Microscope Slide Preparation

Preparing a microscope slide

 Specimens can be viewed under a light microscope; this allows some details of cellular
material to be observed

 Pre-prepared permanent slides can be viewed

o Such slides are produced by cutting very thin layers of tissue which are stained
and permanently mounted on a glass slide for repeated use

 Different methods will be used to view different types of specimen, e.g. temporary slide
preparations can be produced in the school laboratory as described below

Preparing a slide using a liquid specimen

1. Add a few drops containing the liquid sample to a clean slide using a pipette

2. Lower a coverslip over the specimen and gently press down to remove air bubbles

 Coverslips protect the microscope lens from liquids and help to prevent drying
out

Preparing a microscope slide using a solid specimen

1. Use scissors or a scalpel to cut a small sample of tissue, and peel away or cut a very thin
layer of cells from the tissue sample

 The preparation method always needs to ensure that samples are thin enough to
allow light to pass through

2. Place the sample onto a slide

 A drop of water may be added at this point

3. Apply iodine stain

4. Gently lower a coverslip over the specimen and press down to remove any air bubbles

Preparing a microscope slide using onion cells diagram

Tissue from an onion is as a solid specimen, and can be prepared here using iodine stain

Preparing a slide using human cells

1. Brush teeth thoroughly with normal toothbrush and toothpaste

  This removes bacteria from teeth so they don't obscure the view of the cheek
cells

2. Take a sterile cotton swab and gently scrape the inside cheek surface of the mouth for 5-
10 seconds

3. Smear the cotton swab on the centre of the microscope slide for 2-3 seconds

4. Add a drop of methylene blue solution

 Methylene blue stains negatively charged molecules in the cell, including DNA
and RNA

 This causes the nucleus and mitochondria to appear darker than their
surroundings

5. Place a coverslip on top

 Lay the coverslip down at one edge and then gently lower the other edge until it
is flat

 This reduces bubble formation under the coverslip

6. Absorb any excess solution by allowing a paper towel to touch one side of the coverslip

Preparing a microscope slide using cheek cells diagram

Cheek cells can be stained using methylene blue

Staining specimens

 The cytoplasm and other cell structures may be transparent or difficult to

distinguish; stains allow them to be viewed clearly under a light microscope

 As with the type of preparation required, the type of stain used is dependent on the
specimen being viewed

Common microscope stains & uses table

Stain Uses

Iodine Stains starch blue-black, and colours nuclei and plant cell walls pale yellow
Crystal violet Stains cell walls purple

Methylene
Stains animal cell nuclei dark blue
blue

Is not taken up by cells and stains the background red, so providing contrast with any cells
Congo red
present

Drawing Cells

 To record the observations seen under a microscope, a labelled biological drawing is

often made

 Biological drawings are line drawings which show specific features that have been
observed when the specimen was viewed

 There are a number of rules/conventions that are followed when making a biological
drawing

o The drawing must have a title

o The magnification under which the observations shown by the drawing are made
should be recorded if possible

 A scale bar may be used

o A sharp pencil should be used

o Drawings should be on plain white paper

o Lines should be clear, single lines without sketching

o No shading should be used

o The drawing should take up as much of the space on the page as possible

o Well-defined structures should be drawn

o Only visible structures should be drawn, and the drawing should look like the
specimen

o The drawing should be made with proper proportions

o Structures should be clearly labelled with label lines that:

 Do not cross

 Do not have arrowheads

  Connect directly to the part of the drawing being labelled

 Are on one side of the drawing

 Are drawn with a ruler

 Drawings of cells are typically made when visualizing cells at a higher magnification
power, whereas plan drawings are typically made of tissues viewed under lower
magnifications (individual cells are never drawn in a plan diagram)

Plant cell biological drawing

Bacterial cell biological drawings

Animal cell drawing

Magnification Calculations

 Magnification is the number of times that a real-life specimen has been enlarged to give
a larger view/image
 o E.g. a magnification of x100 means that a specimen has been enlarged 100 times
to give the image shown

 The magnification (M) of an object can be calculated if both the size of the image (I),
and the actual size of the specimen (A), is known

