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Microbiology > Visualizing Bacteria > Flashcards

Visualizing Bacteria Flashcards

1
Q

What is a micrometer?

A

Micrometer (µm)

• 0.000 001 m or 10^-6 m

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2
Q

What is a nanometer?

A

Nanometer (nm)

• 0.000 000 001 m or 10^-9 m

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3
Q

What is a light microscope?

A

• Any type of microscope using visible light to observe specimens

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4
Q

What type of microscope uses a series of lenses to magnify a specimen?

A

Compound Light Microscope (LM)

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5
Q

What are the illuminator, condenser, stage, objective lenses, and ocular lenses on a light microscope do?

A
  • Illuminator – light source
  • Condenser – lenses directing light up through specimen
  • Stage – holds microscope slide in position
  • Objective lenses – primary lenses magnifying the specimen, usually 3 of them
  • Ocular lens – eye piece that magnifies the image further
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6
Q

What is total magnification?

A

• Objective lens magnification x ocular lens magnification

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7
Q

What is the strength of the average ocular lens?

A

10x magnification

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8
Q

What is the strength of the average objective lenses?

A

o Low power 10x
o High power 40x
o Oil immersion 100x

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9
Q

Describe resolution

A
  • Also called resolving power
  • Ability of lenses to identify fine detail
  • Ability to distinguish 2 points that are a certain distance apart
  • Most LM have resolution of 0.2 µm
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10
Q

Describe refractive index

A
  • Measure of light bending ability of a medium
  • Hard to see specimens that have the same refractive index medium
  • To increase contrast, stains can be applied which alter the mediums refractive index
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11
Q

Describe immersion oil

A
  • Has same refractive index as glass so it essentially becomes part of the optics
  • Prevents refraction of light away from objective lens improving resolution
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12
Q

Describe bright field illumination

A
  • Field of vision brightly illuminated
  • Usual operation for LM
  • Shows internal structures and outline of transparent microbes
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13
Q

Describe darkfield microscopy

A
  • Used when microbes are invisible to LM and cannot be stained
  • Uses darkfield condenser with opaque disk preventing direct light
  • Only light reflected away from specimen enters objective lens
  • Specimen appears light in a black background
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14
Q

Describe phase contrast microscopy

A
  • One set of light rays from the light source, the other is diffracted (scattered) from a particular structure in the specimen
  • Allows structures to be sharply defined
  • Does not kill the organism
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15
Q

Describe differential interference contrast (DIC) microscopy

A
  • Uses 2 light beams with prisms splitting each beam

* Results in high resolution and brightly coloured, nearly 3D image

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16
Q

Describe fluorescence microscopy

A
  • Fluorescence is the ability of substances to absorb short wavelengths (UV) light and give off longer wavelength (visible) light
  • Some specimens do this naturally, others can be stained with fluorescent dye (fluorochrome)
  • Appear bright on a black background
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17
Q

Describe fluorescent antibody (FA) technique

A
  • Also called immunofluorescence
  • Diagnostic technique
  • Antibodies removed from serum of infected host to a specific antigen
  • Antibodies combined with a fluorochrome
  • Added to a slide of unknown bacteria
  • If the bacterium is the same, the antibodies bind to the bacterium causing it to fluoresce
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18
Q

Describe confocal microscopy (CF)

A
  • Uses short blue wavelength light
  • Specimen stained with fluorochrome
  • Single plane illuminated at a time that provides exceptionally clear 2 D image
  • Can be used with computers to keep scanning constructing 3 D image
  • Can also be used to monitor distribution and concentrations of substances
  • Detail up to depth of <100 µm
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19
Q

Describe two-photon microscopy

A

Two-Photon Microscopy (TPM)

  • Specimens stained with fluorochrome
  • Uses long-wavelength (red) light
  • Detail in tissues up to 1mm (1000 µm) deep
  • Can track cells in real time
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20
Q

Describe Super-Resolution Light Microscopy

A

Super-Resolution Light Microscopy (SRM)

• Uses 2 lasers
o 1 stimulates fluorescent molecules to glow
o 1 cancels out everything but a single desired nanometer
• With the help of a computer, scans nm by nm and puts the images together
• Allows for even a single molecule to be tracked through the cell

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21
Q

Describe scanning acoustic microscopy

A

Scanning Acoustic Microscopy (SAM)

  • Uses soundwaves that reflect off specimens
  • Resolution about 1 µm
  • Used to study living cells on another surface
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22
Q

Describe electron microscopy. What are the 2 types?

