Links to files on the current site



Lab methods Assignments

Supplemental pdfs

Observing living bacteria

By observing living bacteria we can learn some things that are difficult or impossible to learn from a Gram stain. Most importantly, we can observe motility by means of flagellar or gliding mechanisms. It is also far easier to detect the presence of spores in living bacteria rather than in a Gram stain. The resolution in dark field, one of two choices of optics for viewing living bacteria, is far superior to the resolution using bright field optics.
To view living bacteria we have to overcome a couple of challenges.

Problems to overcome

  • A drop of bacteria-laden water under a coverslip begins to dry up immediately, producing distracting currents and limiting the time during which we can make our observations.
  • We have bacteria moving in three, not two dimensions, therefore at a high magnification they will be moving/vibrating in and out of the focal plane.
  • If the concentration of bacteria is too high resolution will be compromised and they are less likely to exhibit motility
  • Not only are many bacterial cells colorless, but individual cells have very little mass and therefore do not absorb enough light in bright field to give us sufficient contrast. That is, they are nearly invisible in a conventional bright field microscope.


  • Prevent evaporation by using a Vaseline® mount to seal the sample between slide and coverslip
  • Use a very small drop of water so that the depth is very shallow
  • Add colony material to the water to produce a concentration gradient of bacterial cells
  • Employ specialized optics – dark field and/or phase contrast – to view living bacteria


Making a wet mount

  1. Pick up some Vaseline®sup> on a forefinger and rub it between finger and thumb to make a film. Hold a single coverslip by the edges in one hand and scrape the film onto one edge to make a small Vaseline® ledge. Repeat for the remaining three edges.

    1. Hold the coverslip toward the edge to be treated, to minimize the risk of breakage. Do not attempt to pick up broken glass with your fingers – coverslips in particular are very thin and the shards are very sharp.
    2. Handle slides and coverslips only by the edges to avoid fingerprints that will obscure the view
    3. Keep the ledge as thin as possible
  2. Set the coverslip down (Vaseline® side up, of course) on the end of a slide so that you can readily pick it up later.

  3. Squirt some water on the bench and then use a loop to place about 30 µl water onto the surface of the slide. How much you need depends on the thickness of your Vaseline® ledge – it is a matter of trial and error.

  4. Aseptically obtain a small amount of colony material on a loop and transfer it to a dry part of the slide off the edge of your water drop. Do not stir in the material.

  5. Place the prepared coverslip onto the drop, Vaseline® side down, and press down the edges using the handle of the loop (not your fingers). Without breaking the coverslip press down enough to spread out the drop and eliminate air bubbles.

    1. As you press down the coverslip the colony material should contact and begin to diffuse into the water.
    2. Repeat for the next isolate to be observed. You can easily fit two wet mounts on the same slide

Fig 1. Preparing a wet mount of living bacteria.

Viewing in dark field

  1. Mount the slide coverslip up onto the microscope stage. Move the condenser turret to the "ph4" position. That position places a phase contrast ring in the light path. Although the ring is designed for phase contrast optics with a 100x objective lens, it provides excellent dark field optics with the low magnification (4x and 10x) lenses.

  2. With the 4x objective lens in place, center the field on the inside edge of the colony material, which should be visible under the coverslip.

  3. Turn up the illumination to maximum and focus (coarse control). In dark field the suspended bacteria will be bright against a dark background, resembling a cloud.

  4. Move to the 10x lens, focus and center again. A final magnification of 100x, should be sufficient with which to observe motility. For cell structures such as spores, parasporal crystals, etc., you will want to go to 400x.

  5. To view at 400x, move the condenser turret to the "DF" position and rotate the 40x objective into place. Focus and center.

    1. If the image is distorted or blurry, the water may be too deep (too thick a Vaseline® ledge), there may be smudges on the coverslip, or the 40x objective may be dirty or have oil on it. If the lens is dirty then have an instructor clean it for you.
    2. If you cannot get an image at 400x then either the light is too low or the condenser may be out of position (too low or off center). Ask an instructor to check the condenser. Do not attempt to raise or lower the condenser yourself.

  6. Dark field is not available with the 100x oil immersion lens.

Viewing in phase contrast

Start with steps 1-4 above. To view at 400x, move the condenser turret to the "ph3" position and rotate the 40x objective into place. Focus and center.

You may need to center the phase ring, using the large phase centering screws; this procedure will be demonstrated during the training session.

As with dark field, a poor image may be due to smudges, a dirty lens, too deep a layer of water, or a misaligned condenser.

What to look for in dark field or phase contrast

Find a part of the field with a modest concentration of bacteria, such that you can see lots of background with some separation among cells and clusters/chains of cells.

True motility is usually by flagellar motion. We may see gliding bacteria, which appear to drift in different directions on the slide. If you suspect gliding bacteria double check that they aren't just drifting on currents.

If bacteria are using flagella to move, you will see translational motion. It may be fast motion with bacteria jetting around in various directions. It may be slower motion with bacteria moving along the field, again in various directions. You may see spinning or tumbling.

If bacteria are merely vibrating, you are seeing Brownian motion, in which molecular motion (temperature) causes the entire cell to move. Brownian motion is especially evident with smaller bacteria, which can be quite active.

If you see only Brownian motion and no evidence of motility, report that motility was not observed. You cannot be certain that you have a nonmotile species because some motile species will drop their flagella in a wet mount.


Some Gram positive rods such as Bacillus species produce spores. Spores will appear as discrete bright objects within the dark (dark field) or blue-gray (phase contrast) cytoplasm of the cell bodies or as bright objects in the field by themselves. They can be spherical or oval in shape, they can be terminal, subterminal, or terminal in position, and the sporangium (cell body bearing a spore) can be swollen or not. All of these characteristics are important in distinguishing species in spore-forming genera.

Created by David R. Caprette, Teaching Professor, Rice University ( 18 Feb 2017 for the course BIOC 318, Laboratory Studies in Microbiology. Please feel free to copy and/or modify these materials for use in your own academic or other nonprofit program. If you don't mind letting me know of such intentions I'll be happy to hear from you.
Last updated 18 Feb 2017