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Biology 2060 > Topic 2: Microscopy & Staining > Flashcards

Topic 2: Microscopy & Staining Flashcards

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1
Q

The quality of an image under the light microscope depends on:

A
  1. Magnification
  2. Contrast
  3. Resolution
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2
Q

Three lenses in a compound microscope

A
  • Condenser
  • Objective
  • Ocular
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3
Q

Magnification

A

The product of the individual magnifications of the ocular and objective lenses

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4
Q

Condenser lense function

A

Focuses the light on the specimen

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5
Q

What minimum magnification is needed to view cells

A

100 x

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6
Q

Resolving power (d)

A

The ability of a lens system to allow you to see two points as being distinct

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7
Q

Do higher or lower values of ‘d’ mean greater resolution

A

Lower

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8
Q

Resolution formula

A 
-d = wavelength/ (2 NA) 
N = refractive index of space between specimen & lens
A = numerical aperture
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9
Q

Resolving power can be maximized by:

A
  • Using a higher magnification

- Use a shorter wavelength of light

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10
Q

Oil immersion

A

The refractive index of oil is much closer to that of glass

- Capture more refracted light

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11
Q

Brightfield

A

Dark objects are visible against a bright background

  • Contrast from light being refracted and absorbed
  • Stain kills specimen
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12
Q

Darkfield

A

Light objects are visible against a dark

  • Only light that is reflected/refracted by the specimen will enter the objective lens
  • Poor detail
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13
Q

Fluorescence Microscopy

A

Adhere fluorescent dyes to the specimen

- Fluorescent substances absorb u.v. light, and emit visible light

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14
Q

Confocal Microscopy

A

Short wavelength light progressively focused at different depths of dyed specimen
- Produces 3-dimensional image

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15
Q

Electron Microscopy

A

Similar to brightfield except that an electron beam is used rather than visible light

  • Wavelength is smaller (magnification is better)
  • Magnification of up to 100,000x
  • TEM & SEM
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16
Q

TEM (Transmission Electron Microscopy)

A

Electron beam passes through specimen then an electromagnetic lens, to a screen/film
- Embedded in special resin

17
Q

SEM (Scanning Electron Microscopy)

A

Electron beams knocks other electrons off the surface of a specimen

  • Specimen usually coated with an electron dense medium
  • Detectors capture the secondary electrons to produce the image
18
Q

Simple Stains

A
  • Use a basic dye
  • Adds contrast based on colour
  • Chromophore has a positive charge
  • Require heat to be fixed
  • Heat causes cells to be distorted
19
Q

What overall charge do bacteria have?

A

Negative

20
Q

What charge do chromophores have?

A

Positive

21
Q

Examples of basic dyes

A

Crystal violet & safranin

22
Q

Negative (acidic) Stains

A
  • Use an acidic dye
  • Chromophore has a negative charge
  • Dye falls to sides of the cells & they appear colourless against a dark background
  • See true size and shape of cells
23
Q

Differential Stains

A

Use simple stains in different ways to visualize specific structure and features of the specimen
- Use mordants & counterstains

24
Q

Mordants

A

Chemicals to modify and enhance the action of the simple stain

25
Q

Counterstains

A

Simple stains that stain structures that weren’t stained the first time
- Makes contrast between these structure clearer

26
Q

Gram Stains

A

Target differing drug resistance characteristics

27
Q

Gram Positive

A
  • Thicker cell wall

- Tend to be killed by Penicillin

28
Q

Gram Negative

A
  • Have an external layer of lipopolysaccharides

- More resistant to antibiotics

29
Q

Counterstain of crystal violey

A

Safranin

30
Q

What color is gram positive bacteria?

A

Purple

31
Q

What colour is gram negative bacteria?

A

Pink/Red

32
Q

Acid-fast Stain

A
  • Used to identify Mycobacteria and Nocardia
  • Have waxy material in cell wall
  • Uses carbol fuschin & counterstains with methylene blue
33
Q

What color do Mycibacteria and Nocardia appear in acid-fast stains?

A

Red

34
Q

Endospore stain

A
  • Bacillus & Clostridium
  • Treat with malachite green
  • Counterstain with safranin
  • Endospores appear green
35
Q

Flagella Stain

A
  • A positive stain from flagella indicates that the specimen cells are motile
  • If flagella present they will appear under brightfield microscopy

Biology 2060 (8 decks)

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