Microbiology and Microscopy Basics

Study Notes

Microbiology

The study of living organisms too small to be seen with the naked eye.
Includes bacteria, viruses, fungi, protozoa, and some algae.
Microorganisms are everywhere, including inside the human body.
Pathogens cause diseases.
Most microorganisms are beneficial.
Microorganisms are used in the production of antibiotics and vaccines, and in insulin production via genetic engineering.

Microscopy

Types of Microscopes
- Bright Field Microscope:
  - Uses visible light to illuminate the specimen.
  - Stained objects appear bright against a dark background.
  - Uses objective lenses with different magnifications: 10x (low power), 40x (high power), and 100x (oil immersion).
- Dark Field Microscope:
  - Illuminates the specimen from the sides.
    - Unstained objects appear bright against a dark background.
    - Used to observe living organisms, especially those that are difficult to stain.
- Phase Contrast Microscope:
  - Creates contrast by enhancing differences in the refractive index of various cell components.
  - Unstained objects appear dark against a brighter background, allowing for visualization of internal structures.
  - Used to observe living cells and tissues.
- Fluorescence Microscope:
  - Uses ultraviolet light to excite fluorescent molecules within the sample.
  - These molecules emit visible light, allowing for visualization of specific structures or substances.
  - Used for immunofluorescence and other types of microscopy that involve fluorescent labeling.
Basic Principles of Microscopy
- Magnification: The ability to enlarge the image of a specimen.
  - Determined by multiplying the magnification of the ocular lens (10x) by the magnification of the objective lens (10x, 40x, or 100x).
- Resolution:
  - The ability to distinguish between two closely spaced objects.
  - Determined by the wavelength of light used and the numerical aperture of the objective lens.
  - Higher resolution means clearer and more detailed images.
- Refraction:
  - The bending of light as it passes from one medium to another with a different density.
  - When viewing specimens under a microscope, refraction can occur when light passes from the slide to the air, which distorts the image.
- Refractive Index:
  - A measure of how much light bends (refracts) when passing from one medium to another.
- Immersion Oil:
  - Used between the objective lens and the slide during oil immersion microscopy.
  - Immersion oil has a refractive index similar to glass, which minimizes light scattering and improves image clarity.
- Illumination:
  - Controlled by the condenser, diaphragm, and light source to provide optimal lighting for viewing.
  - The condenser focuses the light beam onto the specimen.
  - The diaphragm adjusts the amount of light passing through the specimen.
Oil Immersion Lens:
- Used to achieve the highest magnification (100x).
- Requires the use of immersion oil to improve resolution and image quality.
- Procedure:
  - Place a single drop of immersion oil on the slide, directly over the specimen.
  - Carefully lower the oil immersion objective lens into the oil.
  - Adjust the focus using the fine adjustment knob.

Lab Questions

Diaphragm: Controls the amount of light that passes through the specimen and into the objective lens.
Condenser: Concentrates light and enhances image sharpness.
Coarse Adjustment Knob: Used for rapid focusing and locating the specimen in the field of view.
Fine Adjustment Knob: Used to bring the specimen into sharp focus.
Dark-field microscopy is used to obtain a bright object against a dark background.
Phase-contrast microscopy is used to obtain dark objects against a bright background.
Fluorescence microscopy is used to emit visible light by exciting fluorescent molecules, resulting in different shades of orange, yellow, or green.