How do we see what’s in our blood? Magnification Staining
Magnification = observed size (size in picture)
actual size (real size of structure)
You must measure from the edge of the structure and be consistent.
Then divide the number you get by the size of the structure you have been given.
You can rearrange this:
Real size = size of structure in picture
You have measured a chloroplast which is 30mm in length you have been told that the actual size is 0.5um what is the magnification?
First convert the units so they are the same 1um = 0.001mm
30/0.001 = 30000 (o)
Then divide 30000 by 0.5
Magnification = x60000
Definitions - record
Erythrocyte – (red blood cell). They are biconcave discs that transport oxygen and carbon dioxide.
Leucocytes – white blood cells
Platelets – fragments of giant cells called megakaryocytes. They are involved in blood clotting
What is a differential stain?
A differential stain makes some structures appear darker or a different colour to other structures.
For example, in a blood film the nuclei of leucocytes are stained purple. This then allows us to identify different types of leucocytes because their nuclei are different shapes
Spot the difference! More info to follow in lesson 4
Using a haemocytometer
Originally used to count blood cells (as the name suggests). It is now used to count other cells and many types of microscopic particles.
It consists of a thick glass slide with a rectangular indentation that creates a chamber of certain dimensions. This chamber is etched with a grid of lines.
The area bounded by the lines is known
The depth of the chamber is also known.
Therefore, it is possible to count the number of cells in a specific volume of fluid, and thereby calculate the concentration of cells in the fluid overall.
The main divisions separate the grid into 9 large squares. Each square has a surface area of one square mm, and the depth of the chamber is 0.1 mm. Thus the entire counting grid lies under a volume of 0.9 mm-cubed
Counting cells and the Maths
We are counting cells in the centre of the grid.
These are the triple lined squares.
The squares measure exactly 0.2 x 0.2 mm
When the cover slip goes on, the platform is exactly 0.1 mm below the cover slip.
When you view one of the triple lined squares under the microscope the volume is:
0.1 x 0.2 x 0.2 mm = 0.004mm 3
The North-west rule
Sometimes cells lie on top of the triple lines around the edge of the square. If we count them all or miss them all out the cell count won’t be accurate.
If a cell lies on the middle of the triple lines at the top (north) or left hand side (west) of the grid we count it. If it’s on the other two sides we don’t.
Each triple lined square has a volume of 0.004mm 3
We are counting five triple-lined squares so 0.004mm 3 x 5 = 0.02mm 3
Our blood is yeast cells. This is diluted 100 times.
If the number of cells counted is E:
In 1mm 3 of the original yeast sample the number of cells will be:
1 / 0.02 x E X 100
Have a go!!
Dilution factor = 200
Microscopy The light microscope v the electron microscope
Optical microscope Light microscope allows you to see the position of the cell membrane, cytoplasm, and nucleus in eukaryotic cells. Small organelles can not be seen clearly.
Light Microscopy: 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.
Magnification is how much bigger a sample appears to be under the microscope than it is in real life.
Resolution is the ability to distinguish between two points on an image i.e. the amount of detail
The resolution of an image is limited by the wavelength of radiation used to view the sample.
Overall magnification = Objective lens x Eyepiece lens
How do they work?
Uses a beam of electrons focused by electro magnets.
Can investigate the fine (ultra structure of the cell).
There are two types …
Transmission electron microscope.
Scanning electron microscope.
Electromagnets focus an electron beam, this is transmitted through the specimen.
Denser parts of the specimen appear darker as they absorb more electrons (contrast is improved using electron-dense stains).
Provides a high resolution (0.5nm), but only thin sections can be examined.
Scans an electron beam ACROSS the specimen.
Collects reflected electrons in a cathode ray tube to form a TV image.
Shows the surface of a structure, has a greater depth of field and can provide 3-D images.
Has a lower resolution (5-20 nm) than the TEM.
Electrons have a shorter wavelength than light = greater resolution.
Light microscopy has a max resolution of around 200 nm, with the electron microscope it is 0.5 nm.
The maximum useful magnification is higher than with light microscopy
A vacuum is required - this means living specimens cannot be seen.
Preparation and staining can alter or damage cells.
It is expensive, expert training is required and they are not very common.
In general white blood cells defend against pathogens.
They are produced in the bone marrow and mature in the bone marrow and the thymus gland