Test 3

Question 1

. A nanometer is defined as…

Answer 1

10^-9 or one billionth

Question 2

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

Answer 2

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

Contrast is the difference in light absorbance between two objects. Poor contrast gives a high background and makes the visualization of multiple objects difficult.

Question 3

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 3

Iris diaphragm

Question 4

A micrometer is defined as…

Answer 4

one-millionth of a meter and is commonly designated at 10-6

Question 5

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

Answer 5

Total Magnification = Objective x Eyepiece = 60 x 10 = 600x larger

Question 6

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

Answer 6

False, Adherent, flat cells are almost invisible due to the limits on both resolution and contrast

Question 7

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

Answer 7

Virus with a diameter of 0.2 μm
Bacteria with diameter of 24 μm

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

Question 8

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

Answer 8

Fluorescence

Question 9

This type of microscope can provide detailed images of live cells without staining

Answer 9

Phase-Contrast

Question 10

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

Answer 10

dark field

Question 11

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

Answer 11

confocal

Question 12

Identify what type of electron microscope was used to capture the following image and explain your choice

Answer 12

A scanning electron microscope (SEM) was used because this is a 3D detailed shell model. Also, the subcellular structures of the organelles are not visible, a TEM microscope would be needed to do so.

Question 13

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

Answer 13

Purple
Thick

Question 14

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

Answer 14

Pink
Thin

Question 15

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

Answer 15

True

Question 16

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

Answer 16

False, both heat and chemicals kill the cell

Question 17

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 17

Simple stain. You could use any of the following: methylene blue, crystal violet, safranin or fuschin.

Question 18

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 withTB, describe what you would expect to see on the stained slide

Answer 18

If the patient were infected with TB there would be red cells on blue background. The red cells indicate that the patient is infected with TB

Question 19

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

Answer 19

True, Giemsa stains are often used in the clinical setting to aid in the diagnosis of blood parasites

Question 20

Identify what type of electron microscope was used to capture the following image and explain your choice.

Answer 20

Transmission Electron microscope (TEM) was use to capture the following image because there is visualization of subcellular organelles in the specimen even at 20nm resolution. Also it has no 3D detailed shell model

Question 21

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 21

Acid-fast staining

Question 22

Identify what type of microscope was used to capture the following image and explain your choice

Answer 22

Brightfield Microscope, cells are unstained and appear almost invisible

Question 23

Identify the type of microscope used. Explain

Answer 23

Phase contrast microscopes, still lacks stain, but image contains much more detail and certain structures appear visible.

Question 24

Identify the type of microscope used. Explain

Answer 24

Darkfield microscopy, the background is dark, while the sample appears illuminated. Often used for spirochete borrelia specimens - cause of lyme disease

Question 25

What type of microscope is used?

Answer 25

Fluorescence Microscope

A. Adherent expressing GFP

B. Adherent Cell expressing mRFP

c. merged image of A and B for studying specific proteins

Question 26

What type of microscope was used?

Answer 26

Confocal Microscopy

• Increased resolution and contrast compared to standard fluorescence, individual actin filaments are visible.

Question 27

What can poor or low resolution indicate?

Answer 27

Two or more objects may appear as one object instead of separate

Question 28

What happens with poor contrast?

Answer 28

Gives high background and makes visualization of multiple objects difficult. For example, trying to identify 2 dark colored objects at night (low light=low contrast). Easier to view 2 dark objects in the middle of the afternoon.

Question 29

What is one limitation of fixing your sample?

Answer 29

Fixation requires you to kill your sample, a move that is irreversible and therefore you would not be able to determine the motility of a cell. Fixation can also distort the specimen shape an arrangement.

Question 30

Phase-contrast microscopy provided what benefits to imaging?

Answer 30

Phase contrast microscope can provide detailed images of live cells without staining. Phase-contrast microscopes also amplify slight differences between cells and surrounding background making it easier to visualize motility, like swimming or gliding.

Question 31

What is the distinguishing feature of dark field microscopy?

Answer 31

The dark field microscopy reflects light off of the specimen at an angle. The resulting image is an exceptionally dark background and a vibrant specimen. This is advantageous for viewing spirochete borelia, the cause of lyme disease.

Question 32

Unlike brightfield microscope, fluorescence microscopes illuminate samples through what spectrum?

Answer 32

The energy of the incoming light is in the form of the ultraviolet (UV) spectrum.

Question 33

What is the primary difference between TEM and SEM?

Answer 33

During transmission electron microscopy the electron passes through the sample whereas during scanning electron microscopy the electron is reflected off the sample creating a three dimensional ‘shell’ model of the specimen.

Question 34

Gram staining is based on what basic principle?

Answer 34

Gram staining is based on Hans Christian Grams idea that different types of bacteria react differently to various dyes. Thus dividing bacteria into one of two categories: gram-positive or gram-negative.

Question 35

What is a key determinant in a bacteria being Gram-positive?

Answer 35

Gram-positive contain a thick peptioglycan layer and display purple

Question 36

What is the purpose of heat fixing a sample?

Answer 36

Heat fixing is used to remove all water from a sample, ultimately preventing sample loss. Heat fixing ensures the samples tightly adhere to the glass slide prior to staining (and washing) procedures.

Question 37

What is the primary purpose of a wet mount?

Answer 37

Wet mounts are used to view live samples and can therefore be used to observe motility and behavior of the sample.

Question 38

The acid-fast stain is most often used to identify what specific microorganism?