Chapter 3 Microscopy

Question 1

Light Microscope

Answer 1

• Any kind of microscope that uses visible light to observe a specimen
• Compound light microscope - uses two lenses to magnify the image

Question 2

Objective Lens

Answer 2

• Lens closest to the specimen

- Magnifies between 10x-100x

Question 3

Ocular Lens

Answer 3

• Eyepiece

- Magnifies by 10x

Question 4

Calculating Magnification

Answer 4

For a compound microscope: Objective Lens X Ocular Lens= Total Magnification

Question 5

Resolution

Answer 5

• 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

Question 6

Electron Microscope

Answer 6

• 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

Question 7

Transmission (Electron Microscope)

Answer 7

• (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

Question 8

Scanning

Answer 8

(SEM) to see surfaces; less powerful

Question 9

Atomic Force

Answer 9

• (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

Question 10

Clinical Use of the Light Microscope

Answer 10

• 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

Question 11

Steps for a Stain

Answer 11

1) Smear sample on slide
2) Fix with heat
3) Add stain
4) Wash, dry and view

Question 12

How Stains Work

Answer 12

• 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

Question 13

Positive Stains

Answer 13

• Stain will bind to the bacterium
• Bacterium appears coloured
• Background appears clear
• Ex. Crystal Violet

Question 14

Negative Stains

Answer 14

• Will not bind to the bacterium
• Bacterium appears clear
• Background is coloured
• Ex. Nigrosin

Question 15

Gram Stain

Answer 15

• 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

Question 16

Spore Stain

Answer 16

• Stains an internal structure of some bacteria
• Primary stain colours endospores green
• Counterstain (safranin) colours the rest of the cell red (pink)
• Ex. Bacillus anthraces

Question 17

Flagella Stain

Answer 17

• Stains an external structure

- Mordant thickens the flagella so they can be observed under light microscope

Question 18

Acid- Fast Stain

Answer 18

• Detects the presence of a waxy compound in cell wall
• Used to identify the genus Mycobacterium
• Ex. Mycobacterium tuberculosis, Mycobacterium leprae
• Mycobacterium cell wall retains the dye carbon fuschin
• Counterstain with methylene blue stains non acid-fast bacteria and tissue blue

Question 19

Capsule Stain

Answer 19

• Detects a thick layer of polysaccharide outside the cell- capsule
• Negative stain collars the background
• A positive stain colours the cell
• The capsule does not take up most dyes and remains colourless –> Streptococcus pneumoniae