Architecture of biofilms grown in flow cells or in microtiter plates was assessed via confocal laser scanning microscopy (CLSM). CSLM was carried out using a Leica TCS SP5 confocal microscope. Prior to confocal microscopy, biofilms were stained using the BacLight LIVE/DEAD viability stain (Life Technologies) at a 1/1000 dilution in the growth medium. The CLSM images were processed using LAS AF software v2.4.1. Quantitative analysis of the images was performed using the COMSTAT96 (link). For brightfield visualization of biofilm architecture, the samples were viewed by transmitted light using an Olympus BX60 microscope and Olympus UPLNFLN 20× and 40× objectives. Images were captured using a ProgRes CF camera (Jenoptik, Jena, Thuringia) and processed using ProgRes CapturePro 2.7.7 software.
Uplnfln
Manufactured by Olympus
Sourced in Japan
The UPLNFLN is a microscope objective lens designed for use in a variety of laboratory applications. It features a numerical aperture of 0.13 and a working distance of 21.5 mm. The lens is optimized for use with a wide range of biological samples and provides high-quality imaging performance.
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Lab products found in correlation
Baclight live dead viability stain, by Thermo Fisher Scientific (1 mentions)
Tcs sp5 confocal microscope, by Leica (1 mentions)
Bx60 microscope, by Olympus (1 mentions)
Progres cf camera, by Jenoptik (1 mentions)
U mwu2 filter, by Olympus (1 mentions)
Epi fluorescence microscope bx 51, by Olympus (1 mentions)
Nucleopore track etch membrane, by Cytiva (1 mentions)
2 protocols using uplnfln
1
Biofilm Architecture Analysis via CLSM
2
Enumeration and Isolation of Marine Microbes
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Total cells in the water samples (10 mL each) collected from all the discrete depths were stained with 4′,6-diamidino-2-phenylindole (DAPI)17 (link) before counting. Cells stained with DAPI were first fixed with filter sterilized particle free buffered formaldehyde (final concentration, 3.7%) to preserve the cell morphology and improve the staining efficiency. The stained cells were filtered through 0.22 μ black polycarbonate membranes (Nucleopore Track-Etch Membrane, Whatman, Maidstone, UK) and counted under an Olympus epifluorescence microscope (BX 51) with the aid of an Olympus U-MWU2 filter (Excitation 330–385 nm and Emission 420 nm). Counting was done on a Whipple grid with a 100× objective (Olympus UPLNFLN, Tokyo, Japan).
Water samples were spread plated using 100 μL aliquot on pre cooled (4 °C) quarter strength Zobell Marine Agar (ZMA) and incubated at 4 °C for 1–2 weeks. Colonies with unique morphological features were isolated and sub-cultured to obtain pure cultures.
Water samples were spread plated using 100 μL aliquot on pre cooled (4 °C) quarter strength Zobell Marine Agar (ZMA) and incubated at 4 °C for 1–2 weeks. Colonies with unique morphological features were isolated and sub-cultured to obtain pure cultures.
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