Chapter 3: Microscopy And Staining

Question 1

Nanometers (nm)

Answer 1

10^-9

- measure viruses

Question 2

Micrometers (um)

Answer 2

10^-6

- measure bacteria

Question 3

Light properties

Answer 3

Wavelength
Resolution
Light travel

Question 4

Wavelength (lambda)

Answer 4

Length of a light ray

• measure distance between two adjacent peaks or troughs
• average visible light wavelength is .55um

Question 5

Resolution

Answer 5

Ability to separate two objects as distinct

- the shorter the wavelength of light, the better resolution

Question 6

Reflection

Answer 6

Light bounces off the object

Question 7

Transmission

Answer 7

Passes through the object

Question 8

Absorption

Answer 8

Light is absorbed by the object

- luminescence can occur (wavelength is changed and re-emitted by the object)

Question 9

Florescence (glow)

Answer 9

Re-emitted light is only visible when the object is being irradiated (given light)

Question 10

Phosphorescent (glow in the dark)

Answer 10

Light continues to be re-emitted after irradiation stops

Question 11

Refraction

Answer 11

Light bends as it passes through the media

- oil prevents refraction in microscope

Question 12

Diffraction

Answer 12

Light bending around a split

Question 13

Bright field microscopy

Answer 13

Cells are slightly darker on a light background

- difficult to see without staining

Question 14

Dark field microscopy

Answer 14

Cells appear light on a dark background

Question 15

Phase contrast microscopy*

Answer 15

A special condenser and objective lenses are used to distinguish small differences in index of refractions
- can view living organisms

Question 16

Nomarski (differential indifference)*

Answer 16

• same ideas as phase contrast, but focus is on a single level or shorter depth of field
• looks 3D
• can view living organisms

Question 17

Ultraviolet light

Answer 17

Short wavelength to make the object fluoresce

Question 18

Confocal microscopy (UV)

Answer 18

Shows higher resolution than fluorescent

Question 19

Digital microscope

Answer 19

Automatic

Question 20

Electron microscope

Answer 20

Uses electron beams (not light) and electromagnets to help focus on the image
- have shorter wavelengths than visible light rays*

Question 21

Transmission electron microscopy (TEM)

Answer 21

• Provides highest level of magnification
• Prepare super thin slices of the specimen
• can magnify up to 500,000x

Question 22

Scanning electron microscopy (SEM)

Answer 22

• Coat specimen with metal that reacts with electron beam to produce image
• looks 3D

Question 23

Wet mount

Answer 23

• Cover a drop of specimen with coverslip on slide

- Allows you to see a living organism’s size, shape, motility

Question 24

Hanging drop

Answer 24

• Hang a drop of specimen from coverslip into a well on depression slide
• provides better view of motility

Question 25

Smear

Answer 25

- place cells on a glass slide - let them air dry - heat fix them to adhere them to slide - kills cells

Question 26

Acidic stains

Answer 26

- Negatively charged - Stain positively charged glass, not cells - Cells appear white on a dark background - Also called negative stains

Question 27

Basic stains

Answer 27

- Positively charged - Stain negatively charged cells - Cells appear dark on a light background - Also called a direct stain

Question 28

Simple stain

Answer 28

- Uses a single dye | - Shows size, shape, arrangement

Question 29

Differential stain

Answer 29

- Use more than one dye to differentiate cell types

Question 30

Gram stain

Answer 30

- Used to distinguish cells based on cell wall 1. Color everything purple with crystal violet dye (primary stain) 2. Add iodine as a mordant to set the stain 3. Add ethyl alcohol decolorizer 4. Add safranin counterstain to visualize gram negative cells

Question 31

Acid fast bacteria

Answer 31

- mycobacterium - cell wall contains a waxy lipid (mycolic acid) which is hard to stain - look gram positive in a gram stain - diseases like TB, leprosy (take a long time to grow and divide

Question 32

Acid fast stain

Answer 32

1. Primary stain: Carbolfuschin (pink) - use heat to fix stain into cell wall 2. Decolorizer: acid alcohol - wash pink color out of all cells that aren’t acid fast 3. Counterstain: methylene blue Result: acid fast are pink, non acid fast are blue

Question 33

Negative stain

Answer 33

- determined if cells have capsule (not stained by acidic or basic dye) - creates a white ring around stained cell - cells with capsule tend to be more virulent

Question 34

Flagellar stain

Answer 34

Coat flagella with a layer of dye or metal

Question 35

Endospore

Answer 35

- clostridium and bacillus species | - Cell puts a copy of DNA into an endospore when it encounters harsh conditions (heat, lack of food)

Question 36

Schaeffer-Fulton stain

Answer 36

1. Primary stain: malachite green - heat fix 2. Decolorizer: water - washes out everything that isn’t a spire 3. Counterstain: safranin (pink) Result: endospores are green, vegetative cells are pink