Microbiology Chapter 3

Question 1

1 Meter is equivalent to_____inches

Answer 1

39.37 inches

Question 2

1 mm is equal to_____micrometers

Answer 2

1000 Micrometers

Question 3

1 mm is equal to________of a meter

Answer 3

1,000 o=

Question 4

1 micrometer is equivalent to____nanometers

Answer 4

1000

Question 5

1 micrometer is_________of a meter

Answer 5

1/1,000,000,000

Question 6

Typical size of a Eukaryotic Cell

Answer 6

10 micrometers

Question 7

Typical size of a prokaryotic cell

Answer 7

1 micrometer

Question 8

Typical size of a virus

Answer 8

100 nanometers

Question 9

Diameter of a double helix

Answer 9

2 nanometers

Question 10

what is the path of light through a compound microscope?

Answer 10

In the microscope, the light comes from a light source. The light source falls into the mirror with the help of a condenser . It moves in the upward direction, gets reflected, then falls on the specimen and from the specimen this passes upward in the objective lens. The objective lens forms an image.

Question 10

Define Total Magnification

Answer 10

Total magnification: It is a product of the objective and ocular lenses of a microscope. It is the ability to measure the magnification or enhancement of the object by the microscope.

Question 11

Define Resolution

Answer 11

The resolution of a microscope can be defined as the shortest distance between the two points in a specimen.

Question 12

What does it mean when a microscope has a resolution of 0.2 nm?

Answer 12

This means that if two objects are closer than 0.2 μm, they will appear as one object rather than two.

Question 13

Darkfield Microscopy

Answer 13

The specimen appears light on a dark background

Question 14

Phase-contrast Microscopy

Answer 14

provides detailed examination of internal structures of an organism

Question 15

Differential Interference Contrast Microscopy

Answer 15

similar to phase contrast except uses two beams of light

Question 16

Fluorescence Microscopy

Answer 16

organisms are stained with flourochromes, they appear as luminescent. bright objects on a dark background

Question 17

Confocal Microscopy

Answer 17

specimens stained with flourochromes using a short wave length to produce a 3-D image

Question 18

Two-photon Microscopy

Answer 18

similar to confocal except using two photons

Question 19

Scanning Acoustic Microscopy

Answer 19

using sound waves to produce an image

Question 20

Brightfield Illumination

Answer 20

visible against a bright background

Question 21

How are brightfield, darkfield, phase-contrast, and
fluorescence microscopy similar?

Answer 21

They all need a light source

Question 22

Explain how electron microscopy differs
from light microscopy.

Answer 22

Electron microscopes differ from light microscopes in that they produce an image of a specimen by using a beam of electrons rather than a beam of light. Electrons have much a shorter wavelength than visible light, and this allows electron microscopes to produce higher-resolution images than standard light microscopes.

Question 23

SEM Microscopes

Answer 23

to view a surface structure of intact cells and viruses.

Question 23

TEM microscopes

Answer 23

to view an ultrathin section of a specimen (tissue section, molecules).

Question 24

Scanned-probe Microscope

Answer 24

Scanning probe microscopy is a branch of microscopy that forms images of surfaces using a physical probe that scans the specimen

a STM (scanning tunneling Microscope) can view a protein from E.coli and an AFM (Atomic Force Microscopy) can view a perfringoglysin O toxin

Question 25

Staining

Answer 25

a technique which is used to enhance and contrast a biological specimen at the microscopic level.

Question 26

Smear

Answer 26

A thin layer of bacteria that is placed on a tray or a slide for staining

Question 27

Negative Staining

Answer 27

employs the use of an acidic stain and, due to repulsion between the negative charges of the stain and the bacterial surface, the dye will not penetrate the cell. In negative staining, the results yield a clear cell with a dark background.

Question 28

Mordant

Answer 28

a substance that increases the affinity of the cell wall for a stain by binding to the primary stain, thus forming an insoluble complex, which gets trapped in the cell wall. i.e. iodine

Question 29

Why doesn’t a negative stain cause distortion of a cell’s shape?

Answer 29

The advantage of negative staining is that you are able to view the cells without risk of them being damaged or distorted as they might be with a positive stain

Question 30

Explain the purpose of simple staining

Answer 30

Simple staining involves directly staining the bacterial cell with a positively charged dye in order to see bacterial detail, in contrast to negative staining where the bacteria remain unstained against a dark background.

Question 31

Why is fixing necessary for most staining procedures?

Answer 31

This process is called heat fixing the specimen to the slide. Its purpose is to bind the specimen to the slide so that it does not wash off during staining

Question 32

List the steps in preparing a Gram stain,
and describe the appearance of Gram-positive
and Gram-negative cells after each step.

Answer 32

1. smear, dry, fix (kills cells/sticks to slide)
2. crystal violet (primary stain)- rinse
3. Gram’s Iodine- (mordant)- binds CV into cell wall- rinse
4. Decolorize- (95% ETOH)- rinse
5. Counterstain- (red stain)- (basic)- safranin- rinse
6. blot dry
7. observe

Color: Typically, bacteria that are gram-positive appear purple to blue, and bacteria that are
Gram-negative appear pink to red.

Shape: The most common shapes include round (cocci) or rod-shaped (bacilli).

Question 33

Procedure of Gram stain

Answer 33

Crystal violet stain is added over the fixed culture.

The stain is poured off, and the excess stain is rinsed with water.

Iodine solution is used to cover the smear. This step is known as “fixing the dye.”

Iodine solution is poured off, and the slide is rinsed with running water.

A few drops of decolorizer is added to the slide. Decolorizers are often the mixed solvent of ethanol and acetone. This step is known as “solvent treatment.” The slide is rinsed with water in 5 seconds.

The smear is counterstained with basic fuchsin solution for 40 to 60 seconds. The fuchsin solution is washed off with water.

Question 34

Procedure of acid-fast stain

Answer 34

1. Air dry and heat fix a thin film of microorganisms. Allow the slide to cool.
2. Flood the slide with Carbolfuchsin. Steam the slide with a Bunsen burner over the sink. Let the slide set for 5 minutes. Rinse with water.
3. Flood slide with Acid Alcohol for 30 seconds. Rinse with water.
4. Counterstain by flooding the slide with Methylene Blue for 30 seconds. Rinse with water.
5. Dry the slide by putting it between the pages of a book of Bibulous paper.
6. View organisms using the oil immersion objective of your microscope.

Question 34

Which stain would be used to identify microbes in the
genera Mycobacterium and Nocardia?

Answer 34

Acid-fast Staining

Question 35

Capsule Stain

Answer 35

capsule stain- a neg. staining used to make microbial capsules visible.
- used to determine organisms virulence

Question 36

Endospore Stain

Answer 36

special stain that color only certain parts of bacteria

used to differentiate the inclusions of stored material

Question 37

Flagella Stain

Answer 37

The Flagella Stain provides a method for viewing bacterial flagella by employing crystal violet in an alcoholic solution as the primary stain. During the staining procedure, the alcoholic solution evaporates and leaves a precipitate around the flagella, increasing its apparent size to the human eye

Question 38

How do unstained endospores appear? Stained endospores?

Answer 38

Unstained: Refractive under the light.

Stained: Green in a red cell.