Quiz 3

Question 1

What were some of the discoveries/innovations that help us treat or prevent disease?

Answer 1

Improvements in wastewater treatment and protection of clean water sources to limit disease spread

Food protection
- USDA and FDA, measures put in place to screen bacterial counts in food and ensure proper preparation
- Pasteurization: heating beverages quickly to kill most bacteria

Question 2

What happened during the Molecular Biology age of microbiology?

Answer 2

Being able to manipulate DNA opened up new doors for microbiology
- Sequence whole microbial genomes
- Determine how microbes cause disease
- Easily identify new microbial species
- Genetically engineer microbes for our benefit (ex. pest control, medication)

Question 3

What is meant by magnification, resolution? What limits resolution?

Answer 3

magnification : increasing the apparent size of a specimen
resolution: measure of clarity/sharpness
– limited by the wavelength of energy used, quality of lens, and magnification

Question 4

What is contrast?

Answer 4

Contrast: the difference in color or brightness between an object and its background

Question 5

Why is contrast such an important issue for microbiologists (what color are most microbes)?

Answer 5

Most microbes are clear and hard to see

Question 6

Light microscopy
- What is it in general and what is the max magnification?

Answer 6

Light microscopy utilizes lenses to magnify images generated from light passing through a specimen
– max magnification is ~1000x

Question 7

Light microscopy
- Know basics of the brightfield microscope (you will need to know this for lab anyway).

Answer 7

• light sent up through specimen from base
• 4x, 10x, 40x, and 100x objective lenses with 10x ocular lens

Question 8

What is a darkfield microscope and when would it need to be used? What do darkfield images look like?

Answer 8

Darkfield microscopes are used in cases where brightness or staining won’t work
– creates a reverse contrast, with the colorless specimen against a dark background

Question 9

How are phase contrast microscopes different from the others? Know the basics of how they produce images

Answer 9

Phase-contrast microscopes utilize refraction patterns to provide a contrast for the specimen (no fixing or staining required)
– can view live cells

Question 10

How are DIC microscopes different from the others? Know the basics of how they produce images

Answer 10

DIC microscopes are similar to phase-contrast except they use two different light sources and prisms (no fixing or staining needed)
– creates a sort of 3D effect

Question 11

What is fluorescence?

Answer 11

Fluorescence: molecule absorbs UV light at one wavelength and emits visible light at another wavelength
– objects appear to “glow in the dark”

Question 12

What are all the different ways we can use fluorescence in microbiology (e.g. what is immunofluorescence vs GFP tagging)?

Answer 12

Immunofluorescence involves tagging microbes with fluorescent antibodies
GFP tagging involves engineering microbes to express fluorescent proteins

Question 13

Standard fluorescent microscope definition and benefits to using

Answer 13

standard fluorescent microscope: shoot UV light of a specific wavelength at a sample
- all visible light emitted is detected and seen – limited detail/low resolution
- cheap and effective for most microbes

Question 14

confocal microscope definition and what is the major benefit of using confocal microscopes?

Answer 14

confocal microscope: uses a laser to excite a thin section of a thick specimen
- only emitted light from that section glows – higher resolution

Question 15

How does scanning acoustic microscopy work?

Answer 15

Shoot sound waves at a specimen and detect how
they bounce back, penetrate, and refract from the surface
– creates a 3D image

Question 16

How does a transmission EM work and what type of image does it produce?
- What types of specimen can be examined with a TEM and how are those specimen treated prior to visualization?

Answer 16

1) thin sections of specimen are fixed, dehydrated, and coated with heavy metals
2) shoot with an electron beam and measure the electrons that are transmitted through the specimen
– produces high resolution 2D image

note: electron beams go are transmitted through the specimen

Question 17

What is shadow casting and freeze fracturing?

Answer 17

Both are alternative methods to fixation
- Shadow casting: staining with a heavy metal
- Freeze fracturing: super-freezing a specimen

Question 18

How does a scanning EM work and what type of image does it produce?
- What types of specimen can be examined with a SEM and how are those specimen treated prior to visualization?

Answer 18

Specimen is coated with heavy metal and shot with an electron beam, and the electrons bounce off the specimen
- the electron beam forces the release of electrons from the metal ions and the released electrons are then collected and used to produce an image
– produces a 3D image of specimen surface

Question 19

Know the basics of how a scanning probe microscope works and know the two major types (you don’t need to know specific details of how those two differ from one another)

Answer 19

Small probe runs along the surface of a specimen and detects differences in contour, temperature, or current
1) scanning tunnelling microscopy
2) atomic force microscopy

Question 20

Basic steps in staining

Answer 20

1) smear specimen as a thin layer on a slide
2) let air dry and then heat fix
3) flood the slide with dye and let sit
4) wash off excess dye and rinse gently with water

Question 21

Know the basics of staining – basic vs acidic

Answer 21

Basic stains attach to the bacteria
Acidic stains attach to the slide and leave the bacteria colorless

Question 22

Know the basics of staining – simple vs negative

Answer 22

Simple staining - stains everything the same color, gives cells a color against a white background
Negative staining - stains the background instead of the specimen (specimen stays clear)

Question 23

What is a mordant?

Answer 23

A mordant is another chemical added to intensify the color of the stain

Question 24

What are differential stains?

Answer 24

Differential stains are used to distinguish one group of bacteria from another
ex. Gram stains and acid-fast stains

Question 25

Know how to do a Gram stain – what are the four major steps and what occurs in each?

Answer 25

1) stain with crystal violet and iodine
– iodine helps CV clump up
2) add alcohol to decolorize
– stain will come out of cells that have thinner cell walls
3) counterstain those now clear cells with safranin
4) will see purple and pink cells

Question 26

What do Gram + vs – look like after they are stained? Why do the cells stain differently (what is different about their cell walls)?

Answer 26

Gram positive = purple
– thicker cell walls
Gram negative = pink
– thinner cell walls