Microbiology Ch.2 Textbook Learning objectives

Calculate total magnification for a compound microscope.

(10x) (100x) = 1000X

Why do fluorescent dyes emit a different color of light than they absorb?

Because the energy absorption causes electrons to jump to higher energy states , after which they then almost immediately fall back down to their ground states, emitting specific amounts of energy as photons.

Explain why dispersion occurs when white light passes through a prism.

Because the refraction index is different for different frequencies of light

Identify the key differences between brightfield and darkfield microscopy.

Brightfield: A compound microscope with two or more lenses that produce a dark image on a bright background. Darkfield: Is a brightfield microscope that has a small but significant modification to the condenser. A small, opaque disk (about 1 cm in diameter) is placed between the illuminator and the condenser lens. This opaque light stop, as the disk is called, blocks most of the light from the illuminator as it passes through the condenser on its way to the objective lens, producing a hollow cone of light that is focused on the specimen. Shows bright objects on a dark background.

What types of specimens are best examined using confocal or two-photon microscopy?

Confocal Microscopy: Very useful for examining thick specimens such as biofilms, which are examined alive and unfixed. Two- photon Microscopy: Useful for examining thick living cells within intact tissues-brain slices, embryos, whole organs, and even entire animals

Is endospore staining an example of positive, negative, or differential staining?

Differential staining

What are some advantages and disadvantages of electron microscopy, as opposed to light microscopy, for examining microbiological specimens?

Electron Microscopy: Advantages: Has a higher magnifying power. Has better resolution. Can resolve subcellular structures as well as some molecular structures ex: single strands of DNA. Disadvantages: Cannot be used on living specimens. Light Microscopy: Advantages: Can view live and dead specimens. Disadvantages: Lower magnifying power and resolution is not as great.

What color are gram-positive and gram-negative cells, respectively, after the Gram stain procedure?

Gram-postive cells: Purple or Blue color. Gram-negative cells: Pink or Red color.

Which has a higher frequency: red light or green light?

Green light

Why are acid-fast stains useful?

Help with diagnoses of acid-fast bacteria that cause specific illnesses.

Explain the role of Gram's iodine in the Gram stain procedure.

Iodine acts like a mordant ( a substance used to set or stabilize stains or dyes. Iodine acts like a trapping agent that complexes with crystal violet, making crystal violet-iodine complex clump and stay contained in thick layers of peptidoglycan in the cell walls.

Why must fluorochromes be used to examine a specimen under a fluorescence microscope?

To create contrast

What kinds of specimens are best examined using TEM? SEM?

Transmission electron microscope (TEM): Extremely thin and dehydrated specimens Scanning electron microscope (SEM): Specimens that are dried and prepared with fixatives that reduce artifacts the coated with a thin layer of metal such as gold. Samples can be small or large (size of pollen grain)

If an object is transparent, does it reflect, absorb, or transmit light?

Transmits light

Name the device that is used to create thin sections of specimens for electron microscopy.

Ultramicrotome

Name two factors that affect resolution.

Wavelength and Numerical aperture

Name one advantage and one limitation of scanning probe microscopy.

Advantage: Can magnify up to 100,000,000X Limitation:

Explain the role of alcohol in the Gram stain procedure.

Alcohol is a decolorizing agent. If the cell has thick peptidoglycan layers in their cell walls they are less affected to the decolorizing agent; they generally retain the crystal violet dye and remain purple. If the cell has thinner peptidoglycan layers in their cell wall the alcohol washes it out making them colorless again.

How does negative staining help us visualize capsules?

It colors the background blue and but not the capsule so the capsule looks like a halo around each cell by coloring the positive stained body pink/red and the negatively stained background blue.

Compare and contrast the contributions of van Leeuwenhoek, Hooke, and Galileo to early microscopy.

Leeuwenhoek: Was credited as the first person to have created microscopes powerful enough to view microbes. Even though van Leeuwenhoek's microscopes were simple microscopes (as seen in this replica), they were more powerful and provided better resolution than the compound microscopes of his day. Hooke: Published his book Micrographia, observations using a compound microscope. Was the first to discover the structure we know as cells by looking at a sample of a cork. Galileo: He created a microscope which used 2 lenses and used it to view insect parts.

Describe the distinguishing features and typical uses for various types of light microscopes, electron microscopes, and scanning probe microscopes.

Light microscope: Uses light to visualize images. Examples of light microscopes include brightfield microscopes, darkfield microscopes, phase-contrast microscopes, differential interference contrast microscopes, fluorescence microscopes, confocal scanning laser microscopes, and two-photon microscopes. These various types of light microscopes can be used to complement each other in diagnostics and research. Electron microscope: Scanning probe microscope:

What is the main difference between preparing a sample for fluorescence microscopy versus light microscopy?

Light microscopy: Can be thickened with silver particles. Might cause damage to specimen. Fluorescence microscopy: uses fluorescence dyes.

If a light wave has a long wavelength, is it likely to have a low or high frequency?

Low

What are some advantages of phase-contrast and DIC microscopy?

Phase- contrast: Use refraction and interference caused by structures in a specimen to create high-contrast, high-resolution images without staining. DIC microscope: Two beams of light are created in which the direction of wave movement (polarization) differs. Results in high-contrast images of living organisms with a three-dimensional appearance. These microscopes are especially useful in distinguishing structures within live, unstained specimens.

Which has higher magnification, a light microscope or a scanning probe microscope?

Scanning probe microscope Can magnify up to 100,000,000X A light microscope can magnify up to 1,000X

Explain the difference between simple and compound microscopes.

Simple microscope: Light is passed through just one lens Compound microscope: Light passes through two lenses

Differentiate between simple and differential stains.

Simple stains: A single dye is used to emphasize particular structures in the specimen. A simple stain will generally make all of the organisms in a sample appear to be the same color, even if the sample contains more than one type of organism. Differential stains: Distinguishes organisms based on their interactions with multiple stains.

Why is it important to dehydrate cells before examining them under an electron microscope?

The specimen has to be cut very thing but also without damaging it so dehydration is very important.