Using Microscope with Bacteria (Online Module)

Question 1

Magnification, Resolution, Contrast

Answer 1

1) Magnification:
- -The apparent increase in size of an object.
- -Magnification results when a beam of radiation refracts or bends as it passes through a lens and is indicated by a number (“100X”).

2) Resolution:
- -The ability to distinguish between objects that are close together.
- -The higher the resolution, the better the ability to distinguish two objects that are close to one another.
- -Modern microscopes can distinguish between objects as close together as0.2 µm (thus, “limit of resolution for light microscopy” = 200nm).

3) The Resolution Distance is Dependent on
(1) the Wavelength of the electromagnetic radiation and
(2) the Numerical Aperture of the Lens, which is its ability to gather light.
- -Immersion oil: is used to fill the space between the specimen and a lens to reduce light refraction, increasing the numerical aperture and resolution.

4) Contrast:
- -Refers to differences in intensity between two objects, or between an object and its background.
- -Since most microbes are colorless, they are stained to increase contrast.
- -Polarized light may also be used to enhance contrast.

Question 2

Sizes of microbes

Answer 2

Bacteria: 1 micron
Viruses: 50 nm
Rickettsia: 200 nm

Question 3

Microscopy Types

Answer 3

1) Bright-field Microscopes:
- -The field (background) is illuminated and samples are stained to visualize structures.

2) Dark-field light microscopy:
- -Uses scattered light to illuminate the specimen against a dark background

3) Fluorescence microscopy:
- -Samples are labeled with fluorescent markers and illuminated by light of a specific wavelength;
- -Special filters allow only the emitted light wavelength through, an this is perceived as a color by the human eye (one color at a time)

Question 4

Simple Staining

Answer 4

Provides basic and nonspecific contrast between bacteria and background.

Example: Methylene Blue Stain
–Shows metachromatic granules in Corynebacterium.

Question 5

Differential Stains

Answer 5

Differential Stains:
–Use specific dyes and/or methods so that different cells, chemicals, or structures are distinguished.

Example: **Acid-Fast Stain for Mycobacterium.

Gram Stain:

• -The standard differential stain
• -Purple staining Gram Positive vs. Pink staining Gram Negative.

Question 6

Shape Classes of Bacteria

Answer 6

Cocci: Spheres
–Staphylococcus aureus (grapelike clusters)

Bacilli: Rods

• -E. coli
• -Yersinia pestis
• -Pseudomonas aeruginosa

Comma-shaped:
–Vibrio

Spirals:
–Spirochete

Coccobacilli:

• -Intermediate between coccus and bacillus
• -Haemophilus influenzae

Pleomorphic:
–Many forms in one pure culture

Spore-forming:

• -Special class of Bacilli (central, terminal, and subterminal ENDOSPORE location)
• -Clostridium
• -Bacillus

Question 7

Sporulation: Endospore Formation

Answer 7

1) **Dipicolinic Acid and **Calcium form a complex that causes the cortex to contract.
2) The endospore dehydrates as water is expelled.
3) Spores survive adverse conditions.
4) Germination under appropriate environmental conditions; epidemic and virulence potential (hospital; barnyard)
5) Sterilization requires High Heat and Pressure (Autoclave!!)

Question 8

Bacteriai Classes Based on Types of Culture

Answer 8

Simple Culture Requirements:
–Nutrient agar

Fastidious Culture:
–Bacteria that grow only on supplemented media, e.g. Blood Agar

Specialized Culture:

• -Unique Requirements
• -Are rarely cultured outside reference labs

Differential Agar Culture:
–Allows discrimination among bacterial types based on culture appearance using agars containing special ingredients like iron dyes, special sugars, and/or pH indicators.

Impossible Culture:
–Diagnosed by Fluorescence microscopy, serology, or nucleic acid technology.