Laboratory Investigations in Microbiology

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Chapter 3: Microscopy

Because most microorganisms are too small to be seen with the naked eye, microscopes are necessary to view them successfully. There are many kinds of microscopes with a variety of different designs, but all of microscopy accomplishes essentially the same three functions:

Because most bacteria are nearly colorless, contrast is most often provided by staining cells, a topic we will address in the next chapter. Magnification on most light microscopes is limited to ~ 1000x or less - any more and all you will see is simply larger blurry objects!

The most common type of microscopy is bright-field microscopy (Atlas Fig. 3.2A). Here, the entire slide is illuminated and any dark (colored or stained) objects on the slide block light, providing contrast. Unfortunately, most staining and fixation procedures (except for vital stains) kill the cell, making it impossible for the viewer to distinguish live and dead cells.

DarkfieldA second type of microscopy is called darkfield (Atlas Fig. 3.2B); here, the optics are adjusted so that the light source misses the objective lens and the viewer sees only a 'dark field' unless there are objects or cells present. Light is bent (refracted) by these objects, which become visible as bright Phase contrastobjects on a dark background. A third type of microscope, the phase contrast microscope (Atlas Fig. 3.2C), illuminates the entire field, but light passing through objects is slowed down to varying degrees and becomes 'out of phase' with light passing through less dense regions, thus generating contrast. Both the dark-field and phase contrast microscopes are ideal for viewing living, unstained cells. Yet another type of microscope, the fluorescence microscope (Atlas Fig. 3.2D), makes use of special dyes (called fluorochromes) which fluoresce/glow in the dark when illuminated with certain wavelengths of light (often UV). Because these dyes can be linked to certain cell components, this type of microscopy allows the user to visualize parts of cells, such as the bud scars in this image of budding yeasts.

The objective of this lab (no pun intended) is to familiarize you with the basic microscope operations and controls and for you to successfully view (and describe/draw) several microorganisms on prepared slides. In addition to practicing your microscope skills, you will also see representative examples of eukaryotic and prokaryotic microbes: protozoa, algae, fungi, and cyanobacteria. Look for the nucleus, chloroplasts, cilia, etc. on your slides! 

Note: This procedure is part of the Laboratory Skills Test

Materials & Methods

A. The Microscope

1. Take a microscope from the cabinets to the left of the instructor's station. Be sure to always carry microscopes with two hands, one hand supporting the base. 

2. Get a book of lens paper and a small bottle of immersion oil.

3. Clean your microscope each time after using it: Clean lenses/glass surfaces with lens paper only; clean the stage with a Kimwipe or paper towel.

4. Familiarize yourself with the following controls on your microscope:

B. Basic observations
  1. Work individually. 
  2. Choose a slide to view and place in on the stage in the slide holder. Turn on the light (red knob) to maximum. Looking at the stage, try to center the slide in the cone of light coming from the condenser.
  3. Rotate the low power lens (10x) into place. Raise the stage as high as it will go, then lower it 1/4 turn of the coarse focus knob. This will bring you close to being in focus.
  4. Adjust the light level using the iris diaphragm. The iris diaphragm has a setting for each magnification (10x, 40x, 100x).
  5. Slowly turn the coarse focus knob to raise or lower the slide while looking through the ocular lens. Stop when something comes into focus. [It does not matter at this moment whether it is a piece of dust on the slide as long as you actually locate the slide surface!]. You will know the slide is in view if the thing you are focusing on moves when you move the stage controls.
  6. Now move the stage controls back & forth slowly until the colorful  cells come into view. They will likely still be somewhat out of focus. Adjust the focus carefully using the fine focus knob until the image becomes clear.
  7. Change from low power lens to the high-dry lens. Do this by rotating the objective lens into place without touching the focus knobs or stage controls. These microscopes are designed to be par-focal, meaning that an object in focus at one magnification should still be nearly in focus at the next magnification. 
  8. Adjust the light level (iris diaphragm) and move the stage controls slightly to center the cells again. Some slight adjustments in focus will be necessary.
  9. Examine the following organisms under lower power (4x - 40x):
  10. Take digital images of each organism you observe
    1. Remove one eyepiece of your microscope and insert the digital camera in its place. Plug the USB cable into a USB port on your computer. (If prompted, complete the installation instructions for the camera)
    2. For DCM300 camera:
      1. Start the ScopePhoto software. Click on the SD button to select device: DCM300 camera
      2. Click on the TC button to get a live image. Adjust the microscope brightness and/or the image settings if needed.
      3. Click the capture button to take an image. This image appears in a new window. Use the save as function to save the image, as a jpg file, to a pen drive or the computer's thawspace. 
    3. For new cameras:
      1. Start the ToupView software.
      2. Click on the camera name (left side of screen). A live view will appear in the main window.
      3. Use "snap" to capture images.
  11. You can also crop images
    1. Use the SELECT tool to select a part of a captured image
    2. Use the CROP function in the Image menu to crop the selected region
    3. Save the image to your pen drive using Save As.
C. Demonstrations
  1. Take a look through the darkfield microscope (Demo #1). What do you see? How does this differ from the brightfield? What is the darkfield microscope used for?
  2. Examine the cells seen through the phase contrast microscope (Demo #2). How do these cells compare with the darkfield and brightfield microscopes? 
  3. Look at the cells shown with the fluorescence microscope (Demo #3). Describe what you see.
D. Measuring the size of microorganisms

Eyepiece method: Replace one of your regular eye pieces with an eyepiece that has a micrometer scale built into it. The scale units have the following meaning:

Image Fusion Method:  (For use with DCM300 or DCM500 camera) Capture a digital image file of your cells. Open the image file of a micrometer (scale) taken at the same magnification as your digital image (4x, 10x, 40x, 100x). You can find these files on Moodle.  Using the Image fusion function, superimpose the image file with the image of the micrometer.

Example (image on right): The numbers on the micrometer are in millimeters, and each scale division represents 10 micrometers. In this picture, the scale goes up to ~ 0.3 mm = 300 µm. The cell behind it is ~ 360 µm long. 

 

Data table: size of cells

Cell Magnification (lens used) Length of cell (µm)
Paramecium
Closterium
Coprinus (spore)

 

Data Sheet & Review Questions (printable)

© 2003 - 2017 José de Ondarza, Ph.D.