Using a microscope

Awareness level only. This lesson teaches how a microscope works and how to measure with one on safe specimens. In a real lab, handling patient samples, cultures or stains requires the training your employer or institution provides, and you work under supervision until you are signed off.

Why it matters

The light microscope is still one of the most used instruments in biology and clinical labs. Technicians check cell cultures for health and contamination, count cells, read Gram stains and examine blood smears. Doing it well means getting a sharp image quickly without damaging the slide or the lens, and reporting sizes in the right units.

The core idea: magnification and resolution

A compound light microscope uses two lenses in series:

• the eyepiece (ocular), usually 10x
• an objective on the rotating nosepiece, typically 4x, 10x, 40x and 100x

Total magnification = eyepiece × objective. A 10x eyepiece with a 40x objective gives 400x.

Magnification alone is not enough. Resolution is the smallest distance between two points that you can still see as separate. For light microscopes it is about 0.2 µm, limited by the wavelength of light. That is why bacteria (1–5 µm) are visible but viruses (about 0.02–0.3 µm) generally are not. The 100x objective needs a drop of immersion oil between the lens and the slide to reach its best resolution.

Other parts you will use:

• Stage and stage clips (or a mechanical stage) to hold and move the slide
• Coarse focus knob for big movements, fine focus knob for sharpening
• Light source, condenser and iris diaphragm to control brightness and contrast

Worked example: focusing routine

1. Start on the lowest-power objective. It has the widest field and the most room between lens and slide.
2. Watching from the side, raise the stage until the objective is close to the slide.
3. Looking through the eyepiece, focus by moving the stage away from the lens with the coarse knob. Moving away cannot crash the lens into the slide.
4. Centre the part you want, then switch to the next objective. Good microscopes are parfocal: the image stays nearly in focus, so use only the fine knob from now on.
5. Close the diaphragm a little if the specimen looks washed out; unstained cells often need lower light to show contrast.

You will notice that the image is upside down and reversed: move the slide left and the image moves right. A newsprint letter "e" makes this obvious.

Worked example: measuring with the field of view

You can estimate size without special equipment.

1. At 4x, lay a clear ruler on the stage. Suppose 4.5 mm fits across the circle. The field of view is 4.5 mm = 4,500 µm.
2. Field of view shrinks in proportion to objective power. At 10x: 4,500 × (4 ÷ 10) = 1,800 µm. At 40x: 4,500 × (4 ÷ 40) = 450 µm.
3. At 10x you count about 6 onion cells across the field. Each cell is roughly 1,800 ÷ 6 = 300 µm long.

Real labs use a calibrated eyepiece graticule and a stage micrometer for accurate measurements, but the logic is the same: know the size of what you are looking through, then compare.

Remember the units: 1 mm = 1,000 µm, and 1 µm = 1,000 nm.

Care of the microscope

• Carry it with two hands: one on the arm, one under the base.
• Clean lenses only with lens paper, never tissue or your shirt.
• Wipe immersion oil off the 100x objective after use, and never let the 40x objective touch oil.
• Finish on the lowest objective with the stage lowered and the light off.

Common mistakes

• Starting on high power. You will not find the specimen, and you may crack the slide.
• Using the coarse knob at 40x or 100x. It moves too far and can crash the lens.
• Too much light. Unstained cells disappear; close the diaphragm.
• Reporting size without units or magnification. Always say "about 300 µm at 100x total magnification".
• Confusing magnification with resolution. Making an image bigger does not reveal detail smaller than about 0.2 µm.

Quick recap

• Total magnification = eyepiece × objective.
• Resolution of a light microscope is about 0.2 µm.
• Start low, focus away from the slide, then use only the fine knob at higher power.
• Field of view at a new objective = low-power field × (low objective ÷ new objective).
• Estimate size by dividing the field width by how many cells span it.

Try it

Open the Do tab to focus a safe specimen and estimate cell size from the field of view.