How does a light microscope work?
A compound light microscope has two lenses:
- Objective lens - placed near the specimen
- Eyepiece lens – through which the specimen is viewed
The objective lens produces a magnified image which is magnified again by the eyepiece lens.
Illumination is provided by a light under the sample.
What are the types of sample preparation?
- Dry mount
- Wet mount
- Squash slides
- Smear slides
Explain dry mount and wet mount sample preparation.
Dry mount- solid specimens are viewed whole or cut into very thin slices with a sharp blade (sectioning). A cover slip is placed over the sample.
(Eg. Hair, pollen, muscle tissue)
Wet mount- specimens are suspended in a liquid (water or immersion oil). Cover slip is places from an angle. Used for aquatic samples.
Explain squash slides and smear slides sample preparation.
Squash slides- a wet mount is prepared, then a lens tissue is used to gently press down the cover slip. Good for soft samples.
Smear slides- the edge of a slide is used to smear the sample, creating a thin coating on another slide. A cover slip is placed on top. (Cells in blood)
Why is staining used?
In light microscopy, the sample is illuminated from below with a white light:
- image has low contrasts as cells don’t absorb much light.
- resolution is limited due to wavelength and diffraction of light.
- cytosol and other cell structures are often transparent.
Stains increase the contrast as different components within a cell take up stains to different levels. It allows components to become more visible and identifyable.
How do you prepare a sample to stain?
- Place on a slide and air dry.
2. Pass it through a flame to heat fix.
What is differential staining?
Can distinguish between two types of organisms that would otherwise be hard to identify.
or
Differentiate between organelles of a single organism:
- gram stain technique.
- acid-fast technique.
Explain the gram stain technique.
- used to separate bacteria into Gram-positive and Gram-negative.
- Apply crystal violet to specimen, then iodine, which fixes dye.
- Wash the slide with alcohol.
- Gram positive bacteria = retain crystal violet stain and appear blue/purple.
- Gram negative bacteria= lose stain due to thinner cell walls. Counterstained with safranin dye and will now appear red.
Gram positive bacteria are susceptible to the antibiotic pencillin, which inhibits the formation of cell walls.
Explain the acid-fast technique.
-Used to differentiate species of mycobacterium from other bacteria.
- Lipid solvent carries carbolfuchsin dye into sample cells.
- Cells are washed with a dilute acid-alcohol solution
Mycobacterium- retain carbolfuchsin stain = red
Other bacteria- exposed to methylene blue = blue.
What are the stages in the production of pre-prepared slides?
- Fixing- chemicals are used to preserve specimens in as near natural state as possible.
- Sectioning- specimens are dehydrated with alcohols and placed in mould with wax/resin. Can be sliced thinly.
- Staining- treated with multiple stains.
- Mounting- secured to slide with a cover slip on top.
What is magnification and how do you calculate it?
How many times larger the image is than the actual size if the object.
Objective lens allows users to adjust magnification.
Magnification = image size / actual size
What is an eyepiece graticule?
A glass disc marked with a fine scale of 1-100 (no units).
-the scale remains unchanged if objective lens changes but the relative size of divisions increases with magnification.
The scale on the graticule at each magnification Is calibrated using a stage micrometer.
What is a stage micrometer?
A slide with an accurate micro metres (um) scale engraved in it.
The scale marked on the micrometer is usually 100 divisions= 1mm.
So 1 division = 10um
What is electron microscopy?
In electron microscopy, a beam of electrons with a wavelength of less than 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.
- they can produce images with magnifications of up to x500000 and still have a clear resolution.
What are the two types of electron microscopy?
Transmission electron microscope (TEM):
-a beam of electrons is transmitted through the specimen and then focused to produce an image.
similar to light microscopy.
Best resolution with a resolving power of 0.5nm
Scanning electron microscope (SEM):
- beam of electrons sent across the surface of a specimen and the reflected electrons are collected. Three dimensional images of surfaces are produced.
Resolving power is from 3-10nm, so resolution is not as good as TEM.
Explain sample preparation for electron microscopes.
Involves:
- Fixation using chemicals/ freezing.
- Staining with heavy metals.
- Dehydration with solvents
Then:
TEM = set in resin and stained again.
SEM= fractured to expose insides and coated with heavy metals.
How does a laser scanning confocal microscope work?
- Moves a single spot of focused light across the specimen. (Point illumination)
- causes fluorescence ( the absorption and re radiation of light) from the components labelled with a ‘dye’
- Emitted light is filtered through a pinhole aperture. Only light radiated from close to the focal point is detected.
Other key facts about laser scanning confocal microscopes.
- unwanted radiation would cause blurring and reduce the resolution so it doesn’t pass through the pinhole and therefore isn’t detected.
- thin sections of specimen examined = allows high resolution images to be obtained.
- 2D and 3D images can be formed.
- non-invasive: used in the diagnosis of diseases of the eye.
- position of the two pinholes means the light waves from the laser follow the same oath as the radiated light waves from the sample = same focal place (confocal)
What is resolution, how is it limited and how can it be increased?
resolution is the ability to see individual objects as separate entities.
- Limited by the diffraction of light as structures are very close and thus causes light to overlap and as a result detail is lost.
- Increased by using beams of electrons as they have a shorter wavelength.
nucleus and its function?
- contains coded genetic information in the form of DNA molecules.
- DNA directs the synthesis of all proteins required by the cell.
- DNA associates with proteins called histones to form chromatin which then coils and condenses to form chromosomes.
- Also controls the metabolic activities of the cell.
nucleolus and its function?
- It is an area within the nucleus.
- responsible for producing ribosomes.
- composed of proteins and RNA
- RNA is used to produce ribosomal RNA which is then combined with proteins to form the ribosomes necessary for protein synthesis.
mitochondria and its function?
- mitochondria have a double membrane. the inner membrane is highly folded to form structures called cristae. This inner membrane contains enzymes used in aerobic respiration.
- the fluid interior is called matrix.
- they are the site of ATP(energy currency) production during aerobic respiration.
- they are self-replicating, so they can reproduce themselves if the cell requires more energy.
- they are abundant in cells where lots of metabolic activity takes place.
chloroplasts and its function?
- the organelles responsible for photosynthesis in plant cells.
- found in the cells of the green parts of plants.
- They have a double membrane. the fluid enclosed in the chloroplast is called the stroma.
- Contain DNA and ribosomes so they are able to make their own proteins.
- the internal membranes provide a large SA needed for the enzymes,proteins and other molecules necessary in photosynthesis.
Lysosomes and their function?
- Small bags formed from the golgi apparatus. Each is surrounded by a single membrane.
- contain hydrolytic(digestive) enzymes. they are responsible for breaking down waste material in cells, including old organelles.
- important role in the immune system = responsible for breaking down pathogens ingested by phagocytic cells.
- important role in apoptosis(programmed cell death)
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Chapter 2- basic components of living systems35
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Chapter 11- Biodiversity38
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C12- Communicable Disease28
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Chapter 8- Transport In Animals72
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Chapter 9- Transport In Plants52
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Chapter 7- Exchange Surfaces+ Breathing43
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Chapter 6 - Cell Cycle59
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Chapter 10- Classification52
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Chapter 4 - Enzymes33
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3.8 - 3.11 Nucleic Acids32
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Chapter 5 - Plasma Membrane26
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Chapter 3 - Biological Molecules64