A

• Uses a beam of electrons that have a much shorter wavelength than light (~100 000x)
• Can be used for objects smaller than 0.2 µm
• Images are black and white but can be artificially coloured
Transmission Electron Microscopy (TEM)
Scanning Electron Microscopy (SEM)

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23
Q

Describe transmission electron microscopy

A

Transmission Electron Microscopy (TEM)
• Uses ultra thin section of specimen
• Electromagnetic condenser lens focuses electrons in straight line to illuminate specimen
• Specimen placed on copper mesh
• Objects can be magnified up to 10 000 000x
• Transmission electron micrograph – final digital image created
o Appears as light and dark areas depending on electron absorption
• Stains can be used to increase contrast (usually metals)
o Positive staining – metals fixed to specimen
o Negative staining – used to increase opacity of surrounding field
 Helpful for very small viruses, proteins etc.
• Shadow casting – metal sprayed at 45o that creates shadow effect
• Specimen must be fixed, dehydrated, and viewed under high vacuum
• Artifacts – distortions caused by preparation method that appears to be additional structures

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24
Q

Describe scanning electron microscopy

A

Scanning Electron Microscopy (SEM)
• Provides 3D views of specimens
• Scanning electron micrograph – digital image produced
• Uses electrons that knock electrons out of the specimen’s surface
• Useful for studying surface structures
• Objects can be magnified up to 500 000x

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25
Q

Describe scanned-probe microscopy. What are the two types?

A

• Uses probes to examine the surface of a specimen using electric current
• Can be used to map as small as atomic and molecular structures
Scanning Tunneling Microscopy
Atomic Force Microscopy (AFM)

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26
Q

Describe scanning tunneling microscopy

A
  • Uses tungsten probe
  • Shows bumps and depressions of the atoms of the specimen’s surface
  • Can resolve features only 1/100 the size of an atom
  • Special preparation not needed
  • Used to provide details of DNA
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27
Q

Describe atomic force microscopy

A

Atomic Force Microscopy (AFM)
• Metal and diamond probe that move along the surface of a specimen and 3D digital image is produced
• No special preparation required
• Can image biological substance in nearly atomic detail

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28
Q

What is staining?

A

o Colouring microorganisms with dye emphasizing certain structures

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29
Q

How is a specimen fixed onto the slide?

A

• A film (smear) of material containing microorganisms is spread over the slide and allowed to air dry
o Passing through Bunsen burner several times, smear side up or cover slide with methanol for 1 min
o Kills microorganism and preserves it with minimal distortion

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30
Q

What are stains?

A
  • Salts with a positive and negative ion

* Chromophore – one ion that is coloured

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31
Q

Describe basic dyes

A
  • Chromophore is in the cation (+)
  • Attracted to the negatively charged bacterial cell
  • Crystal violet, methylene blue, malachite green, and safranin
  • More common than acidic dyes
32
Q

Describe acidic dyes

A

• Chromophore is in the anion (-)
• Eosin, acid fuchsin, and nigrosine
• Negative staining
o Colours the background
o Repelled by bacterial surface (+ to +) so the background is stained
o Able to observe overall cell shape, size, and capsules

33
Q

What is a mordant?

A

o Chemical added to intensify the stain

34
Q

What are simple stains?

A

• Aqueous or alcohol solution of single basic dye
• Highlights entire organism
• Mordant
o Chemical added to intensify the stain
• Includes methylene blue, carbolfuchsin, crystal violet, and safranin

35
Q

What are differential stains and what are some examples?

A

• React differently with different kinds of bacteria and can be sued to distinguish them

Gram Stains
Acid-Fast Stain

36
Q

How is a slide prepared for gram staining?

