Module 3

Question 1

A nanometer is defined as:

Answer 1

10 -9 or one-billionth of a meter

Question 2

True or False: A nanometer is longer than a micrometer

Answer 2

F

Question 3

Resolution and contrast are two critical factors that influence your ability to see an object. Explain each.

Answer 3

Resolution refers to the distance between two objects at which the objects can still be seen as separate. Poor or low resolution means two or more objects may appear as one.

Contrast on the other hand is the difference in light absorbance between two objects. Poor contrast gives a high background and makes the visualization of multiple objects difficult. For instance, trying to identify 2 dark colored objects at night (low light = low contrast) versus the same 2 objects in the middle of
a sunny afternoon (bright light against 2 dark objects = high contrast).

Question 4

Assuming a fixed ocular, identify the part of the microscope you would adjust to enhance the magnification of a sample.

A. Objective
B. Condenser
C. Iris diaphragm
D. Eye piece

Answer 4

A.

Only the oculars (eyepiece) and the objectives contribute to the magnification of the sample. Since the eyepiece is fixed, only the objectives could be altered.Objective

Question 5

Assuming a constant (non-adjustable) light source power, identify the part of the microscope you would adjust to limit the amount of light entering the microscope.

Answer 5

Iris Diaphram

Question 6

What is the total magnification on (relative to your eye) of a sample imaged with a 60x objective and a 10x eyepiece? Show your math.

Answer 6

60x objective x 10x ocular = 600x magnification

Question 7

True or False: Staining is often required to image a cell that is adherent and flat (thin).

Answer 7

True

Question 8

True or False: A cell that is adherent, flat (thin), and unstained is easily identified using bright field microscopy

Answer 8

False

Question 9

Which of the following could be seen clearly by the unaided eye? Select all that apply.

A. Bacteria with diameter of 24 μm
B. Protozoa with diameter of 150 μm
C. Virus with a diameter of 0.2 μm
D. Skin cell with diameter of 1500 μm

Answer 9

B. Protozoa with diameter of 150 μm
D. Skin cell with diameter of 1500 μm

The unaided eye can, on average, clearly resolve objects > 100 μm

Question 10

Which of the following could NOT be seen clearly by the unaided eye? Select all that apply.

A. Bacteria with diameter of 24 μm
B. Protozoa with diameter of 150 μm
C. Virus with a diameter of 0.2 μm
D. Skin cell with diameter of 1500 μm

Answer 10

A. Bacteria with diameter of 24 μm
C. Virus with a diameter of 0.2 μm

The unaided eye can, on average, clearly resolve objects > 100 μm

Question 11

Label the following unmarked microscope components (numbered arrows) by matching it with the components provided
(letters).

Answer 11

1F- eyepiece
2D- neck
3B- fine adjustment knob
4G- objective
5A- stage
6H- base

Question 12

This type of microscope is best suited for visualizing GFP, RFP, and YFP proteins.

Answer 12

Fluorescence

Question 13

This type of microscope utilizes ultraviolet (UV) light to illuminate stained objects.

Answer 13

Florescence

Question 14

This type of microscope uses a specialized condenser and objective to amplify the slight differences between cells and background.

Answer 14

Phase-contrast

Question 15

This type of microscope enhances contrast between specimen and background but does not permit the visualization of intracellular structures.

Answer 15

Dark Field

Question 16

This type of microscope uses neither halogen nor UV light sources but rather lasers to illuminate stained cells in high resolution.

Answer 16

Confocal

Question 17

This type of microscope is capable of capturing images in multiple focal planes, rendering a specimen in 3-D

Answer 17

Confocal

Question 18

Identify what type of electron microscope was used to capture the following image and explain your choice.
(picture labeled B)

Answer 18

The image was captured using a Scanning Electron Microscope (SEM). The above image shows the trademark ‘shell’ image (no subcellular organelles are visible) reminiscent of SEM. Only TEM is capable of visualizing subcellular substrucutres.

Question 19

Identify what type of electron microscope was used to capture the following image and explain your choice.
(picture labeled A)

Answer 19

The above image is captured via a Transmission Electron Microscope (TEM). Even at 20nm resolution (inset image), subcellular substructures are still visible. The image lacks the outside ‘shell’ only appearance of SEM.

