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MBIO1010 > Lecture Set 1 : Part 3 > Flashcards

Lecture Set 1 : Part 3 Flashcards

1
Q

what are the different types of light microscopy?

A

-bright - field
-phase - contrast
-dark - field
-fluorescence

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2
Q

what is the basis of light microscopy?

A

-compound microscopes (2 lenses) that use visible light to illuminate cells
-microorganisms can be very difficult to view (must improve contrast)

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3
Q

what is bright-field microscopy?

A

-slight differences in contrast make the specimen visible against its surroundings

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4
Q

what are the magnification levels of each lens on a compound microscope?

A

-objective lenses are 10x, 40x, 100x
-ocular lens is 10x-20x (10x in our lab)
-total magnification is calculated by multiply the ocular lens magnification by the objective lens magnification

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5
Q

what is the maximum magnification achievable with a light microscope?

A

-2000x

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6
Q

what is magnification?

A

-the ability to make an image larger

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7
Q

what is resolution?

A

-the ability to distinguish 2 objects as seperate and distinct
-key to microscopy
-the light must pass between 2 points for them to be seen as separate objects
-when wavelength of light decreases, resolution improves (violet is shortest, red is the longest)

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8
Q

what is the limit of resolution for a light microscope?

A

-0.2 micrometers (200nm)
-called the resolving power

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9
Q

how do you improve contrast so that microorganisms can be seen better under the light microscope?

A

-staining

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10
Q

how does staining with dyes work to improve contrast?

A

-dyes are organic compounds that bind to specific cellular materials

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11
Q

what are examples of common stains?

A

-methylene blue (basic)
-safranin (basic)
-crystal violet (basic)
-nigrosin (acidic)

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12
Q

what are the different staining techniques?

A

-simple staining
-gram staining
-acid fast stain
-endospore stain

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13
Q

what is simple staining?

A

-use of one dye to colour the specimen
-2 types of dye that differ in the charge of their chromphore

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14
Q

what is a chromophore?

A

-coloured portion of a dye

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15
Q

what are the 2 types of dye for a simple stain?

A

-basic dye
-acidic dye

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16
Q

how does a basic dye work?

A

-has a positively charged chromophore (positive stain)
-binds to the negatively charged molecules on a cell’s surface (nucleic acids + acidic polysaccharides)
-also tends to stain the entire cell surface because it tends to be negatively charged
-used to stain most bacterial cells

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17
Q

how does an acidic dye work?

A

-has a negatively charged chromophore (negative stain)
-repelled by the cells surface (stains the background)
-does not need to be heat fixed

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18
Q

what is the process of simple staining?

A

-smear is prepared on a slide (air dried)
-once air dried the slide is passed through a small pilot flame (only for basic dyes) (may kill some cells)
-the slide is then flooded with stain
-the stain is then rinsed off and the slide dries (only for basic dyes)

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19
Q

what are differential staining techniques?

A

-gram stain
-acid fast stain
-endospore stain

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20
Q

what is the purpose of the gram stain?

A

-separates bacteria into 2 groups by using a primary stain and a counterstain
-groups are based on their cell wall structure

21
Q

what are the 2 groups of bacteria cells?

A

-gram positive cells
-gram negative cells

22
Q

what are the characteristics of a gram positive cell?

A

-cells retain the primary stain
-thick cell wall that allows them to do so
-cells are stained purple

23
Q

what are the characteristics of a gram negative cell?

A

-cells lose the primary stain
-thin cell wall that cannot retain the primary stain
-take the colour of a counterstain
-cells are stained red or pink

24
Q

what is the purpose of using 2 dyes in the gram stain?

