Oct 11, 2022

Public workspacePrepare agarose pad for microscopy (2022 iGEM)

 Forked from Fluorescence microscopy of Chlamydomonas reinhardtii - mCherry | Chlorophyll
DOI
dx.doi.org/10.17504/protocols.io.8epv5jze5l1b/v1
  • 1Fudan University
Team Fudan iGEM
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DOI: dx.doi.org/10.17504/protocols.io.8epv5jze5l1b/v1
External link: https://doi.org/10.1371/journal.pone.0192433
Protocol Citation: Team Fudan iGEM 2022. Prepare agarose pad for microscopy (2022 iGEM). protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5jze5l1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 10, 2022
Last Modified: October 11, 2022
Protocol Integer ID: 71105
Abstract
This protocol describe the steps to perform fluorescence microscopy for E. coli
Guidelines
Cells should be cultured to a late log phase to increase cell number for microscopy.
Before start
  • Culture cells in liquid media
Cell growth
Cell growth
Grow cells to late-log phase density (e.g. OD600 > 0.6).
Agarose pads
Agarose pads
Prepare a 1% agarose in distilled water, by adding sufficient low-melting agarose to water and dissolve agarose by heating in a microwave for 30-60 seconds.

16h
Prepare a glass slide with a double adjacent slides barrier (see below), forming a wall around the pad slide. Add slides on top of the poured agarose solution (1-1.2 mL) to allow formation of a smooth surface. It takes time for 1% agarose solution to cool down to ~40 degree, and the solution is viscous but not runny.
Wait for 45-60 minutes. Carefully remove adjacent and top slides, leaving the bottom slide.
Critical
Cut the pad into 15x15 mm square pierces, and carefully transfer the pad pierces to other cleared slide (left one agarose pad on the existing glass slide, if it was clean).
Add 2-5 µL bacteria culture onto the agarose pad, and cover it was a 18x18 mm #1.5 square coverslip. Seal with wax if needed.
Microscopy GFP
Microscopy GFP
5m
5m
  • Place the slide onto the microscope stage, with the coverslip facing the objective.
  • Use a laser at 488 nm to excite GFP.
  • Record images.
5m
Analyze the images. For our microscope with 150x objective, the pixel size is 0.0806 µm.