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Microbiology1 > Ch 3: Observic Microorganisms Through a Microscope > Flashcards

Ch 3: Observic Microorganisms Through a Microscope Flashcards

1
Q

One Angstrom (Å) is equivalent to ____ or ____.

A

10^-10 m
0.1 nm

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2
Q

What are the typical magnification ranges for objective and ocular lenses in a compound light microscope?

A

Objectives: 5 - 100x
Oculars: 10 - 20x

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3
Q

What does it mean when a microscope has a resolving power of 0.2 nm?

A

It can distinguish two points ≥0.2 nm

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4
Q

Total magnification = _____?

A

Objective lens magnification x ocular lens magnification

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5
Q

The ultimate limit to the resolution of a light microscope is set by __________?

A

The type of radiation it utilizes

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6
Q

Do shorter or longer wavelengths of light provide greater resolution?

A

Shorter

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7
Q

Resolution = ______

A

Resolution = (0.61*λ) / (n*sinθ)

λ = wavelength of light used

n = refractive index of medium

θ = half the angular width of the cone of rays by the objective lens from a typical point in the specimen

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8
Q

In an electron microscope, with an accelerating voltage of 100,000 V, the wavelength of an electron is ______?

A

0.004 nm

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9
Q

What is the refractive index of air?

A

1

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10
Q

For white light, the wavelength that is commonly assumed is _____.

A

0.53 μm

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11
Q

Define “refractive index”

A

A measure of the light-bending ability of a medium; dependent on how much the speed of light is reduced in the medium

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12
Q

Why does using immersion oil produce a better resolution in a microscope?

A

Immersion oil has the same refractive index as glass, thus does not bend the light

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13
Q

What are the six types of light microscopy?

A
  1. Brightfield
  2. Darkfield
  3. Phase-contrast
  4. Differential interference contrast (DIC/Nomarski)
  5. Fluorescence
  6. Confocal
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14
Q

In _____ microscopy, dark objects are visible against a bright background.

A

Brightfield

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15
Q

True or false. Brightfield microscopy is only useful for stained biological specimens.

A

True. Unstained cells are virtually invisible

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16
Q

What are some benefits of darkfield microscopy?

A

Useful for live organisms not visible with light, can’t be stained, or distorted by staining

Can be used to visualize very thin microorganisms (ex: Treponema pallidum)

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17
Q

Phase-contrast microscopy is useful because ___________

A

internal structures of a cell become more sharply defined, permitting detailed examination of living tissue

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18
Q

What advantage does DIC microscopy have over phase-contrast?

A

Allows for visualization of depth

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19
Q

Both phase-contrast and DIC microscopy _____. (3)

A
  1. Can be used with living cells
  2. Don’t require cell fixation/attachment slides
  3. Don’t require staining
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20
Q

True or false. Fluorescence microscopy takes advantage of fluorochromes which absorb long wavelengths of light (UV or near UV) and emit another, shorter wavelength (visible).

A

False. They absorb short and emit long

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21
Q

Describe the path of light in fluorescence microscopy

A
  1. Barrier filter lets only one wavelength of light through
  2. Beam-splitting mirror reflects light of below one wavelength but transmits light above another
  3. Second barrier filter cuts out unwanted noise before reaching eyepiece
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22
Q

Describe how immunofluorescence works

A

Antibodies conjugated to fluorochromes bind to specific antigens in the specimen of interest

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23
Q

What advantage does confocal microscopy have over standard fluorescence?

A

Illuminate individual planes of light in order to generate a 3D image and visualize depth

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24
Q

What two conditions can cause photobleaching of a specimen stained with a fluorochrome?

A

Exposure to high wavelengths of light

Long exposure times

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25
Q

What is the advantage of two-photon microscopy?

A

Study cells within live tissue up to 1mm deep (1000 μm is the limit for confocals)

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26
Q

Super-resolution fluorescence can increase resolution from 200nm to ____?

A

20 nm

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27
Q

What is the resolution of electron microscopes?

A

0.002 nm

28
Q

True or false. Transmission electron microscopy requires a special preparation that makes it possible to view live cells.

A

False. It is not possible to view live cells in TEM

29
Q

Describe the fixation process for TEM

A
  1. Glutaraldehyde fixes proteins while osmium tetroxide binds and stabilizes proteins and lipids
  2. Dehydrate specimen and permeate it with monomeric resin to form a block of plastic
30
Q

In TEM, elements with higher atomic numbers _____

A

scatter electrons better and produce greater contrast

31
Q

Contrast in TEM can be increased by impregnation with ______

A

Salts of heavy metals

Ex: Ur [92], Pb [82], Os [76]

32
Q

What is the difference between positive and negative staining?

A

Positive: stains sample

Negative: stains everything but the sample

33
Q

When is negative staining preferred? (2)

A
  1. In living cells
  2. Visualization of VERY small samples
34
Q

What is the major difference between SEM vs. TEM?

