Have you ever had a blood test?

How does the nurse (phlebotomist) take the blood?

Why do they tie a tourniquet around the arm?

Why is the area around the vein cleaned?

How do we see what’s in our blood?

MagnificationStaining

Magnification.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 magnification

Example 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 =60000Magnification = 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

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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

The 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 = 0.004mm3

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.

• So…• 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 0.004mm3

• We are counting five triple-lined squares so 0.004mm3 x 5 = 0.02mm3

• Our blood is yeast cells. This is diluted 100 times.

• If the number of cells counted is E:• In 1mm3 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.

Overall magnification = Objective lens x Eyepiece lens

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.

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…

1. Transmission electron microscope.

2. Scanning electron microscope.

TEM

• 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.

Transmission EM

SEM

• 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.

Scanning EM

Scanning EM

Advantages

• 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

Disadvantages

• 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.

Basic informationBasic information• 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

Granulocytes

Why are they called granulocytes?

• Neutrophils – phagocytize antibody coated pathogens

• Basophils – release histamine; may promote the development of T cells

• Eosinophils – kill antibody coated parasites.

They all have small granules in the cytoplasm

LymphocytesLymphocytes

Lymphocytes

• Large, darkly stained nucleus surrounded by a thin layer of cytoplasm.

• Two types, B and T

• B lymphocytes produce antibodies

• T lymphocytes have several functions, including cell destruction

MonocytesMonocytes

Monocytes

• The largest kind of leucocyte• They have a large, bean shaped nucleus

and clear cytoplasm.• They spend 2 to 3 days in the circulatory

system before moving into the tissues.• Once in the tissues they become

macrophages, engulfing microorganisms and other foreign material

What are these?What are these?

Giant sized

Normal sized

PLATELETS- initiate blood clotting

Cell structure – beyond light microscopy

We are examples of Protista!

We are .....

We are ...

Plasma membrane

Cytoplasm

Nucleus

EUKARYOTIC

Animal cell

Cell wallCell membrane

Vacuole

NucleusChloroplast

Starch grain

EUKARYOTIC

Plant cell

The Plasma Membrane

• The entry and exit of substances to cells are controlled by the cell surface membrane or plasma membrane, which surrounds the cytoplasm of a cell.

• The structure is described by the fluid mosaic model, this is basically the same for membranes around cell organelles.

Structure of the plasma membrane

• Understanding of the structure has changed and developed over the past 120 years (see time line).

• The current view held is based on the “fluid mosaic model” of Singer and Nicholson, proposed in 1972.

• “Fluid” indicates the mobility of the components. “Mosaic” reflects the pattern of organisation of the main components.

• All membranes have two fundamental components:

» Lipids (phospholipids and, in animal cells, cholesterol» Proteins

• The selective permeability of the cell membrane is related to the type and distribution of protein and phospholipid molecules present in the membrane.

• The lipids are amphipathic molecules (two sides to their nature – hydrophobic tails – the fatty acid chains and hydrophilic heads.

• The hydrophilic heads are towards the outside and the hydrophobic fatty acid tails form the core of the membrane – hence the bilayer.

• The lipid bilayer is fluid and mobile with the lipid molecules moving within their specific plane of the bilayer. Increasing temp = increasing mobility.

• Protein molecules are embedded in the phospholipid bilayer. Their position in the membrane is determined by their 3D structure

• Cholesterol decreases the permeability and increases the stability of the membrane

The Plasma Membrane

• The phospholipid bilayer allows lipid soluble molecules to pass through but restricts the passage of ions and polar molecules. These can pass through channel proteins (pores) that span the membrane.

• Other protein molecules act as carriers, these help transport ions and polar molecules (e.g. glucose) by facilitated diffusion and active transport.

• Other protein molecules act as specific receptors for hormones (e.g. insulin), which attach to them and so allow the cell to respond.

• Glycoproteins, composed of carbohydrate and protein, are on the outer surface of the membrane and are important in cell recognition, sometimes they act as antigens.

• The proteins are even more mobile. They can migrate through the bilayer so that they can aggregate together, then separate; some can rotate, others can move up/down/across from one plane to the other.

The role of the membranes

• At the surface of the cells:– Separates the cells from their environment

and other cells

• Inside the cells:– Separate the cell into compartments. Allows

complex processes occurring within the cell to be separated.

Cell membrane at approx 240 000 x magnification (electron

microscope)

Cell membrane at a lower magnification

Lighter area = extracellular space

How the membrane becomes more fluid at higher temperatures

Cholesterol

Cholesterol – its function.

• It either increases or decreases membrane fluidity. More cholesterol = more fluid.

Did you know – • organisms can modify their membrane

lipid composition changing membrane fluidity, this compensates for changes in temperature. E.g. housplants.

Extension

• Which organelles are involved in the production of proteins?

• What type of proteins might you find associated with the cell membrane?

• Glycoproteins are very important – why? What is their link to the golgi apparatus? (page 9 of the text book will help)