Microscope Fundamentals
What are the two smallest units of measurement used in microscopy, and what can we observe at each scale?
Micrometers (µm) for bacteria, nanometers (nm) for viruses/proteins
Who first used the term “cell” when observing cork under a microscope?
Robert Hooke
Name two lenses in a brightfield microscope and their functions.
Ocular lens magnifies image, objective lens gathers light and magnifies
What are cationic and anionic dyes, and how do they differ?
Cationic = + charge (stains cell), Anionic = - charge (stains background)
What does the Gram stain target, and what are the steps?
Cell wall differences; crystal violet → iodine → alcohol → safranin
Why do we use microscopes to observe microorganisms?
Microorganisms are too small to be seen with the naked eye
Who first observed living microorganisms, such as protozoa and bacteria?
Anton van Leeuwenhoek
What is the function of the condenser and iris diaphragm?
Condenser focuses light into a cone; iris diaphragm controls light intensity
What are the basic steps of smear preparation?
Spread → air dry → heat fix → stain → rinse → dry → observe
How do Gram-positive and Gram-negative bacteria look after staining?
Gram-positive = purple; Gram-negative = pink
What’s the difference between what the light microscope and electron microscope can view?
Light microscope views bacteria; electron microscope views viruses and structures smaller than 0.2 µm
What is resolving power and how is it calculated?
RP = λ / (2 × NA); it shows how close two points can be and still be seen as separate
Why is immersion oil used at 100X objective?
Prevents light refraction and improves image clarity by matching glass density
What is simple vs. negative staining and an example of each?
Simple stains the cell (example: methylene blue); Negative stains background (example: nigrosin)
What does the acid-fast stain detect and why is it needed?
Detects Mycobacterium and Nocardia; their waxy cell wall resists normal staining
What is total magnification and how do you calculate it?
Total magnification = objective lens × ocular lens
What does numerical aperture represent in microscopy?
The light-trapping ability of the objective lens
What is the full light pathway in a brightfield microscope?
Light source → condenser → specimen → objective lens → ocular lens → eye
Name three types of differential stains.
Gram stain, acid-fast stain, endospore stain
What is the role of mycolic acid in acid-fast staining?
Mycolic acid repels dye unless heated; dye is trapped inside after cooling
What are two ways to increase contrast while viewing a specimen?
Decrease the light or stain the microorganism
What does it mean if the distance between two points is smaller than the RP value?
You will see a blob (not two distinctive points)
What are the coarse and fine adjustment knobs used for?
Coarse = rough focusing on low power; Fine = detailed focusing on high power
Why is staining important even after reducing light?
Some cells are still invisible without staining and staining enhances visibility and contrast
What stains are used in the endospore stain and what’s the key step?
Malachite green (primary), heat application, safranin (counterstain)