Compound Microscope

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A compound microscope is an invaluable tool for viewing microscopic specimens, both biotic and abiotic. In contrast to single-lens microscopes, these use multiple lenses to provide an improved viewing resolution and to allow for illumination techniques.

Parts of the compound microscope

The following image will outline the main parts of the compound microscope:

  • Eyepiece – This is where you look through. It usually provides 10x magnification.
  • Objective Turret – This holds the objectives and allows them to be rotated for different levels of magnification
  • Objective lens – Contains lenses with each separate objective lens providing a different magnification. Standard lenses: low power (3.2x), medium power (10x), high power (40x), oil immersion (100x)
  • Coarse Adjustment Knob – Makes large changes in the focus
  • Fine Adjustment Knob – Makes small changes in the focus
  • Stage – Has brackets to hold slides in place for viewing. Also has a hole for illuminating light to pass through
  • Light source – Usually can be dialed around for varying levels of brightness
  • Condenser & Diaphragm – The condenser focusses the light while the diaphragm controls how much light gets through
  • Stage Control Knob – Allows the stage to shift in four directions

Setting up a slide

There are two ways to set up a slide for viewing depending on the subject of study. As a rule of thumb, almost anything that is dry can be placed directly onto a clean slide under the objective lens of the microscope and viewed. For micro-organisms, a wet mount will have to be prepared:

  • Clean a slide using hot water.

    Do not use soap as it can be toxic to some organisms.

    Dry the slide.

  • Use a pipette to remove a small sample of the liquid containing the organism and place a drop on the centre of the slide. If the organism is not in liquid, add a drop of water. Most micro-organisms thrive in liquid.
  • Place a coverslip on top of the drop. Make sure there are no air bubbles.
  • Blot up any excess liquid and dry the bottom of the slide.
  • The slide is now ready for viewing.

Setting up the compound microscope

  • Place the microscope on a steady, flat surface.
  • Turn the light source on. Keep it at a medium brightness. Too low and it will be hard to see, but too high and certain organisms may die.
  • Place the slide onto the stage and clamp it down with the brackets.
  • Rotate the objective turret so that the low power lens is in place.
  • Rotate the condenser away from the path of the light. It is only used for higher magnifications.
  • Raise the stage as close as possible to the objective lens, but not touching.
  • Use the coarse focus to focus on the specimen. This will move the stage away from the lens.
  • Use the fine focus to further clarify the image.
  • Adjust the eyepieces so your field of view is only one circle.
  • Adjust the contrast using the diaphragm so you can see different parts of the specimen more clearly.
  • Rotate the medium power lens into place. You should only need to use the fine focus to adjust the focus.

Köhler illumination

To provide the best possible image, the lenses must be correctly lined up. When done properly, this will allow for an evenly illuminated field, a bright image without glare, and minimal heating of the specimen.

  • Make sure the specimen is in focus.
  • Rotate the condenser into the path of the light and raise the condenser as far as possible.
  • Close the diaphragm.
  • Focus on the diaphragm using the condenser’s focus knob. A hexagon with purple or blue edges should appear.
  • Open the diaphragm until the entire field is illuminated.
  • The microscope is now set up for Köhler illumination at medium power.
  • For high power, rotate the high power lens into place and repeat steps 1-6.

Oil immersion lens

The oil immersion lens must be used with a layer of immersion oil between the lens and the specimen. The oil has a refractive index similar to glass, which allows for higher magnifications to be used while keeping decent resolution. This is usually only done for non-motile organisms, such as dead ones. At these magnifications levels, it is almost impossible to keep an eye on moving organisms.

  • Focus on the specimen with the high powered lens.
  • Place a drop of oil on the cover slip over the observed area.
  • Rotate the oil immerson lens into place.
  • Bring the lens as close to the slide as possible, then focus by moving away.

    If you focus by moving towards, you may hit the slide

Estimating the real size of the specimen

Create a table like the one above. The data on the magnification of the lenses was obtained by reading the labels on the microscope. The total magnification is the product of the eyepiece and objective lens magnifications. The approximate field diameter is measured using a micrometer (slide with microscopic ruler).

To estimate the size of the specimen, simply estimate how many lengths or widths of the specimen is required to span the field. Divide the field diameter by this number to get the length or width of the specimen.