Continuous Monitoring of Biological Noise in Bacteria Using Time-Lapse Microscopy

Abstract

Source: Chaimovitz, E. A., et al. Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy. J. Vis. Exp. (2021)

This video demonstrates real-time visualization of biological noise in E. coli by tracking fluctuations in GFP fluorescence during bacterial growth and division using time-lapse microscopy.

Protocol

1. Preparing the sample for microscopy imaging
   1. Remove the flask from the water bath. Wipe the outside of the flask clean and let it cool to room temperature (25 °C).
      NOTE: Remove the flask about 3 minutes before the timer ends.
   2. Pour 3 mL of the gel constantly to the plate perimeter in a circular motion. Leave to solidify for a few minutes.
   3. Seal plates with tape and pierce several holes with 25 G needle. Flip all the dishes to prevent water condensation from dripping onto the gel.
   4. Incubate all the dishes at 4 °C for 30 min to allow full solidification while preventing cell mitosis.
      NOTE: Leave samples at 4 °C for successive measurements, no more than one day.
   5. Start the microscope per manufacturer's instructions.
   6. Find the initial focus using the lowest amplification lens and engage the automatic focus system (AFS).
   7. Use oil if needed, drown the lens with oil and spread it carefully by moving the plate with a platform controller (not manually), and AFS can be engaged again.
   8. Use relative cross-section in Z direction, following the default suggestion for Z step cross-sections.
      NOTE: Take bright field images every 5 minutes and fluorescent images every 20 minutes. Sometimes the focus needs to be adjusted during the first 30 minutes. Preheating the microscope incubator box and oil, while refrigerating the sample tends to help reduce the initial loss of focus.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
35mm Glass dish	Mattek	P35G-0.170-14-C	Thickness corresponding with microscope lens.
Agarose	Lonza	5004	LB preperation
Eclipse Ti	Nikon		Inverted microscope
Immersol 518F	Zeiss	4449600000000	Immersion oil
Parafilm tape	Bemis	PM-996	Refered to as tape in text