Microscopy Flashcards
Why are light microscopes good
You can view living organisms
How does a light microscope work
A compound light microscope has two lenses- objective and eyepiece. Objective lens is near specimen, eyepiece lens is where the specimen is viewed. Objective lens produces a magnified image, which is magnified again by the eyepiece lens.
What’s the benefit of having two lenses in a microscope
Allows for much higher magnification and reduced chromatic aberration.
How do you prepare a dry mount sample
Solid specimens cut into thin slices- sectioning. Specimen placed on centre of slide and cover slip placed over the sample. Can also view sample whole.
How do you prepare a wet mount sample
Specimen suspended in water, cover slip placed on from angle. Aquatic samples or other living organisms can be viewed this way.
How do you prepare squash slides
Wet mount first prepare, lens tissue used to gently press down cover slip. Potential damage to cover slip can be avoided by squashing the sample between two microscope slides.
How do you prepare smear slides
Edge of slide is used to smear sample, creating a thin, even coating on another slide, cover slip placed over sample.
Why do you need stains
Stains increase contrast as different components within a cell take up stains to different degrees. The increase in contrast allows components to become visible so they can be identified.
How do you prepare a sample for staining
First placed on slide and allowed to air dry. This is then heat-fixed by passing through a flame. Specimen will adhere to microscope slide and take up stains.
What are the positively charged dyes
Crystal violet or methylene blue are positively charged dyes, which are attracted to negatively charged materials in cytoplasm leading to staining of cell components.
What are the negatively charged dyes
Nigrosin or Congo red are negatively charged and are repelled by the negatively charged cytosol. These dyes stay outside the cell, leaving cells unstained, which then stand out against the stained background.
What are gram stain techniques
Separates bacteria into two groups- gram positive and gram negative.
How do you test for gram positive bacteria
Crystal violet applied to to bacteria specimen on slide, then iodine which fixes dye. Slide is then washed with alcohol. Gram positive bacteria retain the crystal violet stain and will appear blue or purple under a microscope.
How do you test for gram negative bacteria
Gram negative bacteria have thinner cell walls and therefore lose crystal violet stain. They are stained with safranin dye- counterstain. These bacteria appear red.
What are gram positive bacteria susceptible to
Penicillin- Gram negative bacteria have much thinner cell walls which are not susceptible to penicillin.
What is the acid fast technique
It is used to differentiate species of mycobacterium from other bacteria.
How is the acid fast technique carried out
A lipid solvent is used to carry carbolfuchsin dye into the cells being studied dye into the cells being studied. The cells are then washed with a dilute acid-alcahol solution. Mycobacterium are not affected by the acid-alcahol and retain the carbolfuchsin stain, which is bright red. Other bacteria lose the stain and are exposed to a methylene blue stain, which is blue.
What’s fixing, sectioning, staining and mounting.
Fixing- chemicals used to preserve specimens in as near-natural a state as possible.
Sectioning-Specimens are dehydrated with alcohols and then placed in a mould with wax or resin to form a hard block. This can then be sliced thinly.
Staining- Specimens are often treated with multiple stains to show different structures.
Mounting- the specimens are then secured to a microscope slide and a cover slip placed on top.
What is resolution
Ability to see individual objects as separate entities.
What is resolution limited by
Diffraction of light as it passes through samples and lenses. In optical microscopy structures that are closer than half the wavelength of light cannot be seen separately. Resolution can be increased by using beams of electrons with a wavelength thousands of times shorter than light.
What is electron microscopy
In electron microscopes, a beam of electrons with a wavelength of less that 1nm is used to illuminate the specimen. More detail of cell ultrastructure can be seen because electrons have a much smaller wavelength than light waves.
What are the disadvantages to electron microscopes
- They are very expensive pieces of equipment and can only be used inside a a carefully controlled environment in a dedicated space.
- Specimens can be damaged by electron beam and because preparation process is very complex, there is a problem with artefacts.
What are transmission electron microscopes
A beam of electrons is transmitted through a specimen and focused to produce an image
What are scanning electron microscopes
A beam of electrons is sent across the surface of a specimen and the reflected electrons are collected- 3D images produced.