A
  • Heat fixed smear
  • Primary stain – purple dye colours all the cells, then washed off
  • Mordant, iodine, applied and then washed off
  • Decolourizing agent – alcohol or alcohol-acetone solution used to wash slide that removes dye from some species but not others, then slide is rinsed
  • Counterstain – slide stained with safranin, a basic red dye, then washed off providing contrast to the purple
  • Slide is dried and then examined
37
Q

What are the results of a gram stain? What does this mean?

A

• Gram positive bacteria
o Bacteria that retain colour
o Have a thicker peptidoglycan cell wall
o Tend to be killed by penicillin and cephalosporin
• Gram negative bacteria
o Bacteria that loses purple colour so appears pink with counterstain
o Have a layer of lipopolysaccharide as part of the cell wall
 Disrupted by alcohol so loses the purple colour
• Some bacteria do not stain or do not stain well
• Some antibiotic resistance is due to bacterial inactivation of antibiotics

38
Q

What is an acid fast stain? What are the genus of bacteria that can be identified this way?

A 
•	Bind strongly to bacteria with waxy material in their cell walls 
•	Identifies
o	Genus Mycobacterium
	M. tuberculosis – tuberculosis 
	M. leprae – leprosy
o	Genus Nocardia
39
Q

How is the slide prepared for an acid fast stain?

A
  • Red dye carbolfuchsin applied to a fixed smear
  • Gently headed for a few min to enhance penetration
  • Gently heated for a few min to enhance penetration
  • Cooled and washed with water
  • Decolourized with acid alcohol
  • Counterstained with methylene blue
40
Q

What are the results of the acid fast stain?

A
  • Acid fast bacteria appear red

* Non-acid fast bacteria appear blue

41
Q

Describe negative staining for capsules

A
  • Presence of capsules is a means of determining an organism’s virulence
  • Bacteria mixed in a solution of fine colloidal suspension of coloured particles providing a contrasting background
  • Simple stain such as safranin applied
  • Capsules appear as halos surrounding each cell
42
Q

Describe endospore staining and how is the slide prepared to view endospores?

A

Endospore Staining
• Only formed by a few genera
• Resistant, dormant structure formed within a cell protecting bacteria from harsh environmental conditions
• Endospores highly refractive and can be seen under LM but not distinguished from other stored material

Schaeffer-Fulton Endospore Stain
• Heat fixed smear
• Primary stain, malachite green, heated to steaming for 5 min
• Washed for 30 seconds with water
• Counterstain safranin applied
• Endospores appears green within red or pink cells

43
Q

Describe flagella staining

A
  • Carbolfuchsin and a mordant build up flagella until visible
  • Number and arrangement of flagella as diagnostic aids
44
Q

1 micrometer is how many meters?

A

0.000 001 m

45
Q

10^-9 m is equivalent to what?

A

1 nm

46
Q

How many nanometers is 1 micrometer?

A

1000

47
Q

Which type of microscope would be best to use to observe a stained bacterial smear?

A

compound light microscope

48
Q

Which type of microscope would be best to use to observe an unstained bacterial cells in which the cells are small, and no detail is needed?

A

darkfield microscope

49
Q

Which type of microscope would be best to use to observe an unstained live tissue when it is desirable to see some intracellular detail?

A

phase contrast microscope

50
Q

Which type of microscope would be best to use to observe a sample that emits light when illuminated with UV light?

A

Fluorescence microscope

51
Q

Which type of microscope would be best to use to observe intracellular detail of a cell that is 1 micrometer long?

A

Transmission electron microscope

52
Q

Which type of microscope would be best to use to observe unstained live cells in which intracellular structures are shown in colour?

A

differential interference contrast microscope

53
Q

Calculate the total magnification of the nucleus of a cell being observed through a compound light microscope with a 10x ocular lens and an oil immersion lens

A

10 x 100 = 1000x magnification

54
Q

What is the maximum magnification of a compound microscope?

A

1500 x

55
Q

What is the maximum magnification of an electron microscope?

A

10 000 000x

56
Q

What is the maximum resolution of a compound microscope?

A

0.2 micrometers

57
Q

What is the maximum resolution of an electron microscope?

A

10 pm

58
Q

What is one advantage of a scanning electron microscope over a transmission electron microscope?

A

Seeing 3D detail

59
Q

What is a mordant used in the Gram stain? In the flagella stain?

