Rapid estimation of lipids in oleaginous fungi and yeasts using Nile red fluorescence

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 microg/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 microg-lipid/ml-broth.     

Application of the standard addition method for the absolute quantification of neutral lipids in microalgae using Nile red

Microalgae are considered one of the best candidates for biofuel production due to their high content in neutral lipids, therefore, an accurate quantification of these lipids in microalgae is fundamental for the identification of the better candidates as biodiesel source.Nile red is a fluorescent dye widely employed for the quantification of neutral lipids in microalgae. Usually, the fluorescence intensity of the stained samples is correlated to the neutral lipid content determined with standard methods, in order to draw a standard curve and deduce the neutral lipids concentration of the unknown samples positioning their fluorescence intensity values on the curve.Standard methods used for the neutral lipids determination are laborious and often implying solvent extraction and/or other transformation (i.e. saponification or transesterification) of the sample. These methods are also time consuming and may give rise to an underestimation of the lipid content due to variable extraction yields.The approach described in this paper combines the standard addition method and the fluorometric staining using Nile red, avoiding the association of traditional neutral lipids quantification methods to the fluorometric determination. After optimization of instrument parameters and staining conditions, a linear correlation between the fluorescence intensity of each sample stained with the Nile red and its neutral lipids content deduced with the standard addition method was identified. The obtained curve allowed the direct determination of neutral lipids content maintaining a linearity range from 0.12 to 12 μg of neutral lipids per ml of sample, without need of pre-concentration. This curve was then used in the quantification of the neutral lipids content in culture of Skeletonema marinoi (Bacillariophyceae) at different days from the inoculum. This method was also successfully applied on Chaetoceros socialis (Bacillariophyceae) and Alexandrium minutum (Dinophyceae).     

核黄素生物合成研究 1.微量培养中核黄菌(Eremothecium ashbyii)发育过程中的形态变化

从本试验观察到,核黄菌的生长全部过程是自配子或菌丝在培养基中开始发育成营养菌丝,部分菌丝形成配子囊形成配子,部分菌丝衰老自溶。当菌丝发育最盛时,核黄素的形成是最多,衰老时培养基核黄素逐渐丰富起来。核黄菌在有空气和缺乏空气时,它们的发育是有区别的,即是在缺乏空气(表面下生长)时,菌丝是细长不形成配子和产生少量核黄素。     

Microwave-assisted Nile red method for in vivo quantification of neutral lipids in microalgae

In vivo determination of neutral lipids with Nile red fluorescence has been used as a rapid screening method for certain types of microalgae, but has been unsuccessful in others, particularly those with thick, rigid cell walls that prevent penetration of the fluorescence dye into the cell. To solve the problem, a microwave-assisted Nile red staining method for microalgal lipid determination was developed. In a two-step staining protocol, 50 and 60 s were selected as the optimal microwave times for the pretreatment and staining process, respectively. Moreover, several calibration methods for quantitative analysis of neutral lipids in microalgae were investigated and compared with conventional gravimetric methods. Factors that affected the in vivo quantification of cellular neutral lipids were also investigated. Application of the new method for detection and quantification of neutral lipids in a number of green microalgae was demonstrated.     

Estimation of lipids in marine animals and tissues: Detailed investigation of the sulphophosphovanilun method for ‘total’ lipids

A reliable and simple method for the estimation of ‘total’ lipids in the tissue of marine animals is much needed for biochemical-ecological work. As a result of the present investigations the phosphosulphovanillin method used, hitherto, only for blood serum samples, is recommended. The method depends on using a cholesterol standard which has previously been related to gravimetric values obtained on blood sera. The colorimetric values have been compared with gravimetric results on a variety of marine tissues — largely of invertebrates.The data given in the tables may be used to convert colorimetric values, using a cholesterol standard to total lipids. For extended investigations, however, it is recommended that the cholesterol values are first calibrated against gravimetric values for the particular tissue under investigation.A summary of the preparation of reagents and procedure is given.