Microbial Cell Observation Techniques
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Questions and Answers

What is the smallest distance between two objects that can still be distinguished as separate entities?

  • Microscopy
  • Detection
  • Resolution (correct)
  • Magnification
  • What determines the size at which objects become visible to the human eye?

  • The brightness of the light source
  • The distance between the object and the eye
  • The size of the object itself
  • The resolution of the human eye (correct)
  • What is the approximate resolution of the human retina?

  • 150 nm
  • 150 µm (correct)
  • 150 mm
  • 150 pm
  • Which of the following is NOT a factor that affects the resolution of a microscope?

    <p>The color of the object being observed (B)</p> Signup and view all the answers

    What is the relationship between resolution and magnification?

    <p>Resolution and magnification are independent of each other. (B)</p> Signup and view all the answers

    Increasing the numerical aperture of a microscope lens will ____ the resolution.

    <p>increase (D)</p> Signup and view all the answers

    Which of the following techniques can be used to detect the presence of an object without resolving its individual parts?

    <p>Detection (A)</p> Signup and view all the answers

    What is the main difference between detection and resolution?

    <p>Detection refers to the ability to determine the presence of an object, while resolution refers to the ability to distinguish between two objects. (C)</p> Signup and view all the answers

    What does the refractive index of the surrounding medium affect in microscopy?

    <p>The numerical aperture (NA) of the lens (C), The resolution of the image (D)</p> Signup and view all the answers

    Which of the following is NOT a direct factor that limits the resolution of a light microscope?

    <p>The magnification of the objective lens (D)</p> Signup and view all the answers

    What is the minimum resolvable distance (R) in light microscopy?

    <p>Half the wavelength of light used (B)</p> Signup and view all the answers

    Why is immersion oil used in high-power microscopy?

    <p>To increase the numerical aperture of the objective lens (D)</p> Signup and view all the answers

    In a compound microscope, what is the significance of parfocal lenses?

    <p>They allow switching between objectives without re-focusing (C)</p> Signup and view all the answers

    Which of the following correctly describes the relationship between the numerical aperture (NA) of a lens and the resolution of a microscope?

    <p>Higher NA = higher resolution (D)</p> Signup and view all the answers

    What is the primary reason why two wavefronts approaching each other at an angle generate an interference pattern?

    <p>Differences in their phase shifts (B)</p> Signup and view all the answers

    How does the wavelength of light relate to the sharpness of the peak intensity of the point of detail in light microscopy?

    <p>Shorter wavelength = sharper peak (C)</p> Signup and view all the answers

    What is the main advantage of using dark-field microscopy over bright-field microscopy?

    <p>Increased contrast for unstained samples (B)</p> Signup and view all the answers

    How does phase-contrast microscopy enhance contrast for viewing transparent specimens?

    <p>Shifts the phase of light passing through the specimen (D)</p> Signup and view all the answers

    Why is immersion oil typically used with high-power objective lenses in bright-field microscopy?

    <p>To decrease the amount of light lost due to refraction (C)</p> Signup and view all the answers

    Which of the following is a major limitation of using a wet mount preparation for microscopy?

    <p>It provides low contrast between the specimen and its surrounding medium (D)</p> Signup and view all the answers

    What is the fundamental purpose of fixation in sample preparation for microscopy?

    <p>To make the specimen more rigid and easier to handle (C)</p> Signup and view all the answers

    Which of the following is NOT a typical benefit of using a compound microscope?

    <p>Ability to view three-dimensional structures (C)</p> Signup and view all the answers

    In bright-field microscopy, why is it important to have a light source that produces even illumination?

    <p>To minimize artifacts in the image (C)</p> Signup and view all the answers

    How does the numerical aperture (NA) of a lens relate to the angle of the cone of light entering the objective lens?

    <p>NA is directly proportional to the angle (C)</p> Signup and view all the answers

    Which of these statements accurately describes the relationship between resolution and numerical aperture in microscopy?

    <p>Resolution is inversely proportional to numerical aperture, meaning a higher numerical aperture results in higher resolution. (B)</p> Signup and view all the answers

    What type of microscopy is most suitable for observing the internal details of a bacterial cell in two dimensions?

    <p>Transmission electron microscopy (TEM) (C)</p> Signup and view all the answers

    Which type of microscopy is most suitable for observing the surface details of a live bacterium in three dimensions?

    <p>Scanning electron microscopy (SEM) (A)</p> Signup and view all the answers

    Which of the following techniques provides information about the three-dimensional structure of molecules?

    <p>X-ray crystallography (B)</p> Signup and view all the answers

    Which microscopy technique allows for observing the surface of live bacteria in both water and air?

    <p>Atomic force microscopy (AFM) (B)</p> Signup and view all the answers

    What is the main advantage of using a dark-field microscope compared to a bright-field microscope?

    <p>Dark-field microscopy can reveal unstained objects. (D)</p> Signup and view all the answers

    Which of the following correctly describes the relationship between X-ray crystallography and protein structure?

    <p>X-ray crystallography can be used to determine the three-dimensional structure of a protein. (B)</p> Signup and view all the answers

    Which microscopy technique relies on the principle of diffraction to generate images?

    <p>X-ray crystallography (A)</p> Signup and view all the answers

    Study Notes

    Lecture 2: Observing the Microbial Cell

    • The lecture is on observing microbial cells using various microscopy techniques.
    • A 3D image shows Salmonella inside a ruptured vesicle (2 µm).
    • Microscopy techniques are overviewed, including light microscopy, bright-field, phase-contrast, fluorescence, electron microscopy and scanning probe, as well as X-ray crystallography.

