Cell structure

Making a temporary preparation and drawing what you see

A temporary mount is a preparation made on a slide in a few minutes, viewed while the cells are still alive or freshly killed, and thrown away afterwards. To see plant cells, peel a small square of the inner epidermis from a fleshy scale leaf of an onion, Allium cepa, lay it flat in a drop of water and add a drop of iodine in potassium iodide solution, which stains starch grains and makes the nucleus and the cell wall stand out. To see animal cells, scrape the inside of your cheek gently with a clean blunt seeker, smear the cells onto a slide and stain them with methylene blue, which stains DNA so the nucleus appears as a dark blue oval.

Lower the coverslip at an angle of about 45 degrees, supporting it with a mounted needle, so that air and surplus stain are pushed out in front of it. Air bubbles look like thick black rings with a pale centre; if you have trapped them, the preparation has to be remade, because an air bubble is not a structure. A specimen that is too thick is worse than useless, because two or three layers of cells overlap and no detail can be seen at all, and too much stain washes out the contrast instead of improving it.

Start on the lowest power objective, focus with the coarse adjustment, and then change to higher powers and focus only with the fine adjustment, because the coarse focus would drive the objective into the slide. Adjust the iris diaphragm and the lamp so that the image is bright and evenly lit rather than glaring, and keep both eyes open while you work so that you are not straining. Never draw from memory or from a textbook picture: your drawing is evidence of what is in the field of view.

The drawing rules are marked strictly. Use a sharp HB pencil, draw with single unbroken lines and never shade or sketch, and draw the cells large enough to show detail, usually filling at least half the field of view. Rule the label lines with a ruler so that they are straight and do not cross each other, do not put arrowheads on them, and label only the structures you can actually see. Add a title and the magnification, but do not write the function of each structure on the drawing. When you interpret a photomicrograph, work out what a structure is from its shape, its position in the cell and how it has stained, and use the scale bar or the stated magnification to judge its size rather than judging it from the size of the print.

• Air bubble: a thick black ring with a pale centre, which is an artefact and never a cell structure.
• High power: fine adjustment only, or the objective crashes into the slide and cracks it.
• Draw only what is in the field of view, with unbroken pencil lines, straight ruled label lines and a stated magnification.
• Interpret a photomicrograph from shape, position and staining, and read size from the scale bar, not from the size of the printed image.

Magnification and resolution: two ideas that are not the same

Magnification is how many times larger the image is than the object it shows. It has no unit and it is written with a multiplication sign, as in a magnification of x400. You calculate it with magnification = image size / actual size, using the same unit on the top and the bottom. Making an image bigger does not, on its own, reveal any more detail, and that is the point candidates lose marks over every year.

Resolution, or resolving power, is the smallest distance between two points at which they can still be seen as two separate points; closer together than that and the two merge into a single blurred dot. Resolution is fixed by the wavelength of the radiation used to form the image, because a wave cannot be used to detect a detail much smaller than its own wavelength. Light has a wavelength of roughly 400 nm to 700 nm, so a light microscope resolves to about 0.2 µm. That is why you can see the nucleus, the chloroplasts and the cell wall with a light microscope but never a ribosome, which is about 25 nm across, or the two membranes of the nuclear envelope.

An electron microscope forms its image with a beam of electrons, and the wavelength of an electron beam is far shorter than that of light, so the resolution is thousands of times better. A transmission electron microscope (TEM) resolves to about 0.5 nm and magnifies up to about x500000, and it reveals the internal ultrastructure of a cell: the cristae inside a mitochondrion, the cisternae of the rough endoplasmic reticulum, the thylakoid membranes stacked inside a chloroplast. A scanning electron microscope (SEM) scans a fine beam of electrons across the surface of a whole specimen that has been coated with a thin film of metal, and collects the electrons scattered back from it, so the image is a three-dimensional view of the outside. The SEM shows the sculptured surface of a pollen grain or the brush border of the small intestine beautifully, but it cannot show you what is inside a cell, and its resolution of a few nanometres is poorer than that of a TEM.

