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BIOL 2604 > Microscopy > Flashcards

Microscopy Flashcards

1
Q

Who invented Microscopes?

A

-Robert Hook
-Antoni Van Leeuwenhoek invented a better one.

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2
Q

Magnification light path

A

-visualized image
-ocular lens
-intermediate image (inverted)
-objective lens
-condenser lens
-light source

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3
Q

Magnification

A

-the extent to when the image of an object is larger than itself
-the magnification of an image under the microscope is the product of objective and ocular lens

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4
Q

What is the magnification of an 10x ocular and 20x objective?

A

200 magnification

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5
Q

What is the magnification of an 10x ocular and 100x objective?

A

1000 magnification

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6
Q

How big are most bacterial cells?

A

~1 micron = 1 um =1X10-6 m
-at 1000x magnification they would appear to our eye as if they were 1x10-3 m = 1mm

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7
Q

Resolution or resolving power

A

-the degree to which details are retained in magnified images; the ability to distinguish between two points
-resolution = R= 0.5 λ / NA
-λ = wavelength
-NA = numerical aperture (the light gathering ability of the lens)

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8
Q

What is the best resolution with standard microscopes?

A

-1000x magnification is 200nm = 0.2 microns = 0.2um
-most bacterial cells are ~1um, so they can be easily visualized at 1000x magnification

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9
Q

Bright Field Microscope

A

-most common in general microbiology lab
-light is transmitted through the specimen
-contrast between the background and the cells is generated by absorption or scattering of light

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10
Q

3 things about bright field microscopes

A
  1. Bacteria are transparent
    -staining of the cells is generally required
  2. Kills the cells
  3. Dead cells take up stains
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11
Q

Preparing a slide

A
  1. Preparing a smear -> dry in air
    -spread culture in thin film over slide
  2. Heat fixing and staining -> flood slide w/ stain; rinse and dry
    -pass slide through flame to heat fix
  3. Microscopy
    -place drop of oil on slide; examine with 100x objective lens
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12
Q

Simple Stains

A

-stains are generally charged molecules
-basic and acidic dyes
-bacterial cell surface carries a negative charge, therefore basic stains are most commonly used for the most simple staining methods

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13
Q

Basic dyes

A

-cationic
-positively charged
-ex: methylene blue, crystal violet, safranin
-bind to negatively charged cell components
-polysaccharides, nucleic acid, phospholipoids

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14
Q

Acidic dyes

A

-anionic
-negatively charged
-binds to positively charged cell components
-amino groups -> proteins

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15
Q

Differential Stains

A

-Purpose: to tell one type from another
-Example: ~TB
~gram stain (gram stain positive vs negative)
~Acid fast stain (mycobacteria vs other bacteria)
~Endospore stain (spore within mother cell)

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16
Q

Gram Stain Procedure

A
  1. flood the heat-fixed smear with crystal violet
    -results: all cells purple
  2. Add iodine solution for 1 min
    -All cells remain purple
  3. Decolorize with alcohol briefly about 20 seconds
    -Gram positive cells are purple, gram negative cells are colorless
  4. Counterstain with safranin for 1-2 mins
    -Gram positive cells are purple; gram negative cells are pink to red
17
Q

Why the gram stain works?
Gram Positive

A

-peptidoglycan is thick
1. crystal violet + iodine = largo crystal
2. Ethanol dissolves membranes

18
Q

Why the gram stain works?
Gram Negative

A

-peptidoglycan is thin
1. CV +I

19
Q

Dark Field Microscope

A

-The only light reaching the specimen comes from the side
-The light only enters the objective lens if it is scattered by the specimen
-cells are bright against a dark background (no need to stain)
-Can’t see colors/stains
-provides better resolution than bright field, so we can see smaller cells or structure

20
Q

What organism is so skinny it can’t be seen with bright field but only seen in dark field

A

Borrella

21
Q

Plant Contrast Microscopy

A

-special condenser used so that ALL light entering sample area in IN-PHASE
-The phase difference between diffracted rays through cell and direct rays (through medium) appears as a difference in light intensity
-No staining required (both unstained and stained cells are dark against light background
-can view LIVE cells!

22
Q

Differential Inference Contrast Microscopy (DIC)

A
  1. Uses polarized light and a prism to make two separate light beams
  2. The light beams are then combined through the specimen
  3. Gives a 3-dimensional appearance to cells, even bacteria
23
Q

How does Fluorescence Microscopy work?

A
  1. Exit
    2.Emission
  2. Blue light enters and green light comes back up is what you see
24
Q

Fluorescence Microscopy

A

-light source at wavelength causes specimen to fluoresce at a different wavelength
-some cells exhibit autofluorescence
-Fluorescent dyes are used to stain specific cell structure
-Fluorescently tagged proteins can be specifically localized in cells

25
Q

Transmission electron microscope

A

-The electron that pass through the sample are collected to make the image
-has a vaccum

26
Q

Electron Miscroscopy

A

-electron wavelength is 0.004 nm (compared to 500 nm for light)
-Resolution (R) is 0.2 nm (1,000 fold better than light)
-Magnification up to 100,000 x (compared to 1000x for light)
-Transmission vs Scanning

27
Q

Transmission

A

-2D, thin sections of fixed samples =heavy metal stains
-TEM

28
Q

Scanning

A

-3D
-coat cells with heavy metal
-reflects
-SEM

29
Q

Scanning electron microscope

A

-The electron that are scattered back off the sample are collected to produce the image

30
Q

Atomic force

A

-Tiny stylus is scanned across and just above the surface of the sample
-see atoms

31
Q

Atomic Force Microscope

A

-The weak repulsive forces between the atoms of the stylus and the sample cause the styles to deflect up and down
-The deflections are recorded as a surface contour map.
-can be challenging with live/sticky/flexible samples

BIOL 2604 (17 decks)

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