2.1-2.3 Microscopy Flashcards
When was the first type of microscope developed?
16th Century
State the 3 rules of Cell Theory.
-Both plant and animal tissue is composed of cells
-Cells are the basic unit of all life
-Cells only develop from existing cells.
What can microscopes be used for?
Observing living and dead specimens.
What are the names of the two lenses in a compound light microscope?
Eyepiece lens and objective lens.
What is the difference between a light microscope and a compound light microscope?
There is significantly less chromatic aberration and higher magnification in a compound light microscope.
What term should you use instead of “clarity” when discussing microscopy?
“Distinction” and “resolution”
With increased resolution, what do objects become?
“Visibly distinct”, more detail can be seen
What will the method chosen to prepare a sample for examination by a light microscope depend on?
The nature of the specimen and the resolution that is desired.
What is a dry mount?
-Specimens are viewed whole or cut into thin slices with a sharp blade (sectioning).
-Specimen is placed on the slide and a cover slip is placed over the sample.
Give examples of things that can be observed using a dry mount.
Hair, pollen, insect parts, muscle tissue, plants.
What is the name of the mount/slide whereby solid specimens are viewed whole or cut into thin slices?
Dry mount.
What is a wet mount?
-Specimens are suspended in a liquid such as water or an immersion oil.
-Cover slip is placed on at an angle
In a wet mount, why is the cover slip placed on at an angle?
To reduce air bubbles and artefacts.
What is the name of the slide/mount whereby specimens are suspended in a liquid?
Wet mount.
Give examples of specimens that can be viewed using a wet mount.
Aquatic samples, living organisms
What is a squash slide?
-A wet mount is first prepared, then a lens tissue is used to gently press down the cover slip (a microscope slide can also be used instead of a cover slip to prevent damage)
What can squash slides be used for?
Soft samples, for example root tips can be squashed to look at cell division.
What is the name of the mount/slide whereby a lens tissue is used to gently press down the cover slip?
Squash slide.
What is a smear slide?
-The edge of a slide is used to smear the sample to create a thin even coating on another slide
-A cover slip is then placed over the sample
What can smear slides be used for?
Samples of blood, to see all the cells.
What is the name of the mount/slide whereby an edge of a slide is used to spread the sample to create a thin even coating on another slide?
Smear slide.
What are the benefits of wet mounts?
-Can be used for hiehger magnification
-Can allow you to observe behaviour of animals
-Greater resolution because the liquid refracts light
-Can observe natural colour and mobility patterns of animals
What are the problems with wet mounts?
-Greater chance of artefacts being produced (eg air bubbles)
-Slides tend to dry out under the light of the microscope.
What are the benefits of dry mounts?
It is easy to prepare slides
What are the problems with dry mounts?
-Mounts are temporary unless you seal them
-It is harder to see more intricate structures.
Why do specimens need to be thin for sample preparation?
So that light can pass through it and form an image
What is an artefact?
Something observed in a scientific investigation that is not naturally present but occurs as a result of the investigative procedure.
What is brightfield microscopy?
The sample is illuminated from below with white light and observed from above.
What is wide-field microscopy?
The whole sample is illuminated at once.
Why do we stain samples in microscopy? (Short answer)
-Stains increase the contrast of specimens because the different components in the cells absorb the stains to different degrees.
-The increase in contrast allows components to become visible so they can be identified.
Why is it difficult to see specimens under a microscope without staining them first?
-Because cells do not absorb much lught
-The cytosol (aqueous interior) of cells, and other cell structures, are often transparent.
How do you prepare a sample for staining?
-First place it on a slide and allow it to air dry
-Heat-fix it by passing it through a flame
-The specimen will now adhere to the slide and take up stains.
What is differential staining?
Using specific stains to distinguish different types of cell
Describe the process of FIXING in microscopy.
Formaldehyde is used to preserve the specimen
Describe the process of SECTIONING in microscopy.
Specimens are dehydrated and placed in a wax mould, then thinly sliced sith a microtome.
Describe the process of STAINING in microscopy.
Specimens are often treated with multiple stains to show different structures
Describe the process of MOUNTING in microscopy.
The specimens are secured to a microscopy slide and a cover slip is placed on top.
Give two examples of negatively charged dyes
Nigrosin and Congo red.
Explain the process of the NEGATIVE STAIN TECHNIQUE.
Negatively charged dyes are repelled by the negatively charged cytosol so they stay outside of the cell, making the unstained cells stand out against the stained background.
Give two examples of positively charged dyes.
Crystal Violet or Methylene Blue
Explain the process of the POSITIVE STAIN TECHNIQUE.
Positively charged dyes are attracted to the negatively charged materials in the cytoplasm (eg cytosol), which leads to the staining of the cell components.