An equation triangle allows the magnification equation to be easily rearranged

 When carrying out calculations relating to magnification, the following steps should be
followed:

1. Rearrange the equation

o A=I÷M

o M=I÷A

o I=AxM

2. Read and measure the relevant values from the question stem and/or any images
provided

3. Convert any units

4. Substitute numbers into the rearranged equation

5. Consider whether this value makes sense in the context provided

Converting units during magnification calculations

 Cellular structures are usually measured in either micrometers (μm)

or nanometers (nm), while any measurements carried out in an exam with a ruler are
likely to be in millimeters (mm)
 o There are 1000 µm in a mm

o There are 1000 nm in a µm

 When doing calculations all measurements must be expressed using the same units

o It is best to use the smallest unit of measurement shown in the question

o Note that magnification does not have units

 To convert units, multiply or divide depending on whether the units are increasing or
decreasing

o Multiply when converting from a larger unit to a smaller unit

o Divide when converting a smaller unit to a larger unit

Units can be multiplied or divided by a factor of 1000 when converting between mm and µm

Worked Example

A starch grain inside a plant cell was viewed under a microscope at a magnification of x850. The
image of the starch grain captured using the microscope is shown below.
Calculate the actual diameter of the starch grain between points C-D in the image.

Give your answer in μm.

Step 1: Rearrange the equation

We have been asked to calculate actual size, A, so the new equation should be

Actual size = image size ÷ magnification

Step 2: Read and measure relevant values

We need to know the image size and the magnification

The image size has been given in the image as 20 mm

The magnification has been included in the question stem and is x850

Step 3: Convert any units

The actual diameter of a structure inside a cell would normally be measured in μm

The image size has been given in mm, so we need to convert this to μm

1 mm = 1000 μm, so we multiply mm by 1000 to give the value in μm

20 x 1000 = 20 000 μm

Step 4: Substitute number into the equation

Actual size = 20 000 ÷ 850

= 23.53 μm

Step 5: Consider whether the value makes sense

Plant cells measure between 10-100 μm, and the starch grain takes up a large proportion of the
cell in the image. A diameter of around 23 μm could therefore be right for this starch grain

If we had forgotten to convert the units and come up with a value of 0.024 μm, then this step
would show us that this is probably too small and that we must have therefore made a mistake
somewhere

Resolution & Magnification

Magnification

 Magnification is the number of times that a real-life specimen has been enlarged to give
a larger view/image

o E.g. a magnification of x100 means that a specimen has been enlarged 100 times
to give the image shown

 A light microscope has two types of lens which allow it to achieve different levels of
magnification:

o An eyepiece lens, which often has a magnification of x10

o A series of objective lenses, each with a different magnification, e.g. x4, x10, x40
and x100

 To calculate the total magnification of a specimen being viewed, the magnification of

the eyepiece lens and the objective lens are multiplied together:

total magnification = eyepiece lens magnification x objective lens magnification

Resolution

 The resolution of a microscope is its ability to distinguish two separate points on an

image as separate objects; this determines the ability of a microscope to show detail

o If resolution is too low then two separate objects will be observed as one point,
and an image will appear blurry, or an object will not be visible at all

o The resolution of a microscope limits the magnification that it can usefully

achieve; there is no point in increasing the magnification to a higher level if the
resolution is poor

 The resolution of a light microscope is limited by the wavelength of light

o Visible light falls within a set range of light wavelengths; 400-700 nm

 o The resolution of a light microscope cannot be smaller than half the
wavelength of visible light

 The shortest wavelength of visible light is 400 nm, so the maximum

resolution of a light microscope is 200 nm

o E.g. the structure of a phospholipid bilayer cannot be observed under a light

microscope due to low resolution:

 The width of the phospholipid bilayer is about 10 nm

 The maximum resolution of a light microscope is 200 nm, so any points

that are separated by a distance of less than 200 nm, such as the 10 nm
phospholipid bilayer, cannot be resolved by a light microscope and
therefore will not be distinguishable as separate objects

