Microscopy & Staining
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What are the 3 types of microscopes?
- Atomic Force
What is a light microscope?
- Visible light passes through a series of lenses to produce an image.
- Usually magnifies 1000x
What is an electron microscope?
- Microscope that uses electron beams to produce a magnified image.
- Can magnify up to 100,000x
What is an Atomic Force Microscope?
A microscope that uses a probe to move in response to the force between the microscope and specimen to project a map of the bumps and valleys of the specimen.
What is magnification?
Enlarging the size of something as an optical image.
What is resolution?
The ability to measure the angular separation of images that are close together; ie, how "clear" an image is.
What is contrast?
Reflects the number of visible shades in a specimen, with "high contrast" being black and white.
What is resolving power?
- Determines how much detail can be seen.
- Minimum distance between two objects that still appear as separate objects.
What does resolution depend on?
Quality of the lenses and wavelength of illuminating light.
What is the maximum resolving power of most light microscopes?
1 x 10-6
What is immersion oil?
An oil that reduces light refraction, and has nearly the same refractive as glass.
How is higher contrast achieved in microscopy?
What is a scanning electron microscope used for?
Observing surface details of cells.
What is a fluorescent microscope?
Used to observe organisms that are naturally fluorescent, or stained with fluorescent dyes?
What are stains made of?
How do dyes work?
Carry (+) or (-) charge and bind to certain cell structures in a molecule.
How do basic dyes work?
- Carry a positive charge and bond to structures with a negative charge.
- Commonly stain the cell
How do acidic dyes work?
- Carry a negative charge and bond to structures with a positive charge.
- Commonly stain the background.
What are the common basic dyes used?
- Crystal violet
- Methylene Blue
- Malachite Green
What is a simple stain?
Uses one basic stain to increase contrast between specimen and background with no differentiation between cell types.
What is a differential stain?
A stain using a series of reactants to distinguish one bacteria from the other.
What are the two most common types of differential stains?
- Gram stain
- Acid-fast stain
What is a gram stain?
- Most widely used procedure for staining bacteria
- Developed by Dr. Hans Christian Gram
What color are gram stains?
Gram positive - stains purple
Gram negative - stains red or pink
What are the steps in a gram stain?
- Primary Stain - Crystal Violet, Dyes positive and negative cells
- Iodine Treatment - Holds primary dye onto cell
- Decolorizer - removes stain from gram negative cell
- Counter Stain - Safrinin, restains gram negative cell
What is an acid-fast stain?
Stain used to stain organisms that resist convention staining, like Mycobacterium.
What are the steps in acid-fast staining?
- Primary Dye
- Counter Stain
What makes some bacteria difficult to stain conventionally?
High lipid concentration in cell wall prevents uptake of dye.
What does a capsule stain do?
- Stains background
- Allows capsule to stand out around organism
What is an endospore stain?
Uses heat to enhance endospore
What is a flagella stain?
Staining that inscreases the diameter of the flagella, making it more visible.
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