What is Gram stain technique?
Used to separate bacteria into Gram-positive and Gram-negative categories.
Describe the process of the Gram stain technique
-Crystal violet dye is first applied to a bacteria specimen on a slide, then iodine (which fixes the dye),
-The slide is then washed with alcohol
What is the result of a Gram-positive bacteria having the Gram stain technique applied onto it?
-After the wash with alcohol, the bacteria retain the crystal violet stain and appear blue or purple under a microscope
What are Gram-positive bacteria?
A type of bacteria that have a thick cell-wall, and are susceptible to Penicillin (which inhibits the formation of cell walls)
What is the result of a Gram-negative bacteria having the Gram stain technique applied onto it?
-After the wash with alcohol, the bacteria lose the stain.
-They are then stained with a “counterstain” called safranin dye, which will make the bacteria appear red or pink.
What is the counterstain used in Gram stain technique?
Safranin dye
What are Gram-negative bacteria?
Bacteria with a thin cell wall that aren’t susceptible to penicillin
What is Acid Fast Technique?
Used to differentiate species of mycobacterium from other bacteria.
Describe the process of Acid Fast Technique
-A lipid solvent is used to carry carbolfuchsin dye into the cells being studied.
-Cells are washed with a dilute acid-alcohol solution
-Mycobacterium are not affected by the acid-alcohol and retain the stain, which is bright red.
-Other bacteria lose the stain and are exposed to a methylene blue stain
What dye is used in Acid Fast Technique?
Carbolfuchsin dye
What are the cells washed with after the dye is applied in Acid Fast Technique?
A dilute acid-alcohol solution
If mycobacterium is found when using the Acid Fast Technique, what will happen?
The mycobacterium will not be affected by the acid-alcohol, and retain the carbolfuchsin stain which is bright red.
If mycobacterium is NOT found when using the Acid Fast Technique, what will happen?
-The bacterium will be affected by the acid-alcohol, lsoing the carbolfuchsin stain.
-They will then be exposed to a methylene blue stain.
What do we use to stain starch?
Iodine.
Define “Magnification”
The number of times larger an image is compared to the real size of an object
Define “Resolution”
The ability to distinguish between two separate points.
Define “Empty Magnification”
When you magnify an image to make it larger, but it does not appear more defined
Describe the advantages of Light Microscopes
-Cheap, easily accessible, easy to use
-Natural colour of material can be observed
-Living organisms can be observed
-Preparation is quick and simple
Describe the disadvantages of Light Microscopes
-Low resolving power
-Only magnifies objects up to 1500x
Describe the advantages of Electron Microscopes in general
-Can see detail of organelles
-Much better resolution than light microscopes
-Magnifies objects more than 500,000x
Describe the disadvantages of Electron Microscopes in general
-No colour
-Very large and must be operated in special rooms
-Expensive
-Damages the specimen
-Preparation of material is lengthy and complex.
Describe how a Transmission Electron Microscope works.
Fires a beam of electrons though the specimen and produces a 2D image
Describe the resolving power and maximum magnification of a Transmission Electron Microscope.
Resolution: 0.2nm, Magnification 500,000x
Describe how a Scanning Electron Microscope works.
Fires a bean of electrons across the surface of a specimen and produces a 3D image.
Describe the resolving power and maximum magnification of a Scanning Electron Microscope.
Resolution: 0.5-4nm, Magnification = 100,000x
How does an Electron Microscope work?
-A beam of electrons witha wavelength of less than 1nm is used to illustrate the specimen,
-The inside of an electron microscope is a vacuum to ensure the electron beams travel in straight lines.
-Because of the vacuum, specimens need to be treated in very specific ways.
Why do specimens need to be treated in very specific ways for Electron Microscope scanning?
Because the inside of an electron microscope is a vacuum.
Describe how Laser Scanning Confocal Microscopy works.
-Uses lasers to enable the reconstruction of three-dimensional objects
-Shows sets of images obtained at different depths (sectioning)
-Can show fluorescent dyes and tags
Describe how to prepare a specimen for electron microscopy
-Fixation of the specimen using chemicals or freezing
-Stain with heavy metals
-Dehydrate with solvents
-Samples for TEM will be set in resin and may be stained again
-Samples for SEM may be fractured to expose the inside and will then need to be coated with heavy metals.
What is a graticule?
A specialised piece of glass inserted into the microscope, that features a calibrated grid or scale etched into its surface
What are the two types of graticules?
Eyepiece graticule and stage graticule
What is the difference between the two types of graticules?
-The eyepiece graticule has regular divisions that need to be calibrated for each magnification
-The stage graticule shows true lengths
What is 1 stage micrometer unit equal to?
10 micrometers
