1.3 Microscopy

Question 1

can we see the flagellum in motile bacteria under a light microscope?

Answer 1

No its much smaller than the bacteria.

Question 2

the human eye can see organisms greater than _ um

Answer 2

100 um (micrometres)

Question 3

what do compound light micoscopes use to illuminate cells

Answer 3

Visible light.

Question 4

what are the 4 types of light microscopy?

Answer 4

• Dark-field
• Bright-field
• Phase-contrast
• Fluorescence
  (Dark Boston Pizza Friends)

Question 5

How does a bright-field microscope work?

Answer 5

• Slight differences in contrast make specimen visible against surroundings
• Two sets of lenses form the image: objective lens, and ocular lens

Question 6

what does a condensor do

Answer 6

• focuses light into a line.

Question 7

what is a phase-contrast microscope useful for?

Answer 7

• very small and transparent specimens

Question 8

how does a dark-field microscope work?

Answer 8

Makes the speciment appear light against a dark background.

dark-field, like dark background

Question 9

what does a fluorescence microscope need to work

Answer 9

• needs certain molecules that fluoresce
• usually needs stain, sometimes happens naturally

Question 10

what is the objective lens magnification range

Answer 10

10x - 100x

objectives are on the bottom right above the specimen

Question 11

what is the ocular lens magnification range

Answer 11

10x - 20x

most microscopes are 10x some have 20x

ocular lens are at the top what you look through next to ur eyes

Question 12

what’s the total magnification formula

Answer 12

total magnification = ocular X objective

usually ocular is 10x

Question 13

what is the maximum magnification achievable with a light microscope

Answer 13

2000x

only possible with 20x ocular lens.

Question 14

what’s the difference between magnification and resolution

Answer 14

• Magnification is the ability to make an image larger
• Resolution is the ability to distinguish two objects as separate and distinct

Question 15

what must happen for two things to be viewed as separate objects?

Answer 15

Light must pass between them for them to be viewed as separate objects.

Question 16

as wavelength descreases resolution improves or gets worse?

Answer 16

as wavelength decreases, resolution improves.

Question 17

what is the limit of resolution (resolving power) for light microscope

Answer 17

0.2 um

(space between two points that light must pass through, if smaller better resolution/resolving power)

Question 18

what’s the purpose of staining

Answer 18

to improve contrast

Question 19

what are staining dyes

Answer 19

Organic compounds that bind to specific cellular materials.

Question 20

what are 3 examples of common stains

Answer 20

1. Methylene blue (not gonna work with)
2. Safrinin
3. Crystal violet

all work the same

Question 21

what is simple staining

Answer 21

One dye is used to color specimen.
Dye contains chromophore

Question 22

what is chromophore

Answer 22

coloured portion of a dye (usally charged portion

Question 23

what are the two types of simple staining? describe them, which stains cells which stains background?

Answer 23

• Basic dye- positively charged chromophore, binds to negatively charged molecules on cell surface, staining cells.
• Acidic dye-negatively charged chromophore, repelled by cell surface, used to stain background, a negative stain.

Question 24

what are differential stains used for

Answer 24

to learn something about the bacteria, reveal structures and properties

Question 25

How does the Gram stain work

Answer 25

It separates bacteria into 2 groups based on cell wall structure 1. Heat fixed smear is flooded with crystal violet stain for 1 min 2. Iodine solution is added for 1 min (helps violet stick rly well) 3. Stain is decolorized with alchohol briefly (about 20 sec) 4. Counterstain is added (safrinin) for 1-2 min (so it's easier to see gram negative cells that lost their colour) * Gram positive -cells that retain a primary stain, and appear purple * Gram negative -cells that lose a primary stain, take colour of counterstain and appear red or pink.

Question 26

describe how acid fast stains work

Answer 26

* Detect mycolic acid in the cell wall of the genus mycobacterium. * Mycobacterium will retain the primary stain, and appear fushia (pink). * Anything else on slide will appear the color of counterstain (blue).

Question 27

describe how endospore stains work

Answer 27

* Endospores retain primary stain (green) * and the cells appear the colour of the counterstain (pink)

Question 28

How does Phase-contrast microscopy work? and what is it used for

Answer 28

* Phase ring amplifies differences in the refractive index of cell and surroundings, resulting image is dark cells on a light background. * Improves the contrast of a sample without the use of a stain. * Allows for the visualization of live samples.

Question 29

How does dark field microscopy work? What is it used for?

Answer 29

* Specimen is illuminated with a **hollow cone of light** * Only refracted light enters the objective (light that goes through the specimen, if light misses the specimen it also misses the lens. * Specimen appears as a bright object on a dark background. -Used to observe bacteria that don't stain well (don't have a negative charge on surface).

Question 30

How does fluorescence microscopy work? What is it used for?

Answer 30

* light of one colour is emitted when illuminated with another colour of light * Used to visualize specimens that fluoresce (cells may fluoresce naturally or are stained with fluorescent dye (ex: DAPI which binds to DNA) ## Footnote ex: photosynthetic cyanobacteria have chlorophyll that absorbs light at 430 nm (blue-violet), and emits light at 670 nm (red)

Question 31

when is DAPI fluorescent dye used?

Answer 31

* When you're having trouble differentiating bacteria from other things on the slide, all cells would glow (as they have DNA), other things won't glow.

Question 32

How does differential interference contrast (DIC) microscopy work? What's unique about what's visible with DIC

Answer 32

* Uses a polarizer to create two distinct beams of polarized light. * gives structures such as endospores, vacuoles, and granules a 3-D appearance. * and structures not visible by bright-field microscopy are sometimes visible by DIC (like nucleus).

Question 33

How does confocal scanning laser microscopy work? What's unique about what CSLM can do?

Answer 33

* it works by using a computerized microscope coupled with a laser light source to generate a 3D image. * What's unique: -Computer can focus the laser on single layers of the specimen. -resolution is 0.1 um. (better than light microscope)

Question 34

How does an electron microscope work? What's unique about what it can do?

Answer 34

* electron microscopes use electrons instead of photons to image cells and structures * What's unique: -wavelength of electrons is much shorter than for light > higher resolution

Question 35

what are the two types of electron microscopes

Answer 35

* transmission electron microscopes (TEM) * scanning electron microscopes (SEM)

Question 36

How does a transmission electron microscope work?

Answer 36

* Electron beam is focused on specimen by a condensor lens (which is made of magnet not glass) * Electrons that pass through the specimen are focused by two sets of lenses (compound microscope) * Electrons strike a fluorescent viewing screen (since we can't see electrons with our eye)

Question 37

Transmission electron microscopy has high or low magnification and resolution? What's the resolving power?

Answer 37

* 0.2 nm (NANOMETERS, more powerful that brightfield)

Question 38

what are the requirements to view a specimen under a transmisson electron microscope?

Answer 38

* specimen must be very thin (20-60 nm) * must be stained with metals -lead or uranium (unstained cells do a poor job of scattering electrons)

Question 39

what does staining with metals do in TEM? what metals are used for staining? ## Footnote transmission electron microscopy

Answer 39

* metals bind to cell structures to make them more electron dense * which allows for visualization of internal structures * lead or uranium metal is used

Question 40

what's a downside of TEM

Answer 40

The specimen is very manipulated so not alive and not completely accurate as its not in the form it would be found in in nature.

Question 41

is resolution better in light or transmisson electron microscope? why?

Answer 41

Resolution is better in a transmission electron microscope because of shorter wavelength of electrons.

Question 42

How does scanning electron microscopy work? what does it allow for?

Answer 42

* The 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 * it allows for an accurate 3D image of specimen's surface.