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Latvian Science Institute

Advanced Level Biology Notes
BY MR J D MOYO

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FORM 5 - TERM 1
TOPIC 1: CELL STRUCTURE AND FUNCTION.
………………………………………………………………
KEY CONCEPT.

…………… MICROSCOPY.

OBJECTIVES.

……………………………. Calibrate eye piece graticule.

……………………………. Draw and determine linear dimensions of specimens.
…………………………… Distinguish between magnification and resolution.
…………………………… Prepare temporary slides.
………………………………………………………………
“There are more animals living in the scum on the teeth in a man’s mouth than there are men in a whole
kingdom”.
—Antony van Leeuwenhoek.

MICROMETRY.

A micrometre is used as a unit of measurement in micrometry.

In biology very small objects are often observed and also measured.
When measuring cells or parts of cells, the most useful unit is the micrometre (µm).
1µm =1/1000mm.
Smaller structures such as the cell membrane thickness which have smaller sizes are
measured using smaller units known as Nanometres (nm).
1nm=1/1000µm.

NB* Note that centimetres are not units in Biology, nm, µm, & mm are used.

CALIBRATION OF THE EYEPIERCE GRATICULE

An eyepiece graticule is a scale bar that is placed in the eyepiece of a light microscope.
When you look down the microscope, you can see the graticule as well as the specimen.
The graticule is marked in graticule units, so you can use the graticule to measure the
specimen you are viewing in these graticule units.
The graticule units have to be converted to real units, such as mm or µm and this is called
calibration.
To calibrate, a unique slide called a stage micrometer that is marked in a real units scale can be
used.
The smallest markings on a stage micrometer are often 0.01 mm apart.
When viewing a specimen on the stage of a microscope that needs to be measured, take the
specimen off the stage or the microscope and replace it with the stage micrometer.
Focus on it by means of the same objective lens you used for viewing the specimen.
Line up the micrometer scale and the eyepiece graticule scale.
You can do this by turning the eyepiece, and by moving the micrometer on the stage.
Make sure that two large markings on each scale are exactly lined up with each other.

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EXAMPLE

If the 50 mark on the stage micrometer is lined up with the 1.0 mark on the eyepiece graticule.
You should work along towards the right until you see another two lines that are exactly lined
up.
For example, if there is a good second alignment of 68 on the stage micrometer and 9.0 on the
eyepiece graticule.
Hence it would be practical to say:

80 small eyepiece graticule markings = 18 stage micrometer markings

= 18 × 0.01 mm = 0.18 mm
= 180 µm
1 small eyepiece graticule marking = 180/80 = 2.25 µm.

Assuming that we wanted to measure the real width of the plant cell.
After measuring it with the already calibrated eye piece graticule, we discover that it is
23 eyepiece graticule units long.
So its real width would be: 23 × 2.25 = 51.75 µm.

If you want to look at something using a different objective lens, you will have to do the
calibration of eyepiece graticule units all over again using the new lens.

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MICROSCOPY

Microscopy is the study of microscopic structures with the observational aid of a microscope.
Microscopes currently in use are the light microscope and electron microscope.

RESOLUTION AND MAGNIFICATION.

Resolution is the ability of a microscope to distinguish two objects close together rather
than to see them as one object.
Magnification is the number of times an object is enlarged and is calculated as:

Magnification = Image observed/Actual size

E.g. A person makes a drawing of an ant and the width of the actual ant is 5mm while the ant
drawing is 12mm.
Calculate the magnification of the drawing?
Magnification = 12/5 =x2

CALCULATION OF MAGNIFICATION.

Assuming that the actual diameter of a red blood cell is 7µm and you are asked to calculate
the magnification of a red blood cell drawn in a diagram:

Step 1 You are supposed to measure the diameter of the cell in the diagram. Let’s assume
that you find that it is 30mm.
Step2 Considering that you have been given its real size which is 7 µm so we need to
convert 30mm to µm. There are1000µm in a mm, so 30mm =30x1000µm.
Step 3 We can now put the acquired figures in the magnification equation and calculate our
magnification.

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CALCULATING MAGNIFICATION FROM A SCALE BAR.

When given a scale bar, there is no need to measure the erythrocyte as in the previous
example.
We can simply use the scale bar usually given below or above the diagram.
All you need to do is to measure the length of the scale bar and then substitute it’s measured
length and the length that it represents on the scale bar.

Step 1 Measure the scale bar.

Step2 Substitute into the equation: Magnification = length of scale Bar/Length of scale bar it
represents

LIGHT MICROSCOPE.