A

SEM allows for visualization of a whole specimen

35
Q

How does SEM work?

A

An electron gun produces a beam of primary electrons that scans the surface of a whole specimen. A secondary electron is emitted from the surface of the specimen to produce an image

36
Q

What are the advantages of SEM over TEM? (4)

A
  1. Sectioning not required (still must be fixed)
  2. Provides great depth of field
  3. 3D appearance
  4. Smaller, simpler, and cheaper than TEM
37
Q

What are the limitations of SEM? (2)

A
  1. Only surface features can be examined
  2. Lower resolution than TEM (~10 nm)
38
Q

How does scanning tunneling microscopy (STM) work?

A

Uses a sharp probe to scan over the surface of a sample

Electrons flow from surface to probe tip (tunneling)

39
Q

Resolution of STM is not constrained by the wavelength of light or electrons, thus can resolve ______

A

at the atomic level

40
Q

Atomic force microscopy was developed to overcome what drawback to STM?

A

STM can only image conducting or semiconducting surfaces

41
Q

How does atomic force microscopy (AFM) work?

A

A probe is gently forced down onto a specimen. As the probe moves along the surface its movements are recorded

42
Q

True or false. AFM does not require special specimen prep.

A

True

43
Q

How are microbes fixed for brightfield microscopy?

A

Heat

44
Q

In basic dyes, the chromophore is a ____ and typically neutralized by ____

A

Cation

Cl- ion

45
Q

In acidic dyes, the chromophore is ______ and is typically neutralized by ______.

A

Anion

Na+ ion

46
Q

Will bacteria stain with a more acidic dye or a more basic dye?

A

Basic dye

Bacteria are slightly negatively charged at pH 7, thus the colored positive ion of the basic dye is attracted to the bacterial cell

47
Q

Negative staining is useful for determining _____.

A

Overall cell morphology size and shape, as they are highly visible against a contrasting background

48
Q

What is simple staining? Examples?

A

Method of staining of microorganisms with a single basic dye

49
Q

What are some examples of simple stains? (4)

A

Methylene blue

Carbolfusion

Crystal violet

Safranin

50
Q

What is the primary goal by using a simple stain?

A

Highlight the entire microorganism to visualize cellular shapes and basic structures

51
Q

What is differential staining?

A

Staining method that uses more than one chemical stain to differentiate between different organisms

52
Q

What are some examples of differential stains?

A

Gram stain

Acid-fast stain

53
Q

Who developed the Gram stain?

A

Danish bacteriologist Hans Christian Gram

54
Q

Describe the process of Gram staining?

A
  1. Bacteria are fixed
  2. Crystal violet stains peptidoglycan
  3. wash
  4. Iodine treatment complexes with crystal violet
  5. wash
  6. Decolorization with alcohol
  7. Counter stain with safranin
55
Q

Does cystal violet or safarin stain stronger?

A

Crystal violet

56
Q

What is the role of the alcohol washings in both Gram-positive and Gram-negative stains? Do CV-I crystals remain in the sample?

A

Gram-positive: alcohol dehydrates thick peptidoglycan layer; CV-I crystals do not leave

Gram-negative: dissolves outer membrane, leaving behind a thin peptidoglycan layer; CV-I crystals wash out

57
Q

What is mordant?

A

A substance added to a staining solution to hold the stain or coat the specimen to make it thicker and easier to see after staining

58
Q

What are the structural differences between Gram-positive vs. Gram-negative bacteria?

A

Gram-positive: thicker peptidoglycan cell wall

Gram-negative: thinner peptidoglycan cell wall, but have a lipopolysaccharide (LPS) outer membrane

59
Q

Acid-fast stain only binds strongly to ______. Examples?

A

bacteria that have waxy material in their cell wall.

Ex: Mycobacterium (M. tuberculosis, M. leprae) and Nocardia

60
Q

Describe the process of an acid-fast stain.

A
  1. Fixation
  2. Carbol Fuschsin steam 5 min
  3. Cool water wash
  4. Acid alcohol wash
  5. Counterstain with methylene blue
61
Q

What type of staining can reveal the presence of capsulated microbes? Stain examples?

A

Negative staining

Basic dye (Safranin)

62
Q

What is the significance of being able to detect if a microbe is capsulated or not?

A

Capsulated microbes typically cause disease

63
Q

Describe the process for Endospore staining.

A
  1. Stained with Malachite green
  2. steam 5 min
  3. Rinse 30 sec
  4. Counterstain Safranin
64
Q

True or false. Endospores can be detected in a light microscope without staining.

A

True

65
Q

How does a Flagella stain work? Stain example?

A

A mordant is used to coat flagella with stain until thick enough to be seen.

Ex: Carbolfuschin simple stain

Microbiology1 (20 decks)

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