 Electron microscopes have a much higher resolution, and therefore magnification, than
light microscopes as electrons have a much smaller wavelength than visible light

o Electron microscopes can achieve a resolution of 0.5 nm

Resolution of light and electron microscopes diagram

The resolving power of electron microscopes is much greater than that of light microscopes
due to the smaller wavelength of electrons in comparison to visible light
Comparison of light and electron microscopes

 Light microscopes are used for specimens larger than 200 nm

o Light microscopes shine light through the specimen

o The specimens can be living, and therefore can be moving, or dead

o Light microscopes are useful for looking at whole cells, small plant and
animal organisms, and tissues within organs such as in leaves or skin

 Electron microscopes, both scanning and transmission, are used for specimens larger
than 0.5 nm

o Electron microscopes fire a beam of electrons at the specimen

 Transmission electron microscopes (TEM) fire electrons through a

specimen

 Scanning electron microscopes (SEM) bounce electrons off the surface of

a specimen

o The electrons are picked up by an electromagnetic lens which then shows the
image

o Electron microscopy requires the specimen to be dead, meaning that they can
only be used to capture a snapshot in time, and not active life processes as they
occur

o Electron microscopes are useful for looking at organelles, viruses and DNA as
well as looking at whole cells in more detail

Comparing light & electron microscopes table

Electron microscope Light microscope

Large machines that are permanently installed in laboratories Small and portable

Need to create a vacuum for electrons to travel through No vacuum required

Specimen preparation is complex Specimen preparation can be simple

Maximum magnification of x500 000 Maximum magnification of x2000

Maximum resolution of 0.5 nm Maximum resolution of 200 nm

Specimens are always dead Specimens can be living or dead

Calculating Actual Size

 When using microscopes to study biological specimens, it is possible to calculate

the actual size of organisms, cells, and parts of cells

 The actual size of specimens can be calculated using the magnification and
the measured size of an image as follows:

Actual size = image size ÷ magnification

o Magnification is sometimes provided in an exam question stem

o Magnification can be calculated from the scale bar of an image

o Sometimes magnification is calculated from information about the magnification

of the eyepiece lens and the objective lens

 Remember that the equation above is part of the equation triangle from calculating
magnification

Worked Example

Using a scale bar to calculate actual size

A lab technician observed bacterial cells with an electron microscope, and produced the image
below.

The scale bar measures 2 cm in length, and the length of the technician's image of one bacterial
cell measures 7.6 cm.
Use the information provided to calculate the actual length of a bacterial cell in the image.

Step 1: Use the scale bar to calculate the magnification of the image

The equation triangle for magnification tells us that:

Magnification = image size ÷ actual size

The scale bar measures 2 cm = 20 mm = 20 000 μm

The scale bar represents an actual size of 1 μm

Magnification = 20 000 ÷ 1
= 20 000

Step 2: Substitute values into the equation for actual size

Actual size = image size ÷ magnification

The question stem tells us that one cell = 7.6 cm = 76 mm = 76 000 μm

Magnification is ×20 000

Actual size = 76 000 ÷ 20 000

= 3.8

Therefore, the actual length of a bacterial cell in this image is 3.8 μm

Worked Example

Using lens magnification to calculate actual size

A scientist looked at a sample of red blood cells under a light microscope.

The eyepiece lens of the microscope had a magnification of ×10 and the objective lens had a
magnification of ×40.

The scientist produced a photomicrograph of the blood cells, shown below, in which the red
blood cells have an average diameter of 3 mm when measured using a ruler.

Calculate the average diameter of the red blood cells in the sample. Give your answer in
micrometres.