Light microscopes use glass lenses to bend and focus light rays and produces enlarged
images of small objects.
The microscope consists of a metal body or stand composed of a base and an arm.
A light source, either a mirror or an electric bulb located in the base.
The fine and coarse adjustment knobs are located on the arm and can move either the stage
or the nosepiece to focus the image.
The stage is situated halfway up the arm and holds microscope slides by slide clips.
A mechanical stage permits the operator to move the slide smoothly during viewing by use
of stage control knobs.
The sub-stage condenser is mounted beneath the stage and focuses a cone of light onto the
slide.
On the top part of the microscope is the eyepiece containing the eyepiece lens.
The nosepiece holds three to five objectives with lenses of differing magnifying power and
can be rotated to position any objective beneath the body assembly.
The Objective lens has a variable magnification power while the Eye piece lens has a fixed
magnification power.
The total magnification of a light microscope is equal to the product of the objective lens
and the eyepiece lens.
For example, if a 45X objective is used with a 10X eyepiece, the overall magnification of the
specimen will be 450X.

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LIMITATIONS OF THE LIGHT MICROSCOPE.

The resolution of a light microscope increases with a decrease in the wavelength of the light
it uses for illumination.
Hence the resolution of the light microscope is limited by the minimum wavelength in the
visible light region.
An increase in the number of lens of the microscope would only increase magnification but
not increase resolution.
The very best light microscope has a resolution limit of about 0.2 µm but because bacteria
usually are around 1 µm in diameter, only their general shape can be observed.
The detailed internal structure of larger microorganisms also cannot be effectively studied
by light microscopy.

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THE ELECTRON MICROSCOPE.

Electron beams behave like radiation and can be focused just like light waves.
The microscope’s resolution is enormously increased because the wavelength of the
radiation is around 0.005 nm, approximately 100,000times shorter than that of visible light.
The transmission electron microscope has a practical resolution roughly 1,000 times better
than the light microscope.
In the electron microscope, points closer than 0.5 nm can be distinguished, allowing the
useful magnification to be over 100,000X.
The electron microscope is complex and sophisticated.
A heated tungsten filament in the electron gun generates a beam of electrons that is then
focused on the specimen by the doughnut shaped electromagnetic condenser.
The column containing the lenses and specimen must be under a vacuum to obtain a clear
image because electrons are deflected by collisions with air molecules.
The specimen scatters electrons passing through it, and the beam is focused by magnetic
lenses to form an enlarged, visible image of the specimen on a fluorescent screen.
A denser region in the specimen scatters more electrons while electron-transparent regions
are brighter.
The image can be captured on photographic film as a permanent record.

THE LIGHT MICROSCOPE VS THE ELECTRON MICROSCOPE.

Feature Light Microscope Electron Microscope

Highest practical Magnification. About 1,500X Over 100,000X
Best Resolution. 0.2 µm 5 nm
Radiation Source. Visible Light Electron Beam
Medium of travel. Air Vacuum
Type of Lens. Glass Electromagnet
Source of contrast. Differential Light Absorption Scattering of Electrons
Focusing Mechanism. Adjust lens position mechanically Adjust the current to magnetic lens
Method of changing Magnification. Switch the objective lens Adjust the current to magnetic lens
Specimen Mount. Glass slide Metal grid, usually copper
Observation colour Coloured Black and white
Nature of the specimen Can view living specimens Can only view dead specimens

LIMITATONS OF THE ELECTRON MICROSCOPE.

The observations made on the electron microscope are black and white hence the true
colour of the observations will not be known.
The specimen is killed during the staining process with heavy metals and hence the biotic
nature of the tissue cannot be observed.
The electron microscope is very expensive.
Specialists are needed to operate an electron microscope due to its complex nature.
The specimen preparation for viewing is very time consuming and complicated.
The microscope is large in size and hence can be very difficult to transport, e.g. in biological
studies carried out in remote areas like the deep Amazon forests.

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SPECIMEN PREPERATION.

1. PERMANENT PREPARATION

Fixation

This is the preservation of material in a life like condition.

Heat fixation or fixatives like ethanol and acetic acid can be used.
The original shape and structure are maintained.
The tissue hardens so that thin sections can be cut.

Dehydration

Refers to the removal of water.

It’s done to prepare the material for infiltration with stains.
Substances used for dehydration include acetone and ethanol.

Clearing

Alcohols do not mix with some of the common stains hence it is cleared with a clearing
agent.
An example of a substance used as a clearing agent is Xylol.

Sectioning

If a specimen is too thick to allow light to pass through, it is usually cut into very thin
transparent slices to allow light to pass through.
The razor instrument used to cut the slices is called a Microtome.
The slices to be observed should be 8-12µm thick.

Mounting

Involves mounting the specimen on a glass slide and staining with dyes to make some
structures visible.
This is usually done on glass slides.

2. TEMPORARY PREPARATIONS

Stages involved are fixation, staining and mounting.

70% alcohol can be used as fixative.

COMMON STAINS USED IN LIGHT MICROSCOPY.

