-10381 (r = 0.959); n , R. toruloides IFO-0559 (r = 0.861); E , C. curvatus IFO-1159 (r = 0.913); o , M. rammanniana var. angulispora IFO-8187 (r = 0.983); 5 , M. isabellina IFO-7884 (r = 0.980); D , M. nana IFO-8794 (r = 0.873). Plots were taken from the time course data of Fig. 3. Lipid concentration and fluorescence intensity were the calculated values originally contained in 1 ml of culture broth. Values are means of two runs.

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K. Kimura et al. / Journal of Microbiological Methods 56 (2004) 331338 fluorescent stain for intracellular lipid droplets. J. Cell Biol. 100, 965 973. Kamisaka, Y., Noda, N., 2001. Intracellular transport of phosphatidic acid and phosphatidylcholine into lipid bodies in an oleaginous fungus, Mortierella ramanniana var. angulispora. J. Biochem. 129, 19 26. Kamisaka, Y., Noda, N., Sakai, T., Kawasaki, K., 1999. Lipid bodies and lipid body formation in an oleaginous fungus, Mortierella ramanniana var. angulispora. Biochim. Biophys. Acta 1438, 185 198. Kumon, Y., Yokochi, T., Nakahara, T., Yamaoka, M., Mito, K., 2002. Production of long-chain polyunsaturated fatty acids by monoxenic growth of labyrinthulids on oil-dispersed agar medium, 60, 275 280. Lee, S.J., Yoon, B.-D., Oh, H.-M., 1998. Rapid method for the determination of lipid from the green alga Botryococcus braunii. Biotechnol. Tech. 12, 553 556. Murphy, D.J., Vance, J., 1999. Mechanisms of lipid-body formation. T.I.B.S. 24, 109 115. Naganuma, T., Uzuka, Y., Tanaka, K., 1986. Using inorganic elements to control cell growth and lipid accumulation in Lipomyces starkeyi. J. Gen. Microbiol. 32, 417 424. Pomoshchnikova, N.A., Medvedeva, G.A., Levchenko, N.F., Meisel, M.N., Krasovitskii, B.M., 1981. Use of fluorescent technique for detection and quantitative determination of lipids in cells of microorganisms. Microbiology (USSR) 50, 129 134. Pomoshchnikova, N.A., Korotkov, S.A., Galanina, L.A., 1983. Use of fluorescent analysis for study of lipid synthesis by mycelial fungi. Microbiology (USSR) 52, 536 539. Ratledge, C., 1989. Biotechnology of oils and fats. In: Ratledge, C., Wilkinson, S.G. (Eds.), Microbilal Lipids, vol. 2. Academic Press, London, pp. 567 668. Ratledge, C., Wynn, J.P., 2002. The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms. Adv. Appl. Microbiol. 51, 1 51. Sackett, D.L., Wolff, J., 1987. Nile red as a polarity-sensitive fluorescent probe of hydrophobic protein surfaces. Anal. Biochem. 167, 228 234. Suutari, M., Priha, P., Laakso, S., 1993. Temperature shifts in regulation of lipids accumulated by Lipomyces starkeyi. J. Am. Oil Chem. Soc. 70, 891 894. Thakur, M., Prapulla, S.G., Karanth, N.G., 1989. Estimation of intracellular lipids by the measurement of absorbance of yeast cells stained with Sudan Black B. Enzyme Microb. Technol. 11, 252 254. Vijayalakshmi, S., Karthika, T.N., Mishra, A.K.., Chandra, T.S., 2003. Spectrofluorimetric method for the estimation of total lipids in Eremothecium ashbyii fungal filaments using Nile blue and avoiding interference of autofluorescent riboflavin. J. Microbiol. Methods 55, 99 103.

With our protocol, 20 Ag lipids in a 2.1-ml final suspension were detected. In the case of cells with a lower lipid content, the condensation of cells by centrifugation of the culture up to 10 times enabled the lipid measurement. On the other hand, cell suspension which was diluted up to 41 times did not lead to a noticeable error in the case of a higher lipid content. The established method is considered to be applicable to a wide range of microorganisms in a lipid concentrations range of 2 5000 Ag/ml. In the case of cells accumulating lipids more than 20% of dry cell weight, our protocol could detect lipids in the culture broth during the early stage of lipid accumulation. The Nile red addition in high concentration could increase the fluorescence intensity but could not enhance the lipid sensitivity although it would require the attention to the staining period and fading. Previous studies on lipid estimations using the fluorescence of Nile red and Nile blue were applications specialized for some target microbes. On the other hand, our method has been designed for a wide range of microorganisms and represented the applicability of more practical use. A wide range of applications including the mycelial pellet culture has not documented. The method is very rapid and easy compared with the conventional methods, which require complicated procedures such as extraction, purification, and determination of lipids. References
Cole, T.A., Fok, A.K., Ueno, M.S., Allen, R.D., 1990. Use of Nile red as a rapid measure of lipid content in ciliates. Eur. J. Protistol. 25, 361 368. Cooksey, K.E., Guckert, J.B., Williams, S.A., Callis, P.R., 1987. Fluorometric determination of the neutral lipid content of microalgal cells using Nile red. J. Microbiol. Methods 6, 333 345. Fowler, S.D., Brown, W.J., Warfel, J., Greenspan, P., 1987. Use of Nile red for the rapid in situ quantitation of lipids on thin-layer chromatograms. J. Lipid Res. 28, 1225 1232. Greenspan, P., Fowler, S.D., 1985. Spectrofluorometric studies of the lipid probe, Nile red. J. Lipid Res. 26, 781 789. Greenspan, P., Mayer, E.P., Fowler, S.D., 1985. Nile red: a selective