Biology Reference
In-Depth Information
5
AGAROSE SLIDE PREPARATION
An agarose pad on a microscope slide creates a flat surface that is essential for high-
quality imaging and ensures the entire field is in focus. The agarose also draws in the
liquid from the culture resulting in the cells being embedded on the agarose surface,
free of any movement yet able to grow and spread two-dimensionally on the surface.
Pipette 500
L of molten 1.2% agarose (w/v in H
2
O or medium) onto a level glass
microscope slide.
Immediately, drop a coverslip carefully onto the agarose. Hold the coverslip at a 45
angle, allow the bottom edge to just touch the molten agarose and then gently let the
coverslip drop on to the surface so that no air bubbles are trapped. An alternative is to
use a Gene Frame
®
. These are adhesive plastic frames that can be stuck to a slide to
create a space into which a small volume of molten agarose can be pipetted (the
volume depends on the size of the frame used). The molten agarose can then be
immediately overlayedwith the Gene Frame
®
cover.When set, the coverslip can be
carefully peeled off leaving a large flat surface. Due to their low volume, agarose
pads created this way dry out very quickly unless kept in a humidified chamber.
Once solidified, either proceed to applying a sample of bacterial culture or, for
use later in the day, slides may be kept in the fridge wrapped in damp tissue.
Gently remove the coverslip and dispense
m
L of liquid grown culture onto the
centre of the pad. (NB. If cells are at a low OD, a larger volume can be spun down
and resuspended to generate a cell density that is easy to visualise. Cells can also
be counterstained to visualise other components, for example, DAPI for
nucleoids, FM-64 (or similar) for cell membranes (
Johnson
et al.
, 2004
)).
Once the liquid has dried, place a fresh coverslip onto the pad.
5
m
6
IMAGING HARDWARE
6.1
Microscope and camera
For epifluorescence imaging, a suitable microscope (either upright or inverted)
equipped with a Peltier-cooled charge-coupled device (CCD) camera is required.
There are many highly sensitive digital camera systems designed for this purpose,
although we currently use the Hamamatsu Orca-ER. Due to the small size of bacteria
and the often relatively low cellular levels of the fluorescent protein fusion under
investigation, the requirements of the microbial cell biologist are different to those
used to imaging eukaryotic cells. Signal intensity is low and cells are prone to photo-
damage if long exposure times are used. Fluorescence is rapidly quenched, meaning
that minimal illumination should be used prior to image acquisition.
In order to detect the low emission levels from fluorescent proteins, the light
source must be powerful. Often used are high-energy short arc-discharge lamps such
as mercury burners (50-200 W) or xenon burners (75-150 W). These lamps should
not be used beyond their recommended lifetime due to loss of efficiency and an