Stain Use Colours produced

Methylene Blue Staining living cells Dark blue for the nucleus,
Stains light blue in the cytoplasm.
In bacteria the whole cell takes up the stain.
Iodine Staining living plant cells Stains very dark blue on starch grains.
Acidified phloroglucinol Staining lignin Stains Bright red on Lignin.
Acetin orcein Nuclei and chromosomes Stains Red
Light green Staining plant cell walls Stains Green

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……………………………………………………………………………………………………………………………………………………

KEY CONCEPT.

………………….. CELLS

OBJECTIVES.

……………………………. Eukaryotic and Prokaryotic cells.

……………………………. Identify plant and animal cells.
………………..………….. Compare plant and Animal cells.
……………………………. Outline the functions of Organelles.

……………………………………………………………………………………………………………………………………………………..
The era in which workers tended to look at bacteria as very small bags of enzymes has long passed.
—Howard J. Rogers.

PROKARYOTIC CELLS.

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Organisms that lack nuclei are called prokaryotes where ‘pro’ means before and ‘karyon’
means nucleus.
The nuclear material includes a single, circular and double stranded DNA molecule called a
chromosome usually concentrated in a specific region of the cytoplasm, called nucleoid.
A Prokaryote chromosome does not contain histone proteins.
Prokaryotic cells usually have sizes ranging between 1 to 10 µm.
Prokaryotes reproduce asexually by binary fission and endospore formation and sexually
by conjugation.
Infolding of the plasma membrane in some bacteria gives rise to Mesosomes to increase the
plasma membrane’s surface area to allow high rates of aerobic respiration, photosynthesis
and Nitrogen fixation.
Prokaryotes have a cell wall made of peptidoglycans containing a strengthening compound
known as Murein.
In some bacteria, the cell wall is enclosed by the capsule which serves as a protective layer
against attack by phagocytes and helps in regulating the concentration of ions and water
uptake.
Ribosomes of bacteria are 70S type.
Many prokaryotes have extra circular DNA molecules called plasmids which are important
in the production of bacteriocins, stimulation of bacterial conjugation and resistance to
antibiotics.
Some bacteria contain pilli which enable the bacteria to stick firmly to other bacteria and
surfaces and also help in conjugation.
Many bacteria are motile and use flagella for their cellular locomotion.
Reserve materials of bacteria are stored in the cytoplasm as storage granules.
Prokaryotic cells do not contain membranous organelles like mitochondria, golgi apparatus,
lysosomes, endoplasmic reticulum, nucleus and chloroplasts in plants and algae.
Prokaryotic cells do not contain centrioles.

EUKARYOTIC CELLS.

Organisms whose cells possess nuclei are called eukaryotes (‘eu’ means true).
Their DNA lies inside a nucleus.
Eukaryotes include animals, plants, fungi and a group containing most of the unicellular
eukaryotes known as protoctists.
The eukaryotic cells sizes mostly range between 10 to 100 µm.
Eukaryotic cells contain membranous organelles like Mitochondria, Golgi apparatus,
Lysosomes, Endoplasmic reticulum, Nucleus and Chloroplasts in plants and algae.
Most biologists believe that eukaryotes evolved from prokaryotes.
Eukaryotic cells can be divided into two types namely, Plant cells and Animal cells.

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ANIMAL CELLS

A typical animal cell is surrounded by a membrane known as the cell surface or plasma
membrane.
Inside the membrane is a jelly-like fluid known as the protoplasm which contains the
cytoplasm and suspended organelles.
The soluble part of the cytoplasm is known as the cytosol.

PLANT CELLS

Plant cells tend to be uniform in their shape because the cell is bound by a rigid cell wall.
The cells give strength and support to plants due to insoluble cellulose fibres.
Plant cells contain plastids called Chloroplasts which are pigmented to trap sunlight and
produce carbohydrates.

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Figure 1. A plant cell.

STRUCTURE AND FUNCTION OF ORGANELLES IN EUKAYORTIC CELLS.

Organelles allow specialized tasks to be performed namely:

1. Oxidative phosphorylation and generation of energy in the form of ATP molecules by
mitochondria.
2. Protein synthesis in rough endoplasmic reticulum.
3. Lipid and hormone synthesis in the smooth endoplasmic reticulum.
4. Secretion by Golgi apparatus.
5. Degradation of macromolecules in the lysosomes.
6. Regulation of all cellular activities by the nucleus.
7. Organization of spindle apparatus by centrosomes.
8. Trapping of sunlight energy and the production and storage of carbohydrates by the
chloroplasts.

NUCLEUS

Structure:

The nucleus is found in some Eukaryotic cells with a few exceptions e.g. the erythrocytes
and Sieve tube elements.
It is the largest organelle in eukaryotic cell.
It has a spherical structure with a diameter of about 5-7 µm.
The structure is enclosed by a double membrane called nuclear envelope which
compartmentalises chemical reactions taking place in the nucleus.
The Nuclear envelope is sometimes continuous with the endoplasmic reticulum at several
points.

