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Medical Microbiology > Chapter 6 - Role of Microscopy > Flashcards

Chapter 6 - Role of Microscopy Flashcards

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1
Q

What are the three main principles of bright field microscopy?

A

Magnification
- Objective lenses: 10x, 20x, 40x, and 100x
- Ocular lens: 10x
Resolution
- Detail of the magnified object can be observed.
- Immersion oil is used to increase resolution.
Contrast
- Allows objects to stand out from the background.

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2
Q

Köhler Illumination

- purpose

A
  • Properly positions the condenser to ensure that light is focused through the specimen.
  • Provides the maximum illumination and resolution.
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3
Q

Köhler Illumination

- principle

A
  • A path of light is projected from the base of the microscope toward the condenser. The condenser filters the light to remove long wavelengths. The shorter wavelengths pass through the condenser to improve resolution. When the condenser is positioned properly, the light will focus onto the specimen.
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4
Q

Köhler Illumination

- step 1 - 4 in the method

A
  1. Turn on the microscope, and adjust the light source so that it is approximately at a maximum of 50% strength.
  2. Place a microscope slide containing a specimen on the stage, and secure in place with the slide clips.
  3. Adjust the eyepiece for comfort and proper alignment for interpupillary distance.
  4. Using the 10× objective for a total magnification of 100×, focus the specimen.
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5
Q

Köhler Illumination

- step 5 - 8 in the method

A
  1. Adjust each individual eyepiece. To focus the left eyepiece, close the right eye and use the fine focus to adjust the image. Close the left eye and use the Diopter ring on the right eyepiece to adjust the focus for the right eye.
  2. Close the field diaphragm and the condenser aperture. A small circle of light should be visible.
  3. If no light is visible, open the field diaphragm until a circle of light is present.
  4. Adjust the condenser screws as needed to center the light in the field of view.
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6
Q

Köhler Illumination

- step 9 - 12 in the method

A
  1. Adjust the condenser focus knob until the light appears as a sharp circle.
  2. Remove the eyepiece and look down the cylinder. A circle of light should be visible in the center of a dark field.
  3. Open the diaphragm until the circle of light fills three fourths of the field of view.
  4. Place the eyepiece back into the cylinder and record the condenser diaphragm setting for the 10× objective.
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7
Q

Phase Contrast Microscopy

  • what features in an organism does this type of microscopy utilize?
  • how does this type of microscopy work?
A
  • The various organelles show wide variation in refractive index, that is, the tendency of the materials to bend light, providing an opportunity to distinguish them
  • Beams of light pass through a specimen to create different light intensities and greater contrast
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8
Q

Phase Contrast Microscopy

  • is staining necessary?
  • what type of specimens is this type of microscopy useful for observing?
A
  • Staining is not necessary; therefore living organisms can be observed.
  • Is more useful for observing fungi in culture
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9
Q

Fluorescent Microscopy

  • How does this type work?
  • How do objects appear to the observer?
A
  • Fluorochromes are raised to higher energy levels after excitation.
  • Excess energy is released in the form of visible light when molecules return to a normal state.
  • Objects appear brightly lit against a dark background
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10
Q

What is the difference between fluorchroming and immunofluorescence?

A
  • fluorchroming: a fluorescent dye is added to the stain that will bind and fluoresce ALL bacteria
  • immunofluorescence: fluorescent dye is attached to a specific antibody. that antibody attaches to antigens on a target bacteria - ONLY the bacteria that the antibody attaches to will fluoresce
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11
Q

Fluorochromes: Acridine orange stain

  • what organelle does it stain?
  • specific or non-specific dye?
  • what is it used for?
A
  • Stains nucleic acid and is nonspecific.

- Confirms the presence of bacteria in blood cultures when blood cultures are indeterminate.

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12
Q

Fluorochromes: Calcofluor white stain

- what type of specimen is this used for?

A
  • Greatly enhances fungal visibility in tissue and in other specimens.
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13
Q

Fluorochromes: Auramine-rhodamine stain

  • what type of bacteria does it bind to?
  • what do the cells/background look like to the viewer?
A
  • Nonspecifically will bind to nearly all mycobacteria.

- Cells appear bright yellow or orange against a green background.

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14
Q

Dark-Field Microscopy

- how does it work?

A
  • Directing light at an oblique angle achieves contrast.

- Only light that hits the object will be visualized.

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15
Q

Dark-Field Microscopy

  • what qualities on an organism make it a candidate for this type of microscopy?
  • Give an example
A
  • Used with organisms too thin or difficult to grow in culture
  • T. pallidum (syphilis): can’t be grown in culture media. Only way known to culture is in the foot pads of armidillos
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16
Q

Electron Microscopy

  • what does this use to visualize objects?
  • what is the magnification?
A
  • Uses electrons instead of light to visualize small objects.
  • Electromagnetic fields focused by electrons.
  • Magnification is in excess of 100,000x.
17
Q

Electron Microscopy

- TEM vs SEM

A
  • TEM: transition electron microscopy (appears 2-D)

- SEM: scanning electron microscopy (appears 3-D)

18
Q

Gram Stain

  • stains and counterstains
  • what will gram positive and gram negative cells look like?
A 
Stains:
- Primary stain—crystal violet
- Mordant—Gram’s iodine
- Decolorizer—alcohol
- Counterstain—safranin
Results
- Gram-positive organisms will stain deep purple.
- Gram-negative organisms will stain pink to red.
19
Q

Acid-Fast Stains

- what cell feature necessitates this type of staining?

A
  • used to detect bacteria whose cell walls contain mycolic acids (e.g., Mycobacterium).
20
Q

Acid-Fast Stains

  • what are the two methods?
  • what is the main difference between the methods?
A
  • Ziehl-Neelsen stain—Primary stain enters the cell wall by HEATING.
  • Kinyoun stain—Primary stain contains phenol (COLD method).
21
Q

Acid-Fast Stains

  • what color will acid fast positive bacteria stain?
    • what stain used?
  • what are the two most common counterstains?
A
  • Acid-fast organisms will stain pink (carbolfuchsin).
  • Other objects will stain the color of the counterstain.
    • Blue (methylene blue)
    • Green (malachite green)

Medical Microbiology (6 decks)

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