Question 20

Gram-Positive cells appear [answer1] in color due to a [answer2] peptidoglycan layer in the cell wall.

Answer 20

Purple, Thick

Question 21

Gram-Negative cells appear [answer1] in color due to a [answer2] peptidoglycan layer in the cell wall.

Answer 21

Pink, Thin

Question 22

True or False: A Giemsa stain can be used to determine the presence of pathogenic bacteria.

Answer 22

True

Question 23

True or False: The distinguishing characteristic of Gram-Negative bacteria is the presence of LPS in the outer membrane.

Answer 23

True

Question 24

True or False: Following the decolorization step of the Gram stain, Gram-Negative bacteria will appear colorless.

Answer 24

True

Question 25

True or False: If you wish to study the motiity of an organism you cannot heat fix, but you can chemically fix the specimen

Answer 25

False

Question 26

Name one substance capable of chemically fixing cells to a slide.

Answer 26

Any of the following are true: Paraformaldehyde, ethanol or methanol.

Question 27

You suspect a patient may have TB. Once a sample has been obtained, it is sent off to the lab for an acid-fast stain. If the patient were infected with TB, describe what you would expect to see on the stained slide.

Answer 27

I would expect to see red cells (TB+) on a blue background (TB negative).

Question 28

You want to observe the size and shape of a cell. What is the easiest staining technique that you could perform? Name at least one dye you would use during this process.

Answer 28

Simple stainining and crystal violet could be used.

Question 29

True or False: If a patient is suspected of having malaria, a Giemsa stain would be an appropriate differential test to perform.

Answer 29

True Giesma stains are often used in the clinical setting aid in the diagnosis of blood parasites.

Question 30

A human blood sample from a patient with a suspected parasite needs to be stained for diagnosis. Which stain should be selected?

Answer 30

Giemsa

Question 31

Select the type of microscopy used to generate the image: (black background with green glowing cells)

Answer 31

Florescence microscopy

Question 32

You are the lead technician in the Microbiology Department's core Microscopy lab. You are given a list what different scientists would like to observe in their samples. You must match these samples with appropriate type of microscopy. 1. A slide stained with fuchsin simple stain to observe the size of the cells 2. The organelles in a living ciliate 3. An unstained sample that cannot be visualized with bright field because microorganism absorbs very little light 4. Theprotein trafficking of tagged proteins of interest under experimental conditions in a cell 5. Build a 3-dimensional image of a cell A. Bright field B. Phase contrast C. Dark field D. Florescence E. Confocal

Answer 32

1. A 2. B 3. C 4. D 5. E

Question 33

What is the total magnification (relative to your eye) of a sample imaged with a 20x objective and a 15x eyepiece? Show your math.

Answer 33

20 x 15 = 300x magnification

Question 34

A researcher is asked to determine which of two vials contains E coli and which contains Salmonella. Knowing both are Gram-Negative while only one of them is capable of fermenting lactose, which type of media would be best suited: A. Growth media B. Differential media C. Selective media D. Selective and Differential media

Answer 34

B

Question 35

True or False: LB agar is classified as a selective, non-differential media.

Answer 35

False

Question 36

True or False: A Giemsa stain can be used to determine the presence of pathogenic bacteria.

Answer 36

True

Question 37

. True or False: Following the decolorization step of the Gram stain, Gram-Negative bacteria will appear colorless.

Answer 37

True

Question 38

A gram stain has been attempted on an unknown sample and it was found to have a high degree of resistance to decolorization. Given this property, which stain should be attempted next?

Answer 38

Acid-fast staining

Question 39

You want to observe the size and shape of a cell. What is the easiest staining technique that you could perform? Name at least one dye you would use during this process.

Answer 39

Simple stain would be the easiset technique and I would use crystal violet during this process.

Question 40

Name one substance capable of chemically fixing cells to a slide

Answer 40

methanol, ethanol, or paraformaldehyde

Question 41

True or False: Following the decolorization step of the Gram Stain, Gram-Negative bacteria will appear colorless

Answer 41

True