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The protoplasm of the nucleus is the nucleoplasm which contains chromatin and the
nucleolus.
The nuclear envelope has nuclear pores that allow substance exchange between the
nucleoplasm and the cytoplasm.
The hereditary units known as genes are located on the chromosomes which exist as
chromatin during interphase.
Chromosomes contain DNA wound and coiled around proteins known as histones.
The chromatin has two forms namely Euchromatin and Heterochromatin.
Euchromatin is the loosely coiled form of chromatin which takes lighter DNA-stain and is
genetically active, as it is involved in gene transcription and phenotypic expression.
Heterochromatin is the tightly coiled form of chromatin which takes dark DNA-stain and is
genetically inert.
The nucleolus manufactures rRNA and tRNA while the chromosomes manufacture mRNA.

Functions:

The nucleus contains the units of inheritance known as genes.

The Nucleus controls the synthesis of proteins.
By controlling protein synthesis, it controls the cell’s activities.
The Nucleolus assembles ribosomes.

ENDOPLASMIC RETICULUM

Structure:

It is a system of flattened membrane bound sacs called cisternae, forming membranous

tubules and sheets around 40 to 70 nm in diameter.
It is usually continuous with the outer membrane of the nuclear envelope.
There are two different types of ER namely the Rough endoplasmic reticulum and the
smooth endoplasmic reticulum.
The rough ER is studded with ribosomes on its surface and is abundant in cells that either
secrete proteins e.g. the acinar cells of the pancreas and Goblet cells which produce mucus.
The Smooth ER lack ribosomes their surface and are mostly abundant in cells that process
steroids or lipid substances and glycogen e.g. adipose cells, Interstitial cells and
spermatocytes.

Functions:

1. Provides a large surface area for anabolic reactions.

2. Transports materials like proteins and lipids throughout the cell.
3. The Rough ER manufactures and modifies proteins e.g. through glycosylation.
4. The Smooth ER manufactures lipids and steroids.
5. The Smooth ER also synthesizes glycogen.
6. The Smooth ER is involved in the detoxification of drugs.
7. The ER’s network of membranes forms a structural skeleton for maintaining the cellular
shape.
8. The Smooth ER stores calcium ions and releases them when the muscle is stimulated
allowing muscle contraction.

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RIBOSOMES

Structure:

These are very small, dense, rounded and granular particles of ribonucleoprotein.
They are about 25 nm in diameter.
They are found in the mitochondrion, chloroplasts, attached to the membranes of the
endoplasmic reticulum or lying free in the cytoplasm.
They are organelles consisting of a large subunit and a small subunit.
They are made up of nearly equal amounts of protein and ribosomal RNA.
There are two types, the 70s found in prokaryotes and the 80s found in eukaryotes.

Function:

They are responsible for protein synthesis.

They form polysomes during translation were a collection of ribosomes are strung along
messenger RNA.

MITOCHONDRIA

Structure:

Are found in most aerobic eukaryotic cells.

The mitochondrial protoplasm is called the matrix and contains many enzymes and
coenzymes required for energy metabolism.
Mitochondria contain circular DNA coding for the production of mitochondrial proteins like
ATP synthase and cytochromes.
They contain 70S ribosomes for protein synthesis.
Mitochondria are 0.5–2.0 µm in diameter.
They have a double membrane with the inner membrane folded into cristae.
The inner membrane is rough with stalked protein particles like ATP synthase and
cytochromes.
Suspended in the matrix are the phosphate granules which act as the storage compartments
for the inorganic phosphate used in ATP synthesis.
The cristae are the site for oxidative phosphorylation and contain the electron transport
chain needed in aerobic respiration.

Function:

It is the major site for ATP production and this is achieved by substrate level
phosphorylation through the Krebs cycle in the matrix and also through chemiosmosis
along the cristae.

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GOLGI APPARATUS.

Structure:

The Golgi apparatus, like the endoplasmic reticulum, is a complex group of flattened
membrane bound tubules and vesicles. .
Unlike the endoplasmic reticulum it has parallelly arranged vesicles which lack ribosomes.
The simplest unit of the Golgi apparatus is the cisterna.
A group of cisternae is called the dictyosome, and a group of dictyosomes makes up the
cell’s Golgi apparatus.
The margins of each cisterna are gently curved so that the entire dictyosome of Golgi
apparatus takes on a bow-like appearance.
The cisternae at the convex end of the dictyosome comprise of a Cis-face and the cisternae
at the concave end of the dictyosome comprise the trans-face.
The Cis-face of the Golgi is located next to either the nucleus or a specific portion of rough
ER that lacks bound ribosomes and is called transitional ER.
The Trans-face of Golgi is located near the plasma membrane.

