|1. Biochemical Tests|
|These five tests identify the main
biologically important chemical compounds. For each test take a small
amount of the substance to test, and shake it in water in a test tube. If
the sample is a piece of food, then grind it with some water in a pestle
and mortar to break up the cells and release the cell contents. Many of
these compounds are insoluble, but the tests work just as well on a fine
|Chromatography is used to separate pure
substances from a mixture of substances, such as a cell extract. It is
based on different substances having different solubilities in different
solvents. A simple and common form of chromatography uses filter paper.
An Rf value is characteristic of a particular solute in a particular solvent. It can be used to identify components of a mixture by comparing to tables of known Rf values.
Sometimes chromatography with a single solvent is not enough to separate all the constituents of a mixture. In this case the separation can be improved by two-dimensional chromatography, where the chromatography paper is turned through 90° and run a second time in a second solvent. Solutes that didn't separate in one solvent will separate in another because they have different solubilities.
There are many different types of chromatography.
|3. Cell Fractionation|
|This means separating different parts and
organelles of a cell, so that they can be studied in detail. All the
processes of cell metabolism (such as respiration or photosynthesis) have
been studied in this way. The most common method of fractionating cells is
to use differential centrifugation:
A more sophisticated separation can be performed by density gradient centrifugation. In this, the cell-free extract is centrifuged in a dense solution (such as sucrose or caesium chloride). The fractions don't pellet, but instead separate out into layers with the densest fractions near the bottom of the tube. The desired layer can then be pipetted off. This is the technique used in the Meselson-Stahl experiment (module 2) and it is also used to separate the two types of ribosomes. The terms 70S and 80S refer to their positions in a density gradient.
|4. Enzyme Kinetics|
This means measuring the rate of enzyme reactions.
Of all the techniques used in biology microscopy is probably the most important. The vast majority of living organisms are too small to be seen in any detail with the human eye, and cells and their organelles can only be seen with the aid of a microscope. Cells were first seen in 1665 by Robert Hooke (who named them after monks' cells in a monastery), and were studied in more detail by Leeuwehoek using a primitive microscope.
Units of measurement. The standard SI units of measurement used in microscopy are:
Magnification and Resolving Power. By using more lenses microscopes can magnify by a larger amount, but this doesn't always mean that more detail can be seen. The amount of detail depends on the resolving power of a microscope, which is the smallest separation at which two separate objects can be distinguished (or resolved). It is calculated by the formula:
where is the wavelength of light, and n.a. is the numerical aperture of the lens (which ranges from about 0.5 to 1.4). So the resolving power of a microscope is ultimately limited by the wavelength of light (400-600nm for visible light). To improve the resolving power a shorter wavelength of light is needed, and sometimes microscopes have blue filters for this purpose (because blue has the shortest wavelength of visible light).
Different kinds of Microscope.Light Microscope. This is the oldest, simplest and most widely-used form of microscopy. Specimens are illuminated with light, which is focussed using glass lenses and viewed using the eye or photographic film. Specimens can be living or dead, but often need to be stained with a coloured dye to make them visible. Many different stains are available that stain specific parts of the cell such as DNA, lipids, cytoskeleton, etc. All light microscopes today are compound microscopes, which means they use several lenses to obtain high magnification. Light microscopy has a resolution of about 200 nm, which is good enough to see cells, but not the details of cell organelles. There has been a recent resurgence in the use of light microscopy, partly due to technical improvements, which have dramatically improved the resolution far beyond the theoretical limit. For example fluorescence microscopy has a resolution of about 10 nm, while interference microscopy has a resolution of about 1 nm.
Electron Microscope. This uses a beam of electrons, rather than electromagnetic radiation, to "illuminate" the specimen. This may seem strange, but electrons behave like waves and can easily be produced (using a hot wire), focussed (using electromagnets) and detected (using a phosphor screen or photographic film). A beam of electrons has an effective wavelength of less than 1 nm, so can be used to resolve small sub-cellular ultrastructure. The development of the electron microscope in the 1930s revolutionised biology, allowing organelles such as mitochondria, ER and membranes to be seen in detail for the first time.
The main problem with the electron microscope is that specimens must be fixed in plastic and viewed in a vacuum, and must therefore be dead. Other problems are that the specimens can be damaged by the electron beam and they must be stained with an electron-dense chemical (usually heavy metals like osmium, lead or gold). Initially there was a problem of artefacts (i.e. observed structures that were due to the preparation process and were not real), but improvements in technique have eliminated most of these.
There are two kinds of electron microscope. The transmission electron microscope (TEM) works much like a light microscope, transmitting a beam of electrons through a thin specimen and then focussing the electrons to form an image on a screen or on film. This is the most common form of electron microscope and has the best resolution. The scanning electron microscope (SEM) scans a fine beam of electron onto a specimen and collects the electrons scattered by the surface. This has poorer resolution, but gives excellent 3-dimentional images of surfaces.
Comparison of Light and Electron Microscopes