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Preparing Slides With Agarose Supplement To Form Microcolonies

This protocol was adapted from this video protocol from the molecular genetics group at the University of Gronigen.


Bacterial cells are transferred to an agar pad on a microscope slide. A monolayer of cells (all in the same Z-plane) grows between the agar pad and a cover slip, and these cells are immobile. Strips may be cut out of the agar pad to allow the bacteria to grow aerobically.

An immobile monolayer of cells is ideal for obtaining clear confocal microscope images. In the original protocol this technique was used to produce time-lapse films of clonal colonies as they grow from a single cell.


Note: We used the Gronigen protocol essentially unmodified, except that we used LB instead of CMD medium.

  • Preparing Slides With Agarose Supplement To Form Microcolonies
Following Overnight Culture at 37 degrees C, prepare the microscope slide as follows:
  1. Clean two microscope glass slides (e.g. Knittel Glass, 7.6 x 2.6 cm,) with 70% ethanol and water.
  2. Take a gene frame (ABgene; 1.7 x 2.8 cm) and carefully remove one of the plastic foils from the gene frame without causing disassembly of the plastic cover on the other side of the gene frame. (see video protocol for how to do this)
  3. Attach the gene frame in the middle of one of the glass slides by first facilitating contact on just one side, followed by guided attachment of the remaining gene frame with a fingernail. Prevent air bubbles while attaching the gene frame to the glass slide.
  4. Use a microwave to dissolve 150 mg (1.5%) high-resolution low-melting agarose (Sigma) in 10 ml LB. The agarose needs to be fully dissolved to obtain minimal background required for the microscopy experiments. If required, supplement the agarose-CDM with inducer or other compounds at this time (we added Arabinose to 1mM concentration induce reflectin expression via our Reflectin Generator Construct
  5. Transfer 500 μl of the warm agarose-CDM in the middle of the gene frame. Make sure the whole area including (the borders) is fully covered.
  6. The following steps (2.6 - 2.10) have to be carried out quickly to prevent excessive drying of the agarose-LB solution.
  7. Place the second glass slide on the agarose-LB filled gene frame. Try to avoid air bubbles. Place the sandwiched slides in a horizontal position for 45 min at 4°C in the refrigerator to allow the agarose-LB to solidify sufficiently.
  8. Carefully slide off the upper glass slide. Use a razor blade or disposable scalpel to cut out agar strips of ~5 mm width within the gene frame, on which the cells will be grown (Be sure to dispose of this carefully in the sharps bin!). A maximum of three strips can be used per slide, separated by ~4 mm space to either side. These spaces will provide air which is essential for aerobic growth of E.coli (though not essential for E.coli to live). If four different strains need to be followed in time, two strips can be made and be cut in half to result in four small squares. Remove any residual solid medium.
  9. Carefully remove the second and final plastic cover from the gene frame to expose the sticky side of the gene frame
  10. Load 2.5 μl of overnight cell culture for a whole strip, or 1 μl for a small square. Always start on top of the agarose pad and allow the liquid to disperse equally on its assigned growth area by turning the slide up and down. The slide is ready, as soon as the edges of the liquid become corrugated and movement of the liquid is no longer visible when turning the slide.
  11. Place a clean microscope slide cover slip (24 x 50 mm) on the gene frame from one side to the other (avoid air bubbles). Assure complete attachment by applying pressure on the cover slip along the gene frame with your fingernail. If the cover slip is placed on the cells without allowing them to dry long enough, cells tend to grow on top of each other during the experiment. Also be careful not to wait too long before applying the cover slip, since the agarose will then be too dry.


Take Care when heating small volumes of broth and agarose as this can easily boil over. Be sure to dispose of sharps used for scoring the agarose in the sharps bin and take care not to break fragile slides and cover slips. Broken glass cannot be recycled.

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