Functions:

1. Golgi enzymes modify the glycoproteins made in the ER by removing some sugars and
substituting others.
2. Recognition tags, such as phosphate groups can be added to help the Golgi sort molecules
into different batches.
3. Golgi vesicles are the “traffic police” of the cell as they direct metabolic products to their
destinations.
4. In plants it is involved in the secretion of materials of primary and secondary cell walls.
5. They are involved in the formation of the plasma membrane of daughter cells.
6. It is involved in the packaging and exocytosis of pancreatic enzymes, Mucus, collagen and
melanin granules.
7. It is also involved in the formation of lysosomes.
8. Forms the acrosome of spermatozoa and cortical granules of a variety of oocytes.

LYSOSOMES.

Structure:
Lysosomes are tiny membrane-bound vesicles.
The enzymes and membranes of Lysosomes are made by rough ER and processed in the
Golgi apparatus.
They contain a variety of hydrolytic enzymes that remain active under acidic conditions.
The lysosomal lumen is maintained at an acidic pH (around 5) by an ATP-driven proton
pump in the membrane.

Functions:

1. They digest large extracellular particles like foreign proteins, bacteria and viruses.
2. They Digest intracellular substances like stored food e.g. glycogen to supply the cell with
energy.
3. They are responsible for autolysis where they digest the organelles of the cells.
4. They secrete enzymes for extracellular digestion for example in the acrosome reaction
during fertilization.

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VACUOLES.

Structure:

They are temporary membrane bound pockets of cell sap in animal cells.
In plants, it occurs as a permanent feature containing cell sap.
It is surrounded by a membrane called the tonoplast.
The vacuole determines the osmotic properties of a plant cell.
They are structurally and functionally related to lysosomes in animal cells and may contain
a wide range of hydrolytic enzymes.
In addition, they usually contain sugars, salts, acids and nitrogenous compounds such as
alkaloids and anthocyanin pigments.

Functions:

1. It can act as a storage organelle for nutrients and waste products.

2. It can be used as a lysosomal compartment.
3. It is an economical way of increasing the size of plant cells.
4. It acts as a controller of turgor pressure which provides support to young plants.
5. It has homeostatic functions in plant cells as the increased transport of H+ into the vacuole
in acidic environment provides a plant cell with a buffering ability in low pH environments.
6. The vacuoles in the cells of angiosperm petals contain pigments which attract insects during
pollination.
7. Contractile vacuoles in Protozoa allow the cells to control their water content to prevent
bursting in dilute aqueous environments as they do not possess a cell wall.
8. Vacuoles may also contain poisons that protect the plant against herbivores

CENTRIOLES.

Structure:

They are found as a pair inside centrosomes near the nucleus during interphase.
These organelles are present in animal cells only.
They are cylinders of microtubule triplets arranged in a 9 + 0 pattern.

Function:

They pull apart during cell division to produce a spindle made of microtubules which are
involved in chromosome movement.

CILIA AND FLAGELLA.

Structure:

They are outgrowths from cells.

Cilia can beat either in one direction while flagella beat in a wavelike manner.
Flagella are larger than cilia.
Both cilia and flagella have a characteristic 9+2 arrangement of microtubules.

Function:

They are involved in cellular locomotion.

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CHLOROPLAST.

Structure:

Chloroplasts are the photosynthesizing organelles of all photosynthetic eukaryotes.

Chloroplasts are mostly biconcave oval shaped organelles.
They are relatively larger than Mitochondria and measure an average of 7 µm.
Chloroplasts are bound by an envelope made of two membranes which compartmentalise
chloroplast metabolism and allow selective exchange of molecules between chloroplasts
and the cytosol.
The protoplasm of the chloroplasts is known as the stroma which contains enzymes
necessary for the dark reactions of photosynthesis.
Suspended in the stroma are the 70S ribosomes which produce chloroplast proteins.
The chloroplast proteins are coded for by the chloroplast’s circular DNA.
The thylakoids form a membranous network within the stroma and are pigmented with
chlorophyll and accessory pigments for the trapping of sunlight making them the site for the
light dependant reactions of photosynthesis.
The thylakoids fold up at intervals forming the grana to increase the surface area for light
absorption.
Areas of the chloroplast that are not folded up into grana are known as lamellae.

Function:

Traps sunlight energy and uses it to manufacture important Biological molecules like
carbohydrates.

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✓ Qn Compare and contrast the structure of a typical animal and plant cell. [6]

Structure Plant cell Animal cell

Cell wall Cellulose No cell wall
Cell membrane Phospholipid bilayer Phospholipid bilayer
Cytoplasm Fluid with dissolved Fluid with dissolved
substances substances
Vacuole Large central vacuole If present, small and temporary
Shape Regular Irregular
Chloroplast Present Absent
Mitochondrial Present Present
Centrioles Absent Present
Plasmodesmata Present Absent

Qn Compare and contrast the structure of a typical prokaryote and eukaryote cell. [6]

STRUCTURE PROKARYOTE EUKARYOTE

Cell division Binary fission. Mitosis and Meiosis.
No Spindle fibres formed Spindle fibres are formed.
Genetic material DNA is circular and not DNA is linear, often associated
associated with histones with histones
No true nucleus. It is contained in the nucleus.

