Microscopy

Question 1

Simple microscope

Answer 1

Principles: contains a single lens to examine various natural substances for microorganisms
Applications: can see general structures of microbes
Limitations: Crude, hard to use, cannot see much

Question 2

compound/bright field microscope

Answer 2

Principles: has a magnification of 100-1,500x and resolution of 200nm
Applications: can view live or non alive cells
Limitations: Light refraction of images and resolution will lessen at increased magnifications

Question 3

Magnification

Answer 3

Enlarge the image (lens)

Question 4

Resolution

Answer 4

Ability to distinguish between two adjacent items

Question 5

Contrast

Answer 5

Ability to distinguish items from background

Question 6

Numerical aperture (NA)

Answer 6

A measure of light-capturing ability

increased NA= increased resoltuion

Question 7

Physical properties of resolution

Answer 7

Wavelength of light/electrons

shorter wavelength =increased resolution

Question 8

Oil immersion technique

Answer 8

Increases resolution by decreasing light refraction (increases numerical aperture)

Question 9

Basic Dyes from Staining

Answer 9

Crystal violet which binds to DNA

Stains all types of cells

Question 10

Differential Dyes from staining

Answer 10

Gram stain
2 different colors based on characteristics of the cell wall
Gram (+) = purple
Gram (-) = pink

Question 11

Staining A/D

Answer 11

Advantages: increase contrast and differentiate cell types
Disadvantages: kills the cells and can distort their features

Question 12

Phase contrast Microscopy

Answer 12

Principles: refraction index, phase ring, and improved contrast
Applications: viewing live cells
Limitations: can only see so much inside the cell

Question 13

Dark-field microscopy

Answer 13

Principles: light specimen from the side and results in dark background
Applications: viewing live cells, cell motility, and flagella viewing
Limitations: Image is not always clear and cannot see inside cells well

Question 14

Fluorescence microscopy

Answer 14

Principles: visualize light emitting from cells and autofluorescence (from cells) v stained (DAPI stain)
Applications: viewing live and fixed cells, quantifying cell numbers
Limitations: staining kills cells, no clear resolution

Question 15

Differential Interference Contrast (DIC) microscopy

Answer 15

Principles: Polarized light, two beams to one, and enhanced cell structures
Applications: view cellular structures (nucleus, endospores, and vacuoles)
Limitations: can only be used for larger microbial cells

Question 16

Atomic force microscopy

Answer 16

Principles: cantilever, laser detector, and repulsive forces
Applications: viewing live specimens and no fixative or coating
Limitations: cannot see inside cells, only outside of them and image can be distorted easily by fast-moving microbes

Question 17

Confocal scanning laser microscopy

Answer 17

Principles: laser + fluorescence and focusees on different layers
Applications: Thick specimens (ex biofilm) and multilayer image
Limitations: cannot see inside cells very well

Question 18

Electron microscopy

Answer 18

Principles: electron beam, electromagnetic lens, and operates in a vacuum
Applications: increased magnification and resolution, used for viewing inside microbial cells
Limitations: expensive and a lot of prep

Question 19

Transmission electron microscope

Answer 19

Higher resolution, 2D images, and internal features

Thin sectioning

Question 20

Scanning Electron microscope

Answer 20

Lower resolution, 3D images, and external features

heavy metal coating (gold)