Step 1: Calculate the total magnification of the specimen

total magnification = eyepiece lens magnification × objective lens magnification

10 × 40 = 400

Magnification = ×400

Step 2: Convert the image size into μm

1 mm = 1000 μm

3 × 1000 = 3000

Image size = 3000 μm

Step 3: Substitute values into equation for actual size

Actual size = image size ÷ magnification

Actual size = 3000 ÷ 400

= 7.5

Therefore, the average size of a red blood cell in this sample is 7.5 μm

Eukaryotic Cell Structures & Functions

 Cells can be divided into two broad types; eukaryotic and prokaryotic cells

 Eukaryotic cells have a more complex ultrastructure than prokaryotic cells

o The term ultrastructure refers to the internal structure of cells

 The cytoplasm of eukaryotic cells is divided up into membrane-bound compartments

called organelles

Cell organelles

Cell surface membrane

 All cells are surrounded by a cell surface membrane which separates the inside of cells
from their surroundings

 Cell surface membranes controls the exchange of materials between the internal cell
environment and the external environment

o The membrane is described as being partially permeable as it allows the passage

of some substances and not others

 The cell membrane is formed from a phospholipid bilayer and spans a diameter of
around 10 nm
Cell surface membrane diagram

Cell surface membranes separate cell contents from the surrounding environment and control
the passage of substances into and out of cells

Nucleus

 Present in all eukaryotic cells, the nucleus is a large organelle that is separated from the
cytoplasm by a double membrane

 The nucleus contains the DNA, which is arranged into chromosomes

o Chromosomes contain DNA and proteins, which are collectively referred to as

chromatin

 The nuclear membrane is known as the nuclear envelope, and contains many pores

 Nuclear pores are important channels for allowing mRNA and ribosomes to travel out of
the nucleus, as well as allowing enzymes and signalling molecules to move in

 The nucleus contains a region known as the nucleolus, which is the site of ribosome
production

Nucleus diagram
The nucleus of a cell contains the genetic material

Rough & smooth endoplasmic reticulum

 The endoplasmic reticulum (ER) is made up of a series of membranes that form

flattened sacs within the cell cytoplasm

 The ER is linked with the nuclear envelope

 There are two distinct types of ER, with different roles within the cell

o The rough endoplasmic reticulum (RER)

 Continuous folds of membrane that are linked with the nuclear envelope

 The surface of the RER is covered in ribosomes

 The role of the RER is to process proteins that are produced on the
ribosomes

o The smooth endoplasmic reticulum (SER)

 The SER does not have ribosomes on the surface

 It is involved in the production of lipids, and of steroid hormones such as

oestrogen and testosterone
Rough and smooth endoplasmic reticulum diagram

The RER has ribosomes on its outer surface and is continuous with the nuclear envelope, while
the SER lacks ribosomes

Examiner Tips and Tricks

Be sure to always use the full name of the rough and smooth endoplasmic reticulum when you
first refer these structures in an exam; marks are often not awarded for the abbreviations RER
and SER in the absence of the full key terms.

Golgi body

 The Golgi body is often referred to as the Golgi apparatus or the Golgi complex

 It consists of a series of flattened sacs of membrane

 It can be clearly distinguished from the ER, as it is not connected to other membrane-
bound compartments, and it has a distinctive 'wifi symbol' appearance

 Its role is to modify proteins and package them into vesicles

Golgi body diagram

The Golgi body processes proteins and packages them into vesicles

Mitochondria

 Mitochondria (singular mitochondrion) are relatively large organelles surrounded by a

double-membrane

o They are smaller than the nucleus and chloroplasts, but can be seen with a light
microscope

 The inner membrane is folded to form cristae

 Mitochondria are the site of aerobic respiration within eukaryotic cells

 The mitochondrial matrix contains enzymes needed for aerobic respiration

 Small, circular pieces of DNA, known as mitochondrial DNA, and ribosomes are also
found in the matrix

o This allows the production of proteins required for respiration

Mitochondria diagram

Mitochondria have a highly folded inner membrane; this provides a large surface area for
embedded proteins that are involved with aerobic respiration

Ribosomes

 Ribosomes are found in the cytoplasm of all cells or as part of the rough endoplasmic
reticulum in eukaryotic cells

 Each ribosome is a complex of ribosomal RNA (rRNA) and proteins

 80S ribosomes (composed of 60S and 40S subunits) are found in eukaryotic cells

o Smaller, 70S ribosomes (composed of 50S and 30S subunits) are found in
prokaryotes, mitochondria and chloroplasts

 Ribosomes are the site of translation during protein synthesis

Ribosome diagram
Ribosomes are formed in the nucleolus and are composed of almost equal amounts of RNA
and protein