A

-to differentiate cells
-both cells will take on the 2nd stain but purple is darker than red or pink so you will not see it in the gram positive cells

25
how can fluorescence microscopy simplify the gram stain?
-gram stain can be reduced to 1 step as gram positive and gram negative cells will fluoresce different colours when treated with a special chemical
26
when is a gram stain often performed?
-to begin characterization of a new bacterium
27
what is the process of a gram stain?
-flood a heat fixed smear with crystal violet (primary stain) for 1 minutes (all cells are purple) -add an iodine solution for 1 minute (all cells stay purple) -decolourize with alcohol for about 20 secs (gram + cells are purple, gram - cells are colourless) -counterstain with safranin for 1-2 minutes (gram + cells = purple, gram - cells = pink/red)
28
what is the purpose of an acid fast stain?
-detects mycolic acid in the cell wall of the genus mycobacterium -mycobacterium will retain the primary stain (acidic) (fushcia/pink) -anything else on the slide will retain the counter stain (basic)(blue)
29
what is an endospore stain?
-allows for visualization of endospores within cells (internal structures that provide resistance to harsh environmental conditions like heat and radiation) -endospores retain the primary stain (malachite green) -cells retain the counterstain (safranin) -ex: bacillus anthracis spores
30
what is mycolic acid? why must it be detected with a different staining technique?
-long fatty acid chains in the cell wall -make it difficult to gram stain
31
what is phase-contrast microscopy?
-phase ring that amplifies differences in the refractive index of the cell and surroundings -improves the contrast of a sample without the use of a stain -can visualize live samples (no heat fixation) -resulting image is dark cells on a light background
32
what is refractive index?
-how much the path of light going through the material is bent or refracted
33
why would we want to improve contrast without staining?
-staining can distort cells and in some cases kill them
34
what is dark field microscopy?
-does not use staining (used to observe bacteria that do not stain well) -can observe motility easier (better resolution) -specimen is illuminated with a hollow cone of light -only refracted light enters the objective lenses -appears as a bright object on a dark background
35
what is an example of a bacteria that does not stain well?
-treponema pallidum -causative agent of syphilis
36
what is fluorescence microscopy?
-used to visualize specimens that fluoresce -emit light of one colour when illuminated with another colour of light -cells may also fluoresce naturally or after staining with a fluorescent dye
37
what is an example of a cell that naturally fluoresces?
-photosynthetic cyanobacteria (have chlorophyll that allows them to do so) -absorbs light at 430nm (blue-violet) -emits at 670nm (red)
38
what is an example of a fluorescent dye?
-DAPI -specifically binds to DNA
39
what are examples of microscopes that can image cells in 3D?
-differential interference contrast (DIC) microscopy -confocal scanning laser microscopy (CSLM) -scanning electron microscopy (SEM)
40
what is differential interference contrast (DIC) microscopy?
-uses a polarizer to create 2 distinct beams of polarized light -the beams of light pass through the specimen and enter the objective lens where they are recombined into 1 beam -gives structures (endospores, vacuoles, and granules) a 3D appearance -structures that are not visible by bright field can sometimes be visible (nucleus)
41
what is confocal scanning laser microscopy (CSLM)?
-uses a computerized microscope coupled with a laser light source to generate a 3D image -computer can focus the laser on single specimen layers -different layers can then be compiled for a 3D image -resolution is 0.1 micrometers
42
what is electron microscopy?
-use electrons instead of photons to image cells and structures -cells must be dead to view (no live samples) -electromagnets function as lenses -whole system operates in a vacuum (need a specific room) -better resolution (value of these microscopes) -2 types
43
why do electron microscopes have a better resolution?
-wavelength of electrons is much shorter than light
44
what are the 2 types of electron microscopes?
-transmission electron microscopes (TEM) -scanning electron microscope (SEM)
45
why are electron microscopes not more used in classroom?
-expensive -take up lots of space -need specific training
46
what is transmission electron microscopy?
-electron beam focused on a specimen by a condenser lens (magnets not glass) -electrons that pass through the specimen are focused by 2 sets of lenses (magnets) (compound microscope) -electrons strike a fluorescent viewing screen -high magnification -high resolution (0.2 micrometers)
47
what are the specifications of a transmission electron microscope?
-high magnification -high resolution (0.2 micrometers) -specimens must be very thin (20-60nm) -cells must be stained with heavy metals (lead or uranium) or else electrons are poorly scattered -metals bind to cell structures and make them more electron dense which allows for visualization of the internal structures
48
what is the difference between TEM and SEM?
-TEM = e- going through a sample (internal structures) -SEM = e- bouncing off the surface (external structures)
49
what is scanning electron microscopy?
-specimen is coated with a thin film of metal -an electron beam scans the object -scattered electrons are collected by a detector and an image is produced -accurate 3D image of the specimen's surface -wide range of magnifications

MBIO1010 (28 decks)

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