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Observing a Gram stain in a light microscope

The light microscope is arguably the most valuable research tool in the history of biology. A good quality bright field microscope, which is absolutely essential for phenotypic characterization of bacteria, is even more useful when equipped with specialized optics for viewing living bacteria. This document introduces the process of finding a suitable field of Gram stained bacteria using bright field optics, focusing, and optimizing resolution and contrast.

Observing a Gram stain

The following outline is for your reference only. You will definitely need hands-on training to learn to observe bacteria in a light microscope. Please start by reviewing the parts of the microscope (figures 1 and 2). To view a Gram stain:

  1. Mount the slide on the stage with the smear facing the objective lens; if the slide is upside down, you won't be able to focus at high magnification. If you aren't sure which side of the slide bears the smear, either feel for the scratch that you made with the etching tool, scrape over the smear with a loop or stick, or use the microscope at low magnification to check whether or not the cellular material is on the same surface as the scratch that you made.

  2. Rotate the condenser turret to the bright field position ("O" position).

  3. Move the aperture diaphragm knob to the center position (moderate contrast).

  4. Rotate the 4x objective lens into place and turn the illuminator up until light comes through the field lens. Plan to increase the illumination as you move up in magnification. Find an area of the smear with a single, moderately dense layer of cells, and focus using the coarse adjustment. At this magnification (40x total), bacteria will look like dirt on the slide. Bacteria often dry down in concentric rings; if you have trouble finding the bacteria look for the rings, which are usually more densely populated than the rest of the smear, around the edges.

  5. Rotate the 10x objective lens into place, adjust illumination if necessary, focus using the coarse adjustment, and center on an area in which there are plenty of cells with at least some spaces among the cell chains or clusters. A good area will look somewhat "webby."

  6. Carefully rotate the 40x objective into place, adjust illumination if necessary (usually we want the maximum available light), then focus using the fine adjustment and center the specimen. Suggestions for finding the specimen at high magnification:

  7. The depth of field is very shallow at high magnifications. To find the focal plane  you might want to increase contrast by moving the aperture diaphragm knob to the far right position, stopping down the light.

  8. After positioning the objective lens, rotate the fine focus control clockwise a small amount while observing through the eyepieces. If colors become more intense and shapes more condensed, you are focusing in the correct direction. If colors are fading and shapes are becoming more diffuse, switch directions. Note the direction in which to turn the focus knob, to save yourself the trouble next time.

  9. After focusing and centering a suitable part of the specimen in the field, rotate the 40x objective off of the specimen but do not rotate the next (100x objective into place just yet. Instead, place a generous drop of immersion oil directly onto the illuminated part of the smear. Now rotate the 100x objective into place, allowing the lens to contact the oil.

  10. Do not try to interpret a Gram stain at 400x magnification; make observations at 1000x* Even at 400x magnification, neither color nor shape nor size of cells can be reliably observed; or example, with typical resolution of ~1 µm, two cells of < 1 µm diameter lying next to each other will look like one cell.

  11. Again, note the direction in which you need to focus so that you don’t have to rely on trial and error next time. The depth of field is so shallow at 1000x that the specimen may be invisible at first.

  12. Adjust the aperture diaphragm for best resolution, turn up the light to maximum if it is not there already, and begin observing. You need strong light and moderate contrast to make the Gram stain determination (positive or negative).

 

Fig. 1. Nikon Labophot microscope with standard bright field condenser.

Note on viewing with immersion oil

Oil on the 40x lens will render it useless until we clean it off. Therefore, do not go back to using that lens if their is oil on the slide. You can remove the oil by gently blotting it off onto a paper towel, then you may use any objective. You can also use the lower magnification objectives with oil on the specimen because they remain well above the specimen when we are in focus.

Fig. 2. Turret condenser equipped for dark field and phase contrast optics

Interpreting a Gram stain

Here some information that we seek when observing a Gram stain.

  • Is the isolate Gram positive or Gram negative?
  • Do we have a pure isolate?
  • What is the typical cell morphology, including sizes of cells?
  • How are the cells arranged?

Gram Positive or Gram negative?

Purple staining indicates a Gram positive reaction and pink indicates Gram negative. However, Gram positive cells may stain Gram negative if they are older or were overexposed to decolorizing agent. If you have mixed purple and pink staining cells that are otherwise indistinguishable, then you likely have a Gram positive isolate.

Assessing purity

We likely have a pure isolate if the cells all share the same shape, size, and Gram stain reaction (+ or -). We frequently see variation, though. In a pure culture of rods or cocci the cells typically have the same diameter, but rods usually vary in length. Numbers of cells in chains or clusters will usually vary. If you see two or more distinct types of cells then you may have a mixed culture. For example, a pure culture is unlikely to have cells with two different diameters or both rods and cocci. However, irregular species may have both. For some Gram positive species, what looks like a mix of rods and cocci or coccoid cells is actually a mix of rods and spores.

Describe the cell morphology and arrangement

Below are examples of cell types we are likely to find among our unknowns and how the cells might be arranged. The cells of most isolates will look very similar when grown on different media. We will, however, encounter pleiomorphic species, for which the cells may develop different shapes, sizes, and arrangements depending on the growth medium. If you think you have found a pleiomorphic species, double check on fresh media to ensure that you are looking at the same isolate on two different media, and not two different isolates.

Fig. 3. Cell types and arrangements commonly found in Gram stains.

Fig. 4. Some less commonly found bacterial cell types.
Created by David R. Caprette, Teaching Professor, Rice University (caprette@rice.edu) 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