A

In Gram stain, the mordant combines with the basic dye to form a complex that will not wash out of the gram-positive cells.

In a flagella stain, the mordant accumulates on the flagella so they can be seen with a light microscope

60
Q

What is the purpose of a counterstain in the acid-fast stain?

A

Stains the colourless non-acid fast cells so that they are easily seen through a microscope

61
Q

What is the purpose of a decolourizer in the Gram stain? In the acid-fast stain?

A

In the Gram stain, the decolourizer removes the colour from gram-negative cells

In the acid-fast stain, the decolourizer removes the colour from non-acid-fast cells

62
Q

What is a pm?

A

picometer

63
Q

How many pm in a nm?

A

1000

64
Q

How does the gram-positive cells appear after each step of the Gram stain process?

A

crystal violet - purple
iodine - purple
alcohol-acetone - purple
safranin - purple

65
Q

How does the gram-negative cells appear after each step of the Gram stain process?

A

crystal violet - purple
iodine - purple
alcohol-acetone - colourless
safranin - pink/red

66
Q

A sputum stample from Calle, a 30 year old asian elephant, was smeared onto a slide and air dried. The smear was fixed, covered with carbolfuchsin, and heated for 5 minutes. After washing wtih water, acid-alcohol was placed on the smear for 30 seconds. Finally, the smear was stained with methylene blue for 30 seconds, washed with water, and dried.

On examination at 1000x, the zoo veterinarian saw red rods on the slide. What microbe do these suggest?

A

Acid-fast bacteria such as Mycobacterium or Nocardia

67
Q

Assume you stain Bacillus by applying malachite green with heat and then counterstain with safranin. Through the microscope, the green structures are

a) cell walls
b) capsules
c) endospores
d) flagella
e) impossible to identify

A

C

68
Q

Three-dimensional images of live cells can be produced with

a) darkfield microscopy
b) fluorescence microscopy
c) transmission electron microscopy
d) confocal microscopy
e) phase-contrast microscopy

A

D

69
Q

Carbolfuchsin can be used as a simple stain and a negative stain. As a simple stain, the pH is

a) 2
b) higher than the negative stain
c) lower than the negative stain
d) the same as the negative stain

A

B

70
Q

Looking at the cell of a photosynthetic microorganism, you observe the chloroplasts are green in brightfield microscopy and red in fluorescence microscopy. You conclude

a) chlorophyll is fluorescent
b) the magnification has distorted the image
c) you’re not looking at the same structure in both microscopes
d) the stain masked the green colour
e) none of the above

A

A

71
Q

Which of the following is NOT a functionally analogous pair of stains?

a) nigrosin and malachite green
b) crystal violet and carbolfuchsin
c) safranin and methylene blue
d) ethanol-acetone and acid-alcohol
e) all of the above pairs are functionally analogous

A

A

72
Q

Which of the following pairs is mismatched?

a) capsule - negative stain
b) cell arrangement - simple stain
c) cell size - negative stain
d) Gram stain - bacterial identification
e) none of the above

A

E

73
Q

Assume you stain Clostridium by applying a basic stain, carbolfuchsin, with heat, decolourizing with acid-alcohol, and counterstaining with an acidic stain, nigrosin. Through the microscope, the endospores are _____, and the cells are stained ______.

a) red, black
b) black, colourless
c) colourless, black
d) red, colourless
e) black, red

A

D

74
Q

Assume that you are viewing a Gram-stained field of red cocci and blue rods through the microscope. You can safely conclude that you have

a) made a mistake in staining
b) two different species
c) old bacterial cells
d) young bacterial cells
e) none of the above

A

B

75
Q

In 1996, scientists described a new tapeworm parasite that had killed at least one person. The initial examination of the patient’s abdominal mass was most likely made using

a) brightfield microscopy
b) darkfield microscopy
c) electron microscopy
d) phase-contrast microscopy
e) fluorescence microscopy

A

A

76
Q

Which of the following is NOT a modification of a compound light microscope?

a) brightfield microscopy
b) darkfield microscopy
c) electron microscopy
d) phase-contrast microscopy
e) fluorescence microscopy

A

C

Microbiology (7 decks)

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