Microscopy

Question 1

Who invented Microscopes?

Answer 1

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

Question 2

Magnification light path

Answer 2

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

Question 3

Magnification

Answer 3

-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

Question 4

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

Answer 4

200 magnification

Question 5

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

Answer 5

1000 magnification

Question 6

How big are most bacterial cells?

Answer 6

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

Question 7

Resolution or resolving power

Answer 7

-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)

Question 8

What is the best resolution with standard microscopes?

Answer 8

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

Question 9

Bright Field Microscope

Answer 9

-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

Question 10

3 things about bright field microscopes

Answer 10

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

Question 11

Preparing a slide

Answer 11

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

Question 12

Simple Stains

Answer 12

-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

Question 13

Basic dyes

Answer 13

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

Question 14

Acidic dyes

Answer 14

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

Question 15

Differential Stains

Answer 15

-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)

Question 16

Gram Stain Procedure

Answer 16

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

Question 17

Why the gram stain works?
Gram Positive

Answer 17

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

Question 18

Why the gram stain works?
Gram Negative

Answer 18

-peptidoglycan is thin
1. CV +I

Question 19

Dark Field Microscope

Answer 19

-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

Question 20

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

Answer 20

Borrella

Question 21

Plant Contrast Microscopy

Answer 21

-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!

Question 22

Differential Inference Contrast Microscopy (DIC)

Answer 22

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

Question 23

How does Fluorescence Microscopy work?

Answer 23

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

Question 24

Fluorescence Microscopy

Answer 24

-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

Question 25

Transmission electron microscope

Answer 25

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

Question 26

Electron Miscroscopy

Answer 26

-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

Question 27

Transmission

Answer 27

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

Question 28

Scanning

Answer 28

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

Question 29

Scanning electron microscope

Answer 29

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

Question 30

Atomic force

Answer 30

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

Question 31

Atomic Force Microscope

Answer 31

-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