Protein synthesis 70s ribosomes. 80s ribosomes.

No ER present. ER present and ribosomes may
be attached to ER.
Organelles Few organelles. Many organelles.
None are envelope bound. Some are envelope bound.
Cell walls Cell wall made of Cell wall if present may be made
Peptidoglycans with Murein as a of the polysaccharides Chitin
strengthening compound. and Cellulose.
Respiration Aerobic respiration occurs on Aerobic respiration occurs in
the cell surface membrane and the mitochondria.
Mesosomes in some cells.
Photosynthesis May occur in Mesosomes. Occurs in chloroplasts
Nitrogen fixation May occur in Mesosomes. None have the ability
Size 1-10 µm 10-100 µm

CELL FRACTIONATION

To visualise individual organelles under an electron microscope they need to be separated

from the rest of the cell components
There are three steps to doing this:

1. Homogenisation (Breaking Up the Cells)

▪ Homogenisation can be done in several different ways, e.g. by vibrating the cells or
by breaking down the cells up in a blender.
▪ This breaks up the plasma membrane and releases the organelles into solution.

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▪ The solution must be kept ice-cold, to reduce the activity of enzymes that break
down organelles.
▪ The solution should also be isotonic to prevent damage to the organelles through
osmosis.
▪ A buffer solution should be added to maintain the pH.

2. Filtration

▪ The homogenised cell solution is filtered to remove large cell or tissue debris from
the organelles.
▪ The organelles are much smaller than the debris, so they pass through the gauze.

3. Ultracentrifugation (Separating the Organelles)

▪ The cell fragments are poured into a tube.

▪ The tube in put into a centrifuge (a machine that separates material by spinning)
and is spun at a low speed.
▪ The heaviest organelles, like nuclei, sediment at the bottom of the tube forming a
pellet.
▪ The rest of the organelles stay suspended in the fluid above the sediment — the
supernatant.
▪ The supernatant is drained off, poured into another tube, and spun in the
centrifuge at a higher speed.
▪ Again, the heaviest organelles, this time the mitochondria, form a pellet at the
bottom of the tube.
▪ The supernatant containing the rest of the organelles is drained off and spun in the
centrifuge at an even higher speed.
▪ This process is repeated at higher and higher speeds, until all the organelles are
separated out.
▪ Each time, the pellet at the bottom of the tube is made up of lighter and lighter
organelles.

The organelles are separated order of mass (from heaviest to lightest).

This order is usually:

1. Nuclei
2. Chloroplasts
3. Mitochondria
4. Lysosomes
5. Endoplasmic reticulum
6. Ribosomes

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………………………………………………………………
KEY CONCEPT.

……………………….. MOVEMENT OF SUBSTANCES INTO AND OUT OF CELLS.

OBJECTIVES.

……………………………. Describe and explain the cell surface membrane structure.

…………………………… Relate the structure of the membrane to the movement of substances into and out of cells.
………………………………………………………………
MEMBRANES

Structure:

A plasma membrane encloses every type of cell.

It physically separates the cell cytoplasm from its surrounding cellular environment.
It is an ultrathin, elastic, living, dynamic and selective barrier.
It is made up of lipids (phospholipids and cholesterol), proteins and carbohydrates.
It has a thickness of about 7 nm.
All biological membranes in eukaryotic cells are similar in structure and have selective
permeability but differ in other functions.
Biological membranes are well illustrated by the fluid mosaic model.
It is described as ‘fluid’ because both the phospholipids and the proteins can move about by lateral
diffusion mainly in their own layers.
The word ‘mosaic’ describes the pattern produced by the scattered protein molecules on the
surface of the membrane.

Phospholipids

Each phosphor-lipid molecule consists of a polar head containing a phosphate and two fatty acids.
The polar head is hydrophilic and the tails are hydrophobic making the molecules amphipathic.
In aqueous environments, the hydrophilic heads face the external environments and the
hydrophobic fatty acid tails come into close intact to exclude water.
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Phospholipids form the bilayer, which is the basic structure of the membrane and inhibits the
transport of polar molecules.
Some phospholipids can be modified chemically to act as signalling molecules which move about
the phosphor-lipid bilayer, activating other molecules such as enzymes.
Phospholipids may also be hydrolysed to release glycerol-related molecules which diffuse through
the cytoplasm and bind to specific receptors.

