Lecture 4 (Linda Stewart) - Microscopy Flashcards
Evaluate the parts of a light microscope in terms of their contribution to image production and use of the microscope
- Lens - Creates an image by bending light and focusing the light rays on a specific point called the focal point.
- Objective lens - Lens closest to the specimen which produces a magnified image.
- Substage condenser - Focuses a cone of light on the slide.
- Ocular lens - further magnifies the image created by the one time lens.
- Focusing knobs - Move either the stage or the nosepiece vertically to focus the image.
- Mechanical stage clip - Smoothly moves a slide during viewing.
Outline the use of the bright-field microscope, how it creates an image, and the quality of image it produces
- Used to examine both stained and unstained specimens.
- Light from the illuminated specimen is focused by the objective lens, creating an enlarged image within the microscope.
- The ocular lens further magnifies this primary image.
- It forms a dark image against a brighter background.
Outline the use of a dark field microscope, how it creates an image, and the quality of image it creates
- Used to observe living, unstained cells.
- Used to observe internal structures in eukaryotic microorganisms.
- Used to identify bacteria such as Treponema pallidum, the causative agent of syphilis.
- Light reaches the specimen from the sides only.
- The only light reaching the lens has been scattered by the specimen. - An image is formed by the light reflected or refracted by the specimen.
- Produces a bright image of the specimen against a dark background.
Outline the use of the phase-contrast microscope, how it creates an image, and the quality of image produced
- Used to visualise living, unstained microbes.
- Based on the principle that cells differ in refractive index from their surroundings.
- Light passing through the cells differ in phase from light passing through the surrounding liquid.
- Improves the contrast of a sample without using a stain.
- Produces an image of dark cells on a light background.
Outline the use of a differential interference contrast (DIC) microscope, how it creates an image, and the quality of image produced
- Used to observe living, unstained cells.
- Creates image by detecting differences in refractive indices and thickness of different parts of specimen.
- Uses a polariser to create two distinct beams of polarised light that pass through the specimen into the objective where they combine as one. Combined beams are not in phase as their refractive indices are different, enhancing subtle differences in cell structures.
- Structures not visible by bright-field microscopy are sometimes visible by DIC.
- Live, unstained cells appear brightly coloured and three-dimensional.
- Cell walls, endospores, granules, vacuoles, and nuclei are clearly visible.
Outline the use of the fluorescence microscope, how it creates an image, and the quality of image produced
- Fluorochrome-labelled probes, such as antibodies, tag specific cell constituents for identification of unknown pathogens.
- Used for localisation of specific proteins in cells.
- Exposes specimen to ultraviolet, violet, or blue light.
- Specimens are usually stained with fluorochromes.
- Creates a bright image of the specimen resulting from the fluorescent light emitted by the specimen.
Outline the use of the confocal scanning laser microscope (CSLM), how it creates an image, and the quality of image produced
- Used in the study of bio films.
- Uses a computerised microscope coupled with a laser source to generate a sharp, composite 3D image of a specimen.
- The computer can focus the laser on single layers of the specimen.
- Different layers can then be compiled for a three-dimensional image.
- Resolution is 0.1 micrometers.
Recommend a fixation process to use when the microbe is a bacterium or archaeon, and when the microbe is a protist
- Bacteria or archaeon - heat fixation (preserves overall morphology but not internal structures).
- Protists - Chemical fixation (protects fine cellular substructure and morphology).
Plan a series of appropriate staining procedures to describe an unknown bacterium as fully as possible
Gram-Staining
- Used to differentiate whether the bacterium is gram-positive or gram-negative.
Acid-Fast Staining
- Useful for staining members of the genus Myobacterium.
- The cold Ziehl-Neelsen method uses high concentrations of phenol and carbol fuchsin, as well as a wetting agent, to drive carbol fuchsin into mycobacterial cells.
- Once this dye has penetrated, the cells are not easily decolourised by acidified alcohol and are thus called acid-fast.
- Non-acid-fast bacteria are decolourised by acid-alcohol and are therefore stained another colour by a counterstain.
Endospore Staining
- Done by heating the bacterium and using a double-staining technique.
- Bacterial endospore is one colour and the vegetative cell is a different colour.
Capsule Stain
- Used to visualise polysaccharide capsules surrounding bacteria.
- Capsules appear colourless against a stained background (negative stain).
Flagella Staining
- Mordant applied to increase thickness of flagella.
Compare what happens to gram-positive and gram-negative bacterial cells at each step of the gram-staining procedure
Step 1) Apply crystal violet (primary stain) for 1 minute and then water rinse. (All cells stain purple)
Step 2) Apply iodine (mordant) for 1 minute and then water rinse. (All cells remain purple)
Step 3) Apply alcohol (decolouriser) for 10-30 seconds and then water rinse. (Gram-positive cells remain purple and gram-negative cells become colourless)
Step 4) Apply safranin (counterstain) for 30-60 seconds and then water rinse. (Gram-positive cells remain purple and gram-negative cells appear pink/red)
Outline the use of the transmission electron microscope (TEM), how it creates an image, and the quality of image produced
- Enables visualisation of structures at the molecular level.
- Specimen must be very thin (electrons are poor at penetrating) and must be stained (to scatter electrons and improve contrast- osmium acid, permanganate, uranium).
- Electrons scatter when they pass through thins sections of a specimen.
- Transmitted electrons are under vacuum which reduces scatter and produces a clear image.
- Denser regions in the specimen scatter more electrons and appear darker.
- High magnification and resolution (0.2nm) as wavelengths of electrons are much shorter than wavelengths of light.
Outline the use of the scanning electron microscope (SEM), how it creates an image, and the quality of image produced
- Produces a realistic 3D image of specimen’s surface features.
- Can determine actual in situ location of microorganisms in ecological niches.
- Specimen is coated with a thin film of heavy metal e.g. gold.
- An electron beam scans the specimen, causing electrons to be excited from the surface of the specimen.
- Scattered electrons are collected by a detector, and an image is produced.
What is the typical size range for prokaryotes
0.2 micrometers to greater than 700 micrometers
What is the typical size range for eukaryotes
10 to greater than 200 micrometers
What is the significance of a cell being small
Advantages:
- Small cells have a higher surface area to volume ratio than larger cells.
- How fast a cell grows depends on the rate of nutrient exchange per unit cell volume and therefore small cells tend to grow faster than larger cells.
Disadvantages:
- Cellular organisms smaller than 0.15 micrometers are unlikely because of the volume needed to house essential components such as proteins, nucleic acids, and ribosomes.
