Microscopy Flashcards

Explain the function associated with the parts of the microscope.
Explain the role magnification and resolution play in brightfield microscopy.
Describe how oil-immersion increases the resolution for a specimen.
Use the microscope to identify a specimen’s morphology (and arrangement in the case of bacteria).
Calculate total magnification.
Understand how the orientation and movement of the specimen’s image changes when viewed though a compound light microscope.
Demonstrate how to use oil immersion with the 100x objective for bacterial slides.

Microscopes are essential tools in biology, enabling the visualization of tiny objects.
The term microscope translates to “to view the tiny”.
The lab utilizes a compound light microscope.
Light microscopes use light and lenses to magnify the specimen's image.
"Compound" indicates that light passes through the specimen and then through two different lenses.
Objective lens: The lens closest to the specimen.
Ocular lens (eyepiece): The lens nearest to the user’s eye.
Specimens are placed on glass slides.
Types of slides:
- Prepared slide: Permanent, purchased from a science supply company.
- Temporary slide: Fixed and stained in the lab for short-term use.
- Wet mount: Made fresh in the lab for temporary use.
The objective lens is primarily responsible for magnifying the specimen, typically ranging from 4x to 100x.
Total magnification is calculated by multiplying the magnification power of the objective lens by the magnification power of the ocular lens (usually 10x).
Formula for total magnification: $Total Magnification = (Objective Lens Magnification) \times (Ocular Lens Magnification)$
Example: A 40x objective lens with a 10x ocular lens results in a total magnification of 400x.

Light Microscopes rely on light for illuminating samples, requiring appropriate light passage through objective lens.
As magnification increases, the amount of light reaching the ocular lens decreases due to smaller numerical apertures capturing less light.
The field of view becomes smaller which reduces overall brightness.
Increasing the iris diaphragm opening helps increase light intensity with higher magnification objectives.

The 100x objective lens is used to view very small objects, such as bacteria.
Image quality decreases at high magnification, appearing fuzzy or out of focus due to light refraction.
Light is bent by the glass slide, refracting away from the objective, which makes the specimen appear out of focus.
Immersion oil is used to bridge the gap between the slide and the objective, allowing more light to travel into the objective by preventing refraction.
Refractive index: A measure of how well light passes through a substance.
The refractive index of oil is similar to glass, acting like liquid glass.
This results in a better, more focused image.

Set-up
- Microscope is plugged in and the light/power switch is turned on.
- Stage in lowest down position.
- 4x scanning objective in place.
- Microscope slide properly set in stage clips.
- Move microscope slide so light is passing through specimen (is centered over the light).
Visualize the specimen with the scanning (4x) objective
- Raise the stage using the course adjustment knob.
- Once the specimen is in focus, move the stage so that the specimen is in the center of the field-of-view (the circle of light you see in the ocular).
- Adjust the light intensity as needed.
Move the low power objective (10x) lens into place
- Touch nothing - look through the ocular lens; the sample should mostly be in focus already because of the parfocal properties of the microscope.
- If needed, ONLY use the fine adjustment knob to adjust the stage.
- Adjust light intensity as needed.
Move the high power objective (40x) lens into place
- Touch nothing - look through the ocular lens.
- If needed, ONLY use the fine adjustment knob to adjust the stage.
- Adjust light intensity as needed.
Add a small drop of immersion oil to the specimen, and immediately move the 100x oil objective into place
- Only a small amount of oil is needed.
- The oil will touch both the slide and the objective, making a “bridge” between the two.
- Touch nothing - look through the ocular lens.
- If needed, only use the fine adjustment knob to adjust the stage.
- Adjust light intensity as needed.

Specimen is dark: Increase light using the rheostat or adjust the diaphragm.
Debris in the field of view: Clean the slide, ocular, and objective lens with lens paper.
Background light is too bright: Turn down the rheostat.
Always carry the microscope upright with two hands, holding the arm and the base.
If you cannot locate the specimen on scanning, try the low power objective (common with bacterial samples).
Be mindful of the distance between the objective lens and the stage to avoid crashing the stage into the objective.
The slide and the objective should nearly touch only with the oil immersion objective during oil immersion microscopy.

Slides: Remove the slide from the stage and wipe off the oil completely with a Kimwipe; use a little 70% ethanol (EtOH) if the slide appears “greasy”.
100x objective: Use lens paper to wipe off the oil (not a Kimwipe!). Dab a little 70% EtOH onto the lens paper to help remove the oil. Continue to wipe the 100x objective lens with the lens paper until all oil is removed.

Modified from Dr. Timothy Rochbach’s “BIO211-Microscope-Lab Report” Fall 2024.
Wikipedia: https://en.wikipedia.org/wiki/Oil_immersion
Lab: Using a Compound Light Microscope: https://cpb-us-e1.wpmucdn.com/share.nanjing-school.com/dist/3/28/files/2013/02/using-microscope-lab-sq892v.pdf