Proteins

Proteins that are found entrenched inside the membranes, are called intrinsic proteins (or integral
proteins) and sometimes cross the whole membrane, in which case they are known as trans-
membrane proteins.
Intrinsic proteins have hydrophobic and hydrophilic.
Hydrophobic regions stay in the membrane because they are made of hydrophobic amino acids so
they are attracted to the hydrophobic fatty acid tails and are repelled by the aqueous environment
on either side of the membrane.
The hydrophilic regions, made from hydrophilic amino acids, are repelled by the hydrophobic
interior of the membrane and therefore face the aqueous environment inside or outside the cell, or
line hydrophilic pore interiors which pass through the membrane.
All the proteins referred to as transport proteins are intrinsic proteins.
These provide hydrophilic channels or passageways for ions and polar molecules to pass through
the membrane.
There are two types of transport protein: channel proteins and carrier proteins.
A second type of protein molecule is the extrinsic protein (or peripheral protein).
These are found on the inner or outer surface of the membrane and are very easy to extract from
the membrane.
Many are bound to intrinsic proteins.
Some are held in other ways – for example, by binding to molecules inside or outside the cell, or to
the phospholipids.
Other membrane proteins may be enzymes for example, the digestive enzymes found in the cell
surface membranes of the cells lining the small intestine and catalyse the hydrolysis of molecules
such as disaccharides.
Some proteins on the inside of the cell surface membrane are attached to a system of protein
filaments inside the cell, known as the cytoskeleton.
Proteins also play important roles in the membranes of organelles. For example, in the membranes
of mitochondria and chloroplasts they are involved in the processes of respiration and
photosynthesis.

Cholesterol

Cholesterol is a relatively small molecule and like phospholipids, cholesterol molecules have
hydrophilic heads and hydrophobic tails, so they fit neatly between the phospholipid molecules
with their heads at the membrane surface.
Cholesterol is much less common in plant cell membranes and absent from prokaryotes.
At low temperatures, cholesterol increases the fluidity of the membrane.
This is because it prevents close packing of the phospholipid tails.
At high temperatures, cholesterol decreases the fluidity of the membrane.
Cholesterol is also important for the mechanical stability of membranes, as without it, membranes
quickly break and cells burst open.
The hydrophobic regions of cholesterol molecules help to prevent ions or polar molecules from
passing through the membrane.
This is important in the myelin sheath, where leakage of ions would slow down nerve impulses.

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Glycolipids and Glycoproteins.

Carbohydrate oligosaccharide chains are attached to some proteins and lipids on the outer side of
the membrane forming glycoproteins and glycolipids respectively.
The oligosaccharide chains project like antennae forming hydrogen bonds with the water molecules
around the cell stabilising the membrane structure.
The oligosaccharide chains form the glycocalyx.
In animal cells, the glycocalyx is formed mainly from glycoproteins; in plant cells it mainly
comprises glycolipids.
Glycoproteins act as receptor molecules for substances such as hormones and are also associated
with cell recognition e.g. in the formation of antigens like the A,B,O blood groups.
There are three major groups of receptors.
Glycolipids act as cell recognition sites and allow cell to cell adhesion to form tissues.

Functions:

The plasma membrane controls the entry of nutrients and exit of waste products, and generates
differences in ion concentration between the interior and exterior of the cell.
It also acts as a sensor of external signals (for example, hormonal, immunological, etc.) and allows
the cell to react or change in response to environmental signals.

CELL SIGNALLING

Cells can communicate with one another using molecules that interact with cell membranes.
Cell surface membranes contain receptor molecules, into which signalling molecules can fit.
Like enzymes, these receptors are specific, only accepting one type of signalling molecule.
This means that the signalling molecule can only affect cells that have its receptor in their cell
surface membranes and these make up its target cells.
For example, the hormone insulin is a protein that fits into receptors in the cell surface membrane
of liver cells.
When insulin is bound to the receptor, this brings about changes in the cell that result in an
increase of transporter proteins for glucose in the cell surface membrane, causing the cell to take up
glucose.

MOVEMENT OF SUBSTANCES INTO AND OUT OF CELLS

There are five basic cellular transport mechanisms:

1. Diffusion.
2. Facilitated Diffusion.
3. Osmosis.
4. Active Transport.
5. Bulk Transport.

DIFFUSION.

Diffusion is the movement of substances resulting from their random motion down their
concentration gradient.
Due to diffusion, molecules or ions reach an equilibrium situation, where they are uniformly spread
within a specific volume.
The rate at which a substance diffuse across a membrane depends on a number of factors, including
the following:

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1. The concentration gradient steepness:

The differences in the diffusing substance’s concentration on the two sides of the membrane.
2. Temperature:
At high temperatures, molecules and ions have much more kinetic energy than at low
temperatures.
3. The surface area across the membrane:
If the surface area is large, more molecules or ions can cross it at any one moment increasing
the rate of diffusion. Larger cells have a lower surface area to volume ratio.
4. The nature of the substance:
Larger molecules require more energy to move than smaller ones, hence large molecules diffuse
slower than small molecules.
Non-polar molecules diffuse easily through cell membranes than polar ones because as they are
soluble in the non-polar phospholipid tails.
Water molecules although polar, can diffuse across the phospholipid bilayer because they are
small.

Cells rely on diffusion for internal transport of molecules hence cells need to have small sizes to
allow diffusion to occur at a faster rate.

FACILITATED DIFFUSION.

