Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Determination of gallic acid in Phyllanthus emblica Linn. dried fruit powder by HPTLC

Objective : Emblica (Phyllanthus emblica L.), an euphorbiaceous plant, is widely distributed in subtropical and tropical areas of India, China and Indonesia. The fruits possess antimicrobial, antioxidant, anti-inflammatory, analgesic and antipyretic properties. In the current article a new, simple, sensitive, selective, precise, and robust high-performance thin-layer chromatographic (HPTLC) method was developed and validated for the determination of gallic acid in dried fruit powder of Phyllanthus emblica. Materials and Methods : The quantitative determination of gallic acid was performed on TLC aluminium plates pre-coated with silica gel 60F-254 as the stationary phase. The linear ascending development was carried out in a twin trough glass chamber saturated with a mobile phase consisting of toluene: ethyl acetate: formic acid: methanol (3:3:0.8:0.2) at room temperature (25 ± 2°C). Camag TLC scanner III was used for spectrodensitometric scanning and analysis, in the absorbance mode, at 278 nm. Results : The linear regression analysis data for the calibration plots showed good linear relationship with r 2 = 0.99977 in the concentration range of 40 - 240 ng spot−1, with respect to the peak area. According to the guidelines of the International Conference on Harmonization (ICH), the method was validated for precision, accuracy, and recovery. Conclusion : Statistical analysis of the data showed that the method was reproducible and selective for the estimation of gallic acid

Simultaneous determination of related substances of telmisartan and hydrochlorothiazide in tablet dosage form by using reversed phase high performance liquid chromatographic method

Objective : Telmisartan is a potent, long-lasting, nonpeptide antagonist of the angiotensin II type-1 (AT 1 ) receptor that is indicated for the treatment of essential hypertension. Hydrochlorothiazide is a widely prescribed diuretic and it is indicated for the treatment of edema, control of essential hypertension and management of diabetes insipidus. In the current article a new, accurate, sensitive, precise, rapid, reversed phase high performance liquid chromatography (RP-HPLC) method was developed for determination of related substances of Telmisartan and Hydrochlorthiazide in tablet dosage form. Materials and Methods : Simultaneous determination of related substances was performed on Kromasil C 18 analytical column (250 × 4.6 mm; 5΅m pertical size) column at 40°C employing a gradient elution. Mobile phase consisting of solvent A (solution containing 2.0 g of potassium dihydrogen phosphate anhydrous and 1.04 g of Sodium 1- Hexane sulphonic acid monohydrate per liter of water, adjusted to pH 3.0 with orthophosphoric acid) and solvent B (mixture of Acetonitrile: Methanol in the ratio 80:20 v/v) was used at a flow rate of 1.0 ml min−1 . UV detection was performed at 270 nm. Results : During method validation parameter such as precision, linearity, accuracy, specificity, limit of detection and quantification were evaluated, which remained within acceptable limits. Conclusions : HPLC analytical method is linear, accurate, precise, robust and specific, being able to separate the main drug from its degradation products. It may find application for the routine analysis of the related substances of both Telmisartan and Hydrochlorthiazide in this combination tablets