Chapter 3 Microscopy Flashcards
1
Q
Light Microscope
A
- Any kind of microscope that uses visible light to observe a specimen
- Compound light microscope - uses two lenses to magnify the image
2
Q
Objective Lens
A
- Lens closest to the specimen
- Magnifies between 10x-100x
3
Q
Ocular Lens
A
- Eyepiece
- Magnifies by 10x
4
Q
Calculating Magnification
A
For a compound microscope: Objective Lens X Ocular Lens= Total Magnification
5
Q
Resolution
A
- Ability to distinguish fine detail
- Ability to distinguish 2 points a certain distance apart (ex. 4nm)
- Two points can be distinguished if they are at least 4 nm apart
- Light must pass between 2 objects for them to be seen as 2 separate things
- Need light of a short- enough wavelength to fit between them, otherwise will appear 1 object
- Resolution general principle: shorter the wavelength, the better the resolution
6
Q
Electron Microscope
A
- Uses electrons instead of light
- Electrons travel in much shorter waves than light
- Resolving power is greater
- Allows greater magnification (up to 500 00x)
- Allows us to view viruses and internal cell structures
7
Q
Transmission (Electron Microscope)
A
- (TEM) to see internal structure
- Very thin slices can be cut from sample - thin sections
- Samples generally stained with a metal (ex. Osmium, Uranium) to make structures opaque to electrons6
8
Q
Scanning
A
(SEM) to see surfaces; less powerful
9
Q
Atomic Force
A
- (AFM) to see molecules
- Uses this metal probe to scan a specimen revealing bumps depressions
- Human eye can see an object about 0.2mm
- Compound light microscope can view an object about 0.2um
- Electron microscope can view an object about 2nm
10
Q
Clinical Use of the Light Microscope
A
- Use stains to see microorganisms that are colourless
- Stains are composed of positively and negatively charged ions, one of which is coloured - Chromophore
- Simple Stain: Only one dye used to highlight the entire microorganism
11
Q
Steps for a Stain
A
1) Smear sample on slide
2) Fix with heat
3) Add stain
4) Wash, dry and view
12
Q
How Stains Work
A
- Bacteria have a net negative charge on their outer surface
- This charge attracts stains with positively charged chromophores, and repels stains with negatively charged chromophores
13
Q
Positive Stains
A
- Stain will bind to the bacterium
- Bacterium appears coloured
- Background appears clear
- Ex. Crystal Violet
14
Q
Negative Stains
A
- Will not bind to the bacterium
- Bacterium appears clear
- Background is coloured
- Ex. Nigrosin
15
Q
Gram Stain
A
- Differentiates bacteria based on the structure of the cell wall
- Bacteria with a thick cell wall retain the primary stain crystal violet and appear purple = Gram positive –> Ex. Streptococcus Pyogenes
- Bacteria with a thin cell wall lose crystal violet during destaining, take on the colour, take on the colour of the counterstain safranin and appear pink= Gram negative –>E.coli