Facilitated diffusion is the movement of substances down their concentration gradient through
transport proteins in a cell membrane.
Large polar molecules, such as glucose and amino acids and polar molecules such as Na+ cannot
diffuse through the phospholipid bilayer.
These substances can only cross the membrane with the help of transport proteins namely channel
proteins and carrier proteins.
Each transport protein is very specific allowing only one type of substance to pass through it.
Channel proteins are water-filled pores which allow charged substances, usually ions, to diffuse
through the membrane and most channel proteins are gated meaning they can close and open.
Channel proteins have a fixed shape.
Carrier proteins can flip between two shapes.
The rate of facilitated diffusion is affected by how many channel or carrier protein molecules there
are in the membrane, and, in the case of channel proteins, on whether they are open or not.

OSMOSIS.

Osmosis is the movement of water molecules due to their random motion, down their water
potential gradient through a partially permeable membrane.
The concentration of water molecules is described in terms of water potential.
The letter psi, ψ, can be used to symbolise water potential.
Water potential is the tendency of water to move out of a solution.
Water potential depends on two factors namely:
The proportion of water a solution contains in relation to its solutes.
The pressure applied to the solution.
Osmosis occurs until the water potential is the same throughout the system in a state of
equilibrium.
The water potential of pure water at atmospheric pressure is 0 and, in a solution, it has a negative
value.
Solute potential is the extent to which the solute molecules decrease the water potential of the
solution.
Solute potential is 0 for pure water, and has a negative value for a solution.

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The more solute there is, the lower the tendency for water to move out of the solution.
Hence the more negative the solute potential, the lower the water potential.
Solute potential is 0 for pure water, and has a negative value for a solution.
The psi symbol can be used to show the solute potential as ψs.
The contribution of pressure to the water potential of a solution is called pressure potential.
Pressure potential can be shown using the symbol ψp.
For plant cells, water potential is a combination of solute potential and pressure potential.
ψ = ψs + ψp

EFFECTS OF OSMOSIS ON PLANT CELLS

An animal cell placed in pure water takes up water by osmosis.

The volume of the cell increases, and the cell may burst.
Animals and protoctists that live in fresh water have some way of removing excess water from their
cells, so that the cells do not swell and burst through the use of contractile vacuoles.
An animal cell in a concentrated solution loses water by osmosis, and will shrink.
A plant cell in pure water also takes up water by osmosis but it does not burst as it has a cellulose
cell wall outside its cell surface membrane, which prevents the cell from bursting.
Instead, the cell simply becomes swollen and is said to be turgid.
Turgidity helps plant tissues to remain in shape.
A plant cell in a concentrated solution loses water by osmosis, so that the volume of the cytoplasm
and vacuole decrease.
Leaves in which the cells lose their turgidity are no longer supported, and they wilt.
If a great deal of water is lost from a plant cell, then the cell contents shrink so much that the cell
surface membrane pulls away from the cell wall.
This is called plasmolysis.
The membrane often remains attached at points where plasmodesmata link to the next cell.

ACTIVE TRANSPORT.

Active transport is the movement of substances against their concentration gradient with the use of
energy in the form of ATP.
It is achieved through the use of carrier proteins, each of which is specific for a particular type of
molecule or ion.
Unlike facilitated diffusion, active transport requires energy, because movement occurs up a
concentration gradient.
The energy is used to make the carrier protein change shape, transferring the molecules or ions
across the membrane in the process.
An example of a carrier protein used for active transport is the sodium–potassium (Na+/ K+) pump.
Active transport is important in re-absorption in the kidneys, where certain useful molecules and
ions have to be reabsorbed into the blood after filtration into the kidney tubules.
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It is also involved in the absorption of some products of digestion from the gut.
In plants, active transport is used to load sugar from the photosynthesising cells of leaves into the
phloem tissue for transport around the plant, and to load inorganic ions from the soil into root
hairs.

BULK TRANSPORT.

Large molecules such as proteins or polysaccharides, parts of cells or even whole cells may be
transported across the membrane through energy requiring processes.
Endocytosis involves the engulfing of the materials by the cell surface membrane to form an
endocytic vacuole.
It takes two forms:

1. Phagocytosis (cell eating)

This is the bulk uptake of solid material and cells specialising in this are called phagocytes and
the vacuoles formed are called phagocytic vacuoles. An example is the engulfing of bacteria by
the white blood cells.

2. Pinocytosis (cell drinking)

This is the bulk uptake of liquid.

Exocytosis is the reverse of endocytosis and is the process by which materials are expelled from
cells.
For example, the secretion of digestive enzymes from cells of the pancreas.

“I will persist until I succeed. I was not delivered into this world in defeat, nor does failure course in my veins. I
am not a sheep waiting to be prodded by my shepherd. I am a lion and I refuse to talk, to walk, to sleep with
the sheep. The slaughterhouse of failure is not my destiny. I will persist until I succeed”.

O.G. Mandino

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