Both types of electron microscope share one serious limitation: the specimen must be dead, dehydrated and held in a vacuum, so electron microscopy can never show a living process happening. A light microscope works on a living, wet preparation, which is why it is still the first choice for watching cell division in a root tip or the beating of cilia.

Measuring cells: the graticule, the stage micrometer and the equations

An eyepiece graticule is a glass disc printed with a scale of 100 divisions that drops into the eyepiece, so that the scale lies across the image of the specimen. Its divisions are called eyepiece units (e.u.) and they are arbitrary: how much of the specimen one eyepiece unit covers depends on which objective lens is in place, so the graticule has to be calibrated separately for every objective you use. Forgetting to recalibrate is the single commonest reason for a measurement being wrong by a factor of four.

Calibration is done with a stage micrometer, a slide carrying an accurate scale, usually 1 mm divided into 100 divisions, so each division is 0.01 mm, which is 10 µm. Put the stage micrometer on the stage, focus on its scale, rotate the eyepiece until the two scales are parallel, and slide the graticule so that the zero marks coincide. Worked example: at the x10 objective, 40 eyepiece units line up with 0.4 mm on the stage micrometer. 0.4 mm is 400 µm, so one eyepiece unit is 400 / 40 = 10 µm at this magnification.

Change to the x40 objective and the specimen is magnified four times as much, so each eyepiece unit now covers only a quarter of the distance it covered before: 10 / 4 = 2.5 µm. Measure a cell that spans 12 eyepiece units at x40 and its actual length is 12 x 2.5 = 30 µm. Always record which objective a measurement was made with, and always state the unit, because 30 on its own is not an answer.

Two equations cover every calculation in this topic: magnification = image size / actual size, and actual size = image size / magnification. Put both sizes into the same unit before you divide, then convert the answer into whichever unit reads sensibly, a whole cell in µm and an organelle in nm. Worked example: an electron micrograph printed at x15000 shows a mitochondrion 60 mm long, so its actual length is 60 / 15000 = 0.004 mm, which is 4 µm.

The nucleus and the endomembrane system

The nucleus is the largest organelle in most cells and it holds the DNA that controls the cell. The nuclear envelope is a double membrane, two phospholipid bilayers with a narrow space between them, and it is perforated by nuclear pores about 100 nm across. The pores are not simple holes: they are lined by proteins that control what passes, so mRNA and the subunits of ribosomes leave the nucleus and the enzymes needed for DNA replication enter it. Inside the nucleus the DNA is wound around proteins as chromatin, and the nucleolus is the dense region where ribosomal RNA is made and assembled with protein into ribosomal subunits.

The rough endoplasmic reticulum (RER) is a system of flattened, membrane-bound sacs called cisternae, continuous with the outer membrane of the nuclear envelope and studded on its outer surface with ribosomes. Proteins made on those ribosomes are threaded into the cisternae, where they fold and receive their first sugar groups, so the RER is abundant in any cell that exports protein, such as the pancreatic beta cells that secrete insulin or the plasma cells that secrete antibodies. The smooth endoplasmic reticulum (SER) carries no ribosomes: it makes lipids and steroids, and in liver cells its enzymes detoxify drugs and alcohol, which is why the SER takes up a large fraction of the volume of a liver cell.

The Golgi body is a stack of curved, flattened cisternae with vesicles budding from the edges. It receives proteins from the rough endoplasmic reticulum in transport vesicles, modifies them, most commonly by adding short sugar chains to make glycoproteins, and then sorts them into vesicles addressed to different destinations: secretion out of the cell, the cell surface membrane, or a lysosome. The reticulum, the Golgi body and the vesicles are together called the endomembrane system because they are linked by this constant traffic of vesicles.

A lysosome is a single-membrane vesicle containing hydrolytic enzymes such as proteases, lipases and nucleases, held at an acidic pH that suits their activity. The membrane keeps those enzymes away from the cytoplasm, so digestion happens safely inside the vesicle. A neutrophil takes bacteria in by phagocytosis and fuses the vesicle with lysosomes to destroy them, and every cell uses lysosomes to break down worn-out organelles and recycle the molecules for reuse.

