Microscopy and Microorganism Staining

Question 1

What are the 4 types of light microscopy?

Answer 1

1. Bright Field
2. Phase-Contrast
3. Dark-Field
4. Fluorescence

Question 2

What is the magnification of an objective lense?

Answer 2

10x-100x magnification

Question 3

What is the magnification of an ocular lense?

Answer 3

10x-20x magnification

Question 4

How do you get total magnification?

Answer 4

Objective magnification x ocular magnification

Question 5

What is the definition of magnification?

Answer 5

The ability to make an object larger.

Question 6

What is the definition of resolution?

Answer 6

the ability to distinguish two adjacent objects as

separate and distinct.

Question 7

What is the limit of resolution for a light microscope?

Answer 7

0.2 μm

Question 8

True or false: when wavelength increases, resolution increases.

Answer 8

False: when wavelength increases resolution decreases.

Question 9

True or false: light must pass through two points for them to be viewed as separate objects.

Answer 9

True

Question 10

What is simple staining?

Answer 10

One dye used to colour specimen.

Question 11

What is the coloured portion of staining dye called?

Answer 11

Chromophore

Question 12

What are the two types of simple staining dye? What are their charges?

Answer 12

Basic dye- positively charged chromophore

Acidic dye- negatively charged chromophore

Question 13

What does basic dye bind to?

Answer 13

The negatively charged molecules on a cells surface.

Question 14

What does acidic dye bind to?

Answer 14

Acidic dye does not bind to anything, it’s repelled by the cells surface. It stain the background as a “negative” stain.

Question 15

Give an example of a basic dye and acidic dye.

Answer 15

Basic: crystal violet
Acidic: nigrosin

Question 16

Give the five steps of staining.

Answer 16

1.Prepare smear
2. Air fry
3 Heat fix
4. Flush with stain
5. Examine under microscope

Question 17

What is a differential stain?

Answer 17

Stain which separates bacteria into two groups.

Question 18

What type of staining is a gram stain? What does it do?

Answer 18

Differential stain- separates bacteria into two groups based on cell wall structure.

Question 19

What are the two types of gram bacteria? What colour are they?

Answer 19

Gram positive- retain primary stain (purple)

Gram negative- lose primary stain and takes the counter stain colour (red/pink)

Question 20

What type of stain is an acid fast stain? What does it to?

Answer 20

Differential stain- detects mycolic acid in the cell wall of the genus Mycobacterium.

Question 21

The two colours of an acid fast stain are fuchsia and blue. What does each colour mean?

Answer 21

Fuchsia- Mycobacterium that retains primary stain

Blue- anything else on the slide (counter stain)

Question 22

Another differential stain is an endoscope stain. What are the colours, and what do they represent?

Answer 22

Green: endospores that retain primary stain.
Pink: Cells counterstained

Question 23

Give an example of a bacteria that would undergo endospore stain.

Answer 23

Bacillus anthracis

Question 24

What is phase-contrast microscopy?

Answer 24

A type of light microscopy. Phase ring amplifies differences in the refractive index of cell and surroundings.

Question 25

What is a benefit to phase-contrast microscopy?

Answer 25

Can view live samples

Question 26

What is dark-field microscopy?

Answer 26

A type of light microscopy where specimen are illuminated by a hollow cone of light. Only refracted light enters the object.

Question 27

What is one reason we use dark-field microscopy? Give an example of a bacteria that requires it.

Answer 27

Used to observe specimen that don’t stain well. | Example: Treponema pallidum (syphilis)

Question 28

What is fluorescence microscopy?

Answer 28

A type of microscopy that visualizes bacteria that fluoresce. The bacteria emit light of one colour when illuminated with another colour of light.

Question 29

What is differential interference contract (DIC) microscopy used for? How does it work?

Answer 29

Used to image cells in three dimensions. Polarizer creates two distinct beams of polarized light which give structures such as endospores, vacuoles, and granitas a 3-D structure.

Question 30

What type of microscopy is Confocal scanning laser microscopy (CSLM)? How does it work?

Answer 30

Three dimensional microscopy- uses a computerized microscope and lasers to generate a 3-D image by layering multiple images.

Question 31

What is the resolution of CSLM?

Answer 31

0.1 um

Question 32

Electron microscopes use electrons instead of photons to view cells. Why is the resolution higher?

Answer 32

Electrons have a shorter wavelength then light.

Question 33

What are the two types of electron microscopes?

Answer 33

Transmission electron microscopes (TEM) and Scanning electron microscopes (SEM)

Question 34

What is the magnification and resolution of TEM? How thin must the specimen be?

Answer 34

Magnification and resolution: 0.2 nm | Cells must be 20-60 nm thin

Question 35

What must be done to a specimen before it undergoes TEM?

Answer 35

Must be stained with metals like lead or uranium.

Question 36

What must specimen do before undergoing SEM?

Answer 36

Be coated with a thin film of heavy metal (e.g., gold)