Vesicles

 Vesicles are small, membrane-bound sacs used by cells for transport and storage

 They can be pinched off the ends of the Golgi body; these are known as Golgi vesicles

 They can fuse with the cell surface membrane to allow exocytosis, or bud from the
membrane during endocytosis

Vesicle diagram

Vesicles carry out transport and storage of substances within cells

Lysosomes

 Lysosomes are specialised vesicles which contain hydrolytic enzymes

 Hydrolytic enzymes break down biological molecules, e.g.

o Waste materials, such as worn-out organelles

o Engulfed pathogens during phagocytosis

o Cell debris during apoptosis (programmed cell death)

Lysosome diagram

Lysosomes contain hydrolytic enzymes for the breakdown of biological molecules

Centrioles

 Centrioles are hollow fibres made of microtubules

 Two centrioles at right angles to each other form a centrosome, which organises
the spindle fibres during cell division

 Note that centrioles are not found in flowering plants and fungi

Centrioles diagram
Centrioles are involved with the movement of chromosomes during cell division

Microtubules

 Microtubules are hollow tubes made of tubulin protein

o α and β tubulin proteins combine to form dimers, which are then joined into
protofilaments

o Thirteen protofilaments in a cylinder make a microtubule

 Microtubules make up the cytoskeleton of the cell

o The cytoskeleton is used to provide support and movement of the cell

Microtubules and cytoskeleton diagram

Microtubules are tubes of protein that are involved with the structure of cell cytoskeletons

Cilia

 Cilia are hair-like projections made from microtubules

 They can be found of the surface of some cells where they Allow the movement of
substances over the cell surface

o E.g. ciliated epithelial cells in the airways waft mucus away from the lungs

Cilia diagram
Ciliated cells form ciliated epithelium in the airways

Microvilli

 Microvilli are cell membrane projections that increase the surface area for absorption

 Microvilli are found in parts of the body that carry out absorption, e.g.

o The lining of the small intestine

o The kidney tubules

Microvilli diagram
Microvilli increase the surface area of the cell surface membrane

Examiner Tips and Tricks

Be careful not to confuse microvilli with villi. Villi are much larger structures made up of several
layers of cells, while microvilli are found on the surfaces of individual cells. Microvilli will be
present on the outermost layer of cells that make up the villi!

Cell wall

 Cell walls are outside cell surface membranes and offer structural support to some types
of cell

o Structural support is provided by the polysaccharide cellulose in plants, and by

chitin in fungi

 Cell walls are freely permeable and do not play a role in controlling the movement of
substances into and out of cells

Cell wall diagram

Plant cell walls contain cellulose

Chloroplasts

 Chloroplasts are larger than mitochondria, and are also surrounded by a double-
membrane

 Membrane-bound compartments called thylakoids stack together to form structures

called grana

 Grana are joined together by lamellae

 Photosynthetic pigments such as chlorophyll are found in the membranes of the

thylakoids, where their role is to absorb light energy for photosynthesis

 Chloroplasts contain small circular pieces of DNA and ribosomes used to synthesise
proteins needed in chloroplast replication and photosynthesis

Chloroplast structure diagram

Chloroplasts are found in the green parts of a plant

Plasmodesmata

 Plasmodesmata are bridges of cytoplasm between neighbouring plant cells

 They allow the transfer of substances between plant cells

Plasmodesmata diagram
Plasmodesmata mean that the cytoplasm of neighbouring plant cells is continuous; this
allows substances to move easily between cells, e.g. sucrose can move easily from the
surrounding cells into the phloem

Large permanent vacuole

 Large permanent vacuoles are found in plant cells, where they store cell sap and
provide additional structural support to cells

o Vacuoles are sometimes found in animal cells, but these will be small and
temporary

 Vacuoles are surrounded by the tonoplast, which is a partially permeable membrane