Mitochondria, chloroplasts, ribosomes and ATP

A mitochondrion has two membranes: a smooth outer membrane and an inner membrane folded into cristae, which enclose the matrix. Aerobic respiration is divided between those compartments, with the link reaction and the Krebs cycle in the matrix and the electron transport chain and oxidative phosphorylation on the cristae, where the carrier proteins and enzymes that make most of the ATP are embedded. Folding the inner membrane into cristae gives a large surface area for those reactions, so a muscle fibre or a liver cell with a high demand for ATP has mitochondria with many cristae, whereas a slowly metabolising cell has fewer.

Mitochondria contain small circular DNA and 70S ribosomes, and they divide by splitting in two, so a cell never has to build one from scratch. In the cytoplasm of a eukaryotic cell the ribosomes are 80S, larger and richer in RNA and protein, while the ribosomes inside mitochondria and chloroplasts are 70S, like bacterial ribosomes. That difference has a medical consequence: the antibiotic chloramphenicol binds to 70S ribosomes and blocks protein synthesis in bacteria, and because our mitochondria use 70S ribosomes it can also interfere with protein synthesis in the mitochondria of the bone marrow, which is why it is only used for serious infections and carries a risk of damaging blood cell production.

A chloroplast is also a double-membrane organelle, but it contains a third membrane system, the thylakoid membranes stacked into grana, suspended in a fluid called the stroma. The light-dependent reactions of photosynthesis take place on the thylakoid membranes and the light-independent reactions take place in the stroma, and the stroma also holds starch grains and the enzymes of carbon fixation. Like mitochondria, chloroplasts contain small circular DNA and 70S ribosomes and can divide, which is why a cell that grows more chloroplasts does not need to make the DNA of every one of them from the nucleus.

Cells use ATP from respiration to drive all their energy-requiring processes: active transport by carrier proteins, the synthesis of proteins, DNA and other polymers, muscle contraction, the beating of cilia and the pulling of spindle fibres in cell division. ATP is the universal energy currency because hydrolysing its terminal phosphate group releases a small, usable quantity of energy exactly where it is needed. Every peptide bond a ribosome makes costs ATP, which is why a protein-secreting cell is full of both ribosomes and mitochondria.

Cell surfaces, walls, vacuoles and cell specialisations

The cell surface membrane is a phospholipid bilayer about 7 nm thick with proteins embedded in it. It is partially permeable, so it controls what enters and leaves the cell, and its proteins carry out transport, act as receptors for hormones and act as antigens for cell recognition. At 7 nm it is far below the resolution of a light microscope, so in an electron micrograph you see it as a single thin line, and a light microscope does not show it at all. Outside it, a plant cell has a cell wall of cellulose microfibrils embedded in a matrix of other polysaccharides. The wall is freely permeable, so it does not control exchange; its job is strength, and resisting the outward pressure of the cell contents so that the cell does not burst when water enters by osmosis.

Neighbouring plant cells are cemented together by a thin layer of calcium pectate called the middle lamella, and they are joined by plasmodesmata, narrow channels that pass through the walls. Each channel is lined with cytoplasm and a thin tube of membrane, so water, ions and small molecules can move directly from one cell to the next without crossing two membranes, which is how substances spread through a plant tissue. A mature plant cell also has a large permanent vacuole filling much of the cell, bounded by a membrane called the tonoplast. The vacuole holds cell sap, a solution of sugars, amino acids, salts and often pigments such as anthocyanin, and it is the vacuole pushing outwards on the wall that gives the cell its turgor and supports a non-woody plant. The tonoplast controls what moves between cytoplasm and cell sap, so the vacuole can store ions and release them when the cell needs them.

