Quantifying Biogenic Silica (bSi) Deposition Rates Adapted Method v1

Abstract

This method can be used to assess and quantify the rate of silica deposition (bSi) over time in diatoms to determine their rate of frustule synthesis. This protocol has been adapted for the processing of both cultures as well as environmental samples when inoculated with PDMPO [2-(4-pyridyl)-5-((4-(2dimethylaminoethylaminocarbamoyl)methoxy)phenyl)oxazole], which is a fluorescent dye that is co-deposited with silica during frustule biosynthesis in a 3230:1 Si:PDMPO (mol:mol) ratio.

Elevated pH Conditions Associated With Microcystis spp. Blooms Decrease Viability of the Cultured Diatom Fragilaria crotonensis and Natural Diatoms in Lake Erie

“…Quantitative assessment of epifluorescence microscopy data was achieved by randomized scoring of 100 F. crotonensis filaments per pH treatment: analyses included chlorophyll a fluorescing cells per filament, PDMPO fluorescing cells per filament, and the proportion of filaments demonstrating ≥one PDMPO fluorescing cells. Total Si deposition was quantitatively measured fluorometrically after HCl-Milli Q lysis to remove unincorporated PDMPO from the silica deposition vesicle (SDV), followed by frustule digestion with hot-NaOH ( Saxton et al, 2012 ; Zepernick et al, 2019 ). After 48 h of growth, 20 mL of culture was collected on a 0.2-μm nominal pore-size 47-mm diameter polycarbonate filter and subjected to HCl-Milli Q lysis.…”

“…Samples were flash frozen in liquid N 2 and stored at −80°C until further processing. Quantitative Si deposition analyses were performed using hot-NaOH frustule digestion, fluorometry, and subsequent calculations using a fresh standard curve ( Supplementary Figure 5 ; Zepernick et al, 2019 ). Total silica deposited per bottle after 48 h (μmol) was normalized to chlorophyll a concentration (μg/L) ( Saxton et al, 2012 ).…”