The growth of the cultures was monitored by measuring the total cellular volume using a Beckman Coulter Counter Multisizer 3 until decline in the biomass was observed for all culture bottles9 29 . As explained elsewhere9 , the use of the Coulter counter provides a precise and reliable way of monitoring growth rates. The growth rates were determined on different days, between days 5 to 13, during the log-linear stage of the growth curve. Determination of intracellular quotas was done by harvesting Trichodesmium cells while on the exponential phase of the growth curve (indicated in Fig. 1 ). The cells were filtered onto acid washed polycarbonate filters (25 mm with 5 μm pore size), washed with ultrapure Milli-Q water and subsequently decomposed before analysis9 10 (link). The elemental composition was determined using HR-ICPMS (Element XR, Thermo Scientific).
Counter multisizer 3
Manufactured by Beckman Coulter
Sourced in United States
The Beckman Coulter Counter Multisizer 3 is a laboratory instrument designed for accurate particle size and count analysis. It utilizes the principle of electrical sensing zone technique to measure the size and number of particles suspended in an electrolyte solution.
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Lab products found in correlation
6 protocols using counter multisizer 3
1
Monitoring Growth and Cellular Composition in Trichodesmium
2
Growth Monitoring and Metal Quotas of S. kawagutii
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The growth of S. kawagutii was monitored every other day by measuring cell density per ml using a Beckman Coulter Counter Multisizer 3 equipped with a 100 μm aperture tube until decline in biomass was observed. The growth rates and intracellular metal quotas were evaluated while the cultures were in the exponential phase of growth. The intracellular metal quotas were determined using cells harvested by filtration onto acid-washed polycarbonate filters (25 mm with 2 μm pore size) during the light phase of the light:dark regime. The filtered cells were washed with ultrapure water and digested using concentrated HNO3 prior to elemental analysis using HR-ICPMS (Element XR, Thermo Scientific).
3
Determining Cell Number and Volume
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Cell number and volume were determined using the Coulter Counter® Multisizer 3 (settings: aperture 30 µm; kd 41; current 800 µA; gain 16; sizing threshold 0.3 µm) after adding 50 μl cell culture to 10 ml ISOTON II electrolyte.
4
Cell Counting Using Coulter Counter
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Cell number and volume were determined using the Coulter Counter® Multisizer 3 (settings: aperture 30 µm; kd 41; current 800 µA; gain 16; sizing threshold 0.3 µm) after adding 50 µl cell culture to 10 ml ISOTON II electrolyte.
5
Copper and Sulfite Resistance Assay
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Copper resistance in presence or absence of SO2, was assessed by a drop-test for three wild type strains with different CUP1 copy number in their genome (Oak-Rom 3-2, LMD17 and L1374), and their counterpart engineered to over-express CUP1 (see below). Triplicates of these strains were grown overnight at 28 °C in 5 mL of YPD. Cells were then counted with an electronic particle counter (Multisizer 3 counter; Beckman Coulter), washed with PBS and resuspended in sterile PBS to obtain 107 cells/mL. Three successive 1/10 dilutions were prepared and 1.5 µL of each dilution was spotted on synthetic must having the same composition of the one used for the fermentations, gelled with 20 g/L agar. According to the tested modalities, copper (0, 0.5, 1, 6, 12 mM) and sulfite (0, 40, 60 mg/L SO2) were added to the media to evaluate their effect. Agar plates were incubated at 28 °C for 72 h and growth was assessed by visual examination.
6
Chondrogenic Differentiation of BMMNCs and BMSCs
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Before seeding, freshly harvested BMMNCs and P3 BMSCs were counted using a Multisizer 3 Counter (Beckman Coulter, Boulevard Brea, CA, USA) to confirm approximately equivalent cell numbers. Briefly, cells were mixed with neutralized collagen solution at a concentration of 107 cells/ml. Gelation occurred after 10 min at 37 °C. Cell-hydrogel composites were cultured in chondrogenic defined medium containing α-MEM (Gibco, Shanghai, China), 10% FBS, 1% Insulin-Transferrin-Selenium Solution (Gibco), 100 nM dexamethasone (Sigma Aldrich, Billerica, MA, USA), 50 μg/ml ascorbic acid (Sigma) and 10 ng/ml TGF-β1 (Prope Tech, Rocky Hill, NJ, USA). The medium was replaced every 3 days. Samples were harvested at time points of 7, 14 and 21 days for various analyses and characterizations.
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