Large permanent vacuoles store cell sap inside plant cells

Prokaryotic v Eukaryotic Cells (Cambridge (CIE) A Level Biology): Revision Note

Animal & Plant Cells (Cambridge (CIE) A Level Biology): Revision Note

Electron Micrographs: Animal Cells

 Exam questions will not always contain neat diagrams of cellular structures, but may
instead present images taken using microscopes

o Such images are known as micrographs

 It is possible to identify organelles in micrographs of animal cells on the basis of

their shape, location, and size relative to other organelles, e.g.

o The nucleus will always be the largest organelle

 o Mitochondria are the next largest, and are often cylindrical with a folded inner
membrane

 Note that mitochondria are not always cylindrical, but can also be
circular; their shape will depend on their age, and on the angle at which
they were sliced during specimen preparation

o RER will be near the nucleus, and ribosomes can sometime be seen

o Lysosomes and vesicles will be smaller than mitochondria

Exam questions may present micrograph images of cells

Structural Features of Typical Prokaryotic Cells

 Animal and plant cells are eukaryotic cells, whereas bacterial cells are prokaryotic

 Prokaryotes have a cellular structure that is distinct from eukaryotes:

o Their genetic material is free in the cytoplasm and is circular

 Eukaryotic genetic material is packaged as linear chromosomes in the

nucleus
 o Prokaryotes lack membrane-bound organelles

 This means that they do not have any internal structures surrounded by
membrane, e.g. a nucleus or mitochondria

o They are many times smaller than eukaryotic cells

 Prokaryotic cells are usually 1-5 μm in diameter, while eukaryotic plant

cells can be 10-100 μm across

o Their ribosomes are structurally smaller (70 S) in comparison to those found in

eukaryotic cells (80 S)

o Their cell walls are made of peptidoglycan rather than cellulose or chitin

 Prokaryotes are always unicellular, while eukaryotic animal and plant cells can function
together in multicellular organisms
Prokaryotic cells have no internal membrane-bound structures, and are smaller than
eukaryotic cells

Prokaryotic v Eukaryotic Cell Structures

Comparing prokaryote and eukaryote cell structure table

Feature Prokaryotes Eukaryotes

Size 0.5-5 μm Up to 100 μm

Genetic Circular chromosome in the Linear chromosomes in the nucleus

 cytoplasm
material Associated with histone proteins
Not associated with proteins

Binary fission Mitosis or meiosis

Cell division
No spindle fibres involved Chromosomes are separated by spindle fibres

Ribosomes 70S 80S

No membrane-bound Multiple membrane-bound organelles, e.g. a nucleus,

Organelles
organelles mitochondria, chloroplasts

Cell wall Made of peptidoglycan Made of cellulose in plants, or chitin in fungi

The Vital Role of ATP (Cambridge (CIE) A Level Biology): Revision Note

The Vital Role of ATP

 All organisms require a constant supply of energy to maintain their cells and stay alive

 This energy is required

o In anabolic reactions to build larger molecules from smaller molecules

o To move substances across the cell membrane in active transport, or to move

substances within the cell

o In animals, energy is required

 For muscle contraction

 In the conduction of nerve impulses

 In all known forms of life, ATP from respiration is used to transfer energy in all energy-
requiring processes in cells; this is why ATP is known as the universal energy currency

o Energy is released from ATP when it is broken down to ADP and inorganic
phosphate

o This process is reversed during respiration to make ATP and maintain a supply
of energy

 Adenosine Triphosphate (ATP) is a nucleotide

o The monomers of DNA and RNA are also nucleotides

ATP is the energy currency of cells

Viruses (Cambridge (CIE)

Key Features of Viruses

 Viruses are non-cellular particles that infect living cells

o Note that viruses are not cells, and they are not considered to be living
organisms, so are referred to as 'particles'

 They are much smaller than prokaryotic cells, with a diameter of 20-300 nm

 Structurally they have

o A nucleic acid core made of either DNA or RNA

o A protein coat called a capsid

 Some viruses have an outer layer called an envelope; this forms from the membrane
phospholipids of the host cell in which they were produced

 Viruses can only reproduce by infecting living cells and using their protein-building
machinery to produce new viral particles

o Viruses use attachment proteins on their surface to bind to and infect their host
cells

Virus structure diagram

Basic virus structure includes a protein capsid and a nucleic acid core