    Resolving Power of the Human Eye

    • Resolution is the smallest distance at which two objects can be separated and still be distinguished.
    • The resolution of the human retina is about 150 µm (1/7 mm).
    • This is limited by the distance between foveal cones and neuron clusters.

    Detection vs. Resolution

    • Detection is the ability to determine the presence of an object.
    • Magnification increases the apparent size of an image to resolve smaller separations between objects.
    • Samples (Rhodospirillum rubrum and Oenococcus oeni) are shown in different magnification states under a light microscope.

    Microbial Size

    • Microbes vary greatly in size across a few orders of magnitude.
    • Eukaryotic microbes (protozoa, algae, fungi): 2-2,000 μm.
    • Prokaryotic microbes (bacteria, archaea): 0.2-10 μm.
    • Viruses: 5-1000 nm.

    Microbial Shape

    • Prokaryotic cell structures are generally simpler than eukaryotic ones.
    • Examples include filamentous rods, rods, spirochetes, cocci in chains.
    • Eukaryotic microbes have diverse shapes, including amoeba proteus and Trypanosoma brucei.

    Microscopy at Different Size Scales

    • Different types of microscopy (human eye, light, scanning electron, transmission electron., cryo-electron, atomic force, X-ray crystallography) are suited for different sizes of objects.
    • Scales ranges from millimeters to angstroms.

    Light Properties

    • Electromagnetic radiation is composed of electric and magnetic waves.
    • Waves exist in different wavelengths (spectrum).
    • Visible light is a small part of the electromagnetic spectrum with a wavelength range of 400-750 nm.
    • For resolution, contrast between the object and the medium and small wavelengths relative to the object are required.

    Light Interactions with Objects

    • Absorption: photon energy is acquired by the object.
    • Reflection: wavefront bounces off.
    • Refraction: light bends as it enters a medium with a different speed.
    • Scattering: wavefront interacts with an object smaller than the light wavelength.

    Magnification by the Lens

    • Magnification uses refraction – the bending of light rays.
    • Parabolic lenses refract parallel light rays to intersect at a focal point.

    Limitations of Light Microscopy

    • Light rays create wavefronts, which interfere with the image's resolution.
    • The sharpness of the peak intensity of the point of detail is limited by wavelength.
    • Shorter wavelengths leads to sharper images.
    • Light microscopy can resolve objects at half the wavelength of visible light or about 200 nm.

    Bright-Field Microscopy

    • Bright-field microscopy generates a dark image of an object over a light background.
    • Magnification is achieved using objective lenses.
    • Numerical aperture (NA) affects resolving power by controlling the angle of the light cone entering the objective lens (higher NA = higher resolution). Immersion oil increases the refractive index.
    • The total magnification is the product of the magnification of the ocular lens and the objective lens.

    Compound Microscope

    • A compound microscope uses multiple lenses correcting for aberrations.
    • An ocular lens together with an objective lens provides total magnification.

    Dark-field Microscopy

    • Dark-field microscopy visualizes microbes as bright light halos against dark background.
    • Light scattered by the sample reaches the objective lens, making small objects visible.

    Phase-Contrast Microscopy

    • Phase-contrast microscopy superimposes refracted and transmitted light, shifted out of phase, to highlight refractive index differences.

    Fluorescence Microscopy

    • In fluorescence microscopy, the specimen absorbs light of a specific wavelength and emits light of a longer wavelength, called fluorescence.
    • Color filters are used to isolate excitation and emission light.
    • Fluorophores (molecules that fluoresce when excited) exhibit specific properties.

    Fluorescent Proteins

    • Fluorescent protein fusions are used to monitor gene expression, protein location, and movement in the cell.
    • The technique allows for localization.

    Fluorescence In Situ Hybridization (FISH)

    • FISH uses the specificity of DNA/RNA probes to create color visualization of specific regions or molecules within the specimen at the cellular level.
    • FISH allows for simultaneous visualization of multiple targets.

    Electron Microscopy

    • Electron microscopy relies on electrons instead of light for observation.
    • Transmission Electron Microscopy (TEM): electrons pass through the specimen revealing internal structures.
    • Scanning Electron Microscopy (SEM): electrons scan the specimen surface revealing external features in 3D.

    Cryo-Electron Microscopy and Tomography

    • Cryo-EM freezes specimens to prevent damage, reducing the need for staining.
    • Cryo-electron tomography reconstructs 3D images from multiple cryo-EM images of a rotated specimen.

    Scanning Probe Microscopy

    • Scanning probe microscopy (SPM) uses a sharp tip to observe nanoscale surface features, like the interactions of molecules.
    • The atomic force microscope (AFM) is an example.

    X-ray Crystallography

    • X-ray crystallography analyzes the diffraction pattern of X-rays scattered from atoms in a crystal to determine their position.
    • X-ray data are digitally analyzed to visualize sophisticated molecular models.

    Other Stains

    • Different types of stains are used for specific targeting of microbial structures, including acid-fast, spore, and negative stains.

    Preparing Specimens for Microscopy

    • Wet mounts are a simple technique but may lack contrast.
    • Fixation and staining are used to enhance contrast and preserve cell structures for visualization.

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    Description

    This quiz covers various microscopy techniques used for observing microbial cells, including light microscopy, electron microscopy, and X-ray crystallography. It also examines the concepts of resolving power and detection in relation to microbial sample analysis. Test your understanding of these critical microbiological methods and concepts.

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