Chapter 2- Basics of Living Systems Flashcards
How do you prepare a dry mount in sample prep?
- Solid specimens are either viewed whole or cut into thin slices (sectioning)
- Specimen is placed on the centre of slide and a coverslip is placed over a sample.
How do you prepare wet mount in sample prep?
- specimens are suspended in liquid e.g. water/immersion oil
- cover slip is placed on from an angle (45º)
- e.g. aquatic sample + living organisms
How do you prepare squash slides in sample prep?
- wet mount is first prepared
- lens tissue is used to gently press down cover slip
- use 2 microscope slides to avoid damage to cover slip
- good for soft sample
- e.g. root tip for cell division
How do you prepare a smear slides in sample prep?
- edge of slide is used to smear sample
- creates thin, even coating
- cover slip is then placed over sample
- e.g. blood smear slide.
Why do we use staining?
- The cytosol (aqueous interior) of cells and other cell structures are often transparent
- Stains increase contrast (different components in cell take stains to different degrees.)
- The increase in contrast allows components to become visible so they can be identified.
How do positively charged dyes like “Crystal Violet” and “Methylene Blue” work?
-The positively charged dye is attracted to negatively charged materials in cytoplasm, leading to staining of cell components.
How do negatively charged dyes like “Nigrosin” or “Congo Red” work?
The negatively charged dye is repelled by the negatively charged cytosol.
These dyes stay outside cells, leaving the cells unstained, which then stand out against the stained background.
=Negative Stain Technique.
What is Fixing, Sectioning, Staining and Mounting in sample prep?
Fixing- chemicals like formaldehyde are 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 be sliced thinly with a microtome.
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.
Describe what magnification is briefly:
Magnification is how many times larger the image is than the actual size of the object being viewed.
What is resolution in microscopy?
Resolution is the ability to see individual objects as separate entities.
It is limited by the diffraction of light, as it passes through samples.
What is the calculation for magnification?
Magnification = image size / actual size
What is an eyepiece graticule?
- An eyepiece graticule is a glass disc marked with a fine scale of 1 to 100.
- The scale has no units and remains unchanged whichever objective lens is in place.
- The relative size of the divisions increases with each increase in magnification.
- The scale on the graticule at each magnification is calibrated using a stage micrometer.
What is a stage micrometer?
- A stage micrometer is a microscope slide with a very accurate scale in μm.
- the scale marked in the micrometer slide is usually 100 divisions = 1nm, so 1 division = 10 μm.
How does a Transmission Electron Microscope (TEM) work?
In a transmission electron microscope, a beam of electrons is transmitted through a specimen and focused to produce an image.
Resolution of 0.5 nm
How does a Scanning Electron Microscope work?
In a scanning electron microscope, a beam of electrons is sent across the surface of a specimen and the reflected electrons are collected.
Resolution = 3-10 nm
Describe sample prep for electron microscopes:
Inside of electron microscope is a vacuum, so electron beams travel in a straight line.
- Fixation using chemicals.
- Freezing
- Staining with heavy metals
- dehydration with solvents
- TEM- samples set in resin and stained again
- SEM- fractured to expose the inside and will then need to be coated with heavy metals.
Why do we use fixation when preparing samples for electron microscopy?
Fixation stablises sample and prevents decomposition.
Why do we use dehydration when preparing samples for electron microscopy?
Dehydration prevents vaporisation of water in vacuum, as vaporisation would damage sample.
Why do we embed sample in resin when preparing samples for electron microscopy?
Embedding allows thin slices to be obtained
Why do we stain samples with heavy metals when preparing samples for electron microscopy?
Staining with heavy metals creates contrast, in electron beams.
Describe differences between an Light microscope and electron microscope:
LM- inexpensive buy/easy to operate
EM- expensive/difficult to operate
LM- small + portable
EM- large, needs installation
LM- simple sample prep
EM- complex sample prep
LM- sample prep does not lead to distortion
EM- sample prep often distorts material
LM- vacuum not required
EM- vacuum required
LM- natural colour sample seen
EM- black and white images produced (can be coloured digitally)
LM- up to x2000 magnification
EM- over x500,000 magnification
LM- resolution is 200nm
EM- resolution of TEM is 0.5nm and SEM is 3-10nm
LM- specimen can be living/dead
EM- specimen dead
What is an artefact?
- An artefact is a visible structural detail caused by processing the specimen + not a feature of specimen.
- in both Light + Electron Microscopy.
- E.g. bubbles trapped under cover slip.
Describe structure and function of: Nucleus
S:
- largest organelle
- spherical
- chromatin
- surrounded by nuclear membrane
- nucleolus inside
F:
- contains genetic material
- nucleolus makes RNA and ribosomes
- contains instructions for making proteins.
Describe structure and function of: Rough and Smooth Endoplasmic Reticulum
S:
- flattened membrane bound sacs- cisternae
- which are continuous with outer nuclear membrane
- contains ribosomes (only RER)
F:
RER- transports proteins made on attached ribosomes
SER- involved in making lipids
