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Abstract A flow cytometry method for enumerating marine heterotrophic bacteria and phytoplankton in a living or preserved sample using a low power solid state near‐ultraviolet laser is described. The method uses Hoechst 34580 to stain DNA in microbial cells in seawater samples. This stain is optimally excited at 375 nm unlike the similar Hoechst 33342, which requires ~ 350 nm excitation only available on more expensive lasers. Phytoplankton abundances from the Hoechst 34580 method are comparable to those of unstained samples and when analyzed by the Hoechst 33342 staining method. With this new method, nonpigmented marine bacteria and phytoplankton abundances are obtained simultaneously in a single sample as the Hoechst emission wavelength (~ 450 nm) is well separated from the emission wavelengths of chlorophyll and phycoerythrin fluorescence. Bacteria abundances are similar between this new method and those obtained with established Hoechst 33342 and SybrGreen I methods. Precision estimates (coefficient of variation) on populations with abundances near ~ 105cells mL−1are 1–3%, increasing to 3–9% at lower cell concentrations of 103cells mL−1. The Hoechst 34580 method is simple, requiring no heating or pretreatment with RNAse, can be used on unpreserved and formaldehyde‐preserved cells, and is amenable to at‐sea use with portable, compact, low power‐requiring flow cytometers.
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From Bioreactor to Bulk Rheology: Achieving Scalable Production of Highly Concentrated Circular DNA
Abstract DNA serves as a model system in polymer physics due to its ability to be obtained as a uniform polymer with controllable topology and nonequilibrium behavior. Currently, a major obstacle in the widespread adoption of DNA is obtaining it on a scale and cost basis that accommodates bulk rheology and high‐throughput screening. To address this, recent advancements in bioreactor‐based plasmid DNA production is coupled with anion exchange chromatography producing a unified approach to generating gram‐scale quantities of monodisperse DNA. With this method, 1.1 grams of DNA is obtained per batch to generate solutions with concentrations up to 116 mg mL−1. This solution of uniform supercoiled and relaxed circular plasmid DNA, is roughly 69 times greater than the overlap concentration. The utility of this method is demonstrated by performing bulk rheology measurements at sample volumes up to 1 mL on DNA of different lengths, topologies, and concentrations. The measured elastic moduli are orders of magnitude larger than those previously reported for DNA and allowed for the construction of a time‐concentration superposition curve that spans 12 decades of frequency. Ultimately, these results can provide important insights into the dynamics of ring polymers and the nature of highly condensed DNA dynamics.
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- PAR ID:
- 10576389
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Advanced Materials
- Volume:
- 36
- Issue:
- 35
- ISSN:
- 0935-9648
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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