Centrioles are two short cylinders lying at right angles to one another, each built from nine triplets of microtubules, and they organise the spindle fibres during nuclear division in animal cells. Microtubules also form part of the cytoskeleton, act as tracks along which vesicles are carried, and form the core of cilia, whose 9+2 arrangement of microtubules produces a whipping beat that sweeps mucus and trapped dust up the airways. Microvilli are finger-like folds of the cell surface membrane, each supported by actin filaments, which greatly increase the surface area of the cell for absorption; the epithelial cells of the small intestine and of the proximal tubule of a kidney nephron are packed with them.

Comparing typical plant and animal cells, both are eukaryotic, so both have a cell surface membrane, a nucleus with a nuclear envelope and pores, mitochondria, ribosomes, rough and smooth endoplasmic reticulum, a Golgi body and lysosomes. Four things separate them. A plant cell has a cellulose cell wall and a large permanent vacuole, and an animal cell has neither, although it may form small temporary vacuoles. A plant cell that photosynthesises has chloroplasts, and an animal cell never does. Most plant cells have no centrioles and store carbohydrate as starch, whereas animal cells have centrioles and store carbohydrate as glycogen. Animal cell surface membranes also contain cholesterol, which plant membranes generally do not.

Prokaryotic cells: the bacterial cell

A prokaryotic cell has no nucleus and no membrane-bound organelles of any kind. Its DNA is a single circular molecule, not wound around protein, lying free in the cytoplasm, and there are no mitochondria, no chloroplasts, no endoplasmic reticulum, no Golgi body and no lysosomes. What a bacterium does have is cytoplasm packed with 70S ribosomes, a cell surface membrane, and in almost all species a cell wall made of peptidoglycan, a polymer of sugar chains cross-linked by short chains of amino acids.

A typical bacterium is unicellular and generally 1-5 µm in diameter, roughly ten times smaller than a typical eukaryotic cell. Many bacteria also carry plasmids, which are small extra circles of DNA that can be passed from one cell to another and often carry genes for antibiotic resistance, and some have a slime capsule outside the wall, pili for attachment, or one or more flagella made of the protein flagellin for movement.

Compare a bacterium with a eukaryotic cell and the differences follow from having no nucleus. A prokaryote keeps its DNA, its ribosomes and its enzymes in one compartment, so transcription and translation can happen at the same time on the same piece of DNA, whereas a eukaryote separates them by the nuclear envelope, with transcription in the nucleus and translation in the cytoplasm. A prokaryote has circular DNA and 70S ribosomes; a eukaryote has linear chromosomes wound around histone proteins inside a nucleus and 80S ribosomes in the cytoplasm. A bacterial wall is peptidoglycan, a plant cell wall is cellulose and an animal cell has no wall at all.

That peptidoglycan wall is unique to bacteria, and it is a good target for drugs. The antibiotic penicillin prevents the cross-links forming between the sugar chains, so the wall cannot be repaired as the cell grows and the bacterium bursts under its own osmotic pressure. Human cells have no wall, so they are untouched, which is exactly what a selective toxicity needs. Lysozyme, an enzyme in tears, saliva and mucus, attacks the same wall by breaking the bonds between the sugar units, and it is part of the body's first line of defence against bacteria getting into the tissues.

Viruses: non-cellular structures

A virus is not a cell and is not made of cells. Every virus consists of a nucleic acid core, which is either DNA or RNA but never both, surrounded by a capsid, a coat made of protein. The capsid is built from many copies of one or a few protein subunits arranged in a repeating pattern, which is why a virus can be constructed from a very small amount of genetic information. Some viruses have an extra outer envelope made of phospholipid, picked up from the membrane of the host cell as the virus leaves, with virus-coded proteins such as glycoprotein spikes projecting from it. A virus is far smaller than a bacterium, measured in nanometres rather than micrometres, and it is far below the resolution of a light microscope.

Because a virus has no cytoplasm, no ribosomes and no machinery for making ATP of its own, it carries out none of the processes of a living cell. It does not respire, it does not synthesise proteins and it cannot reproduce by itself. A virus is an obligate parasite: it can only replicate inside a host cell, where it uses the host's ribosomes, ATP and enzymes to copy its nucleic acid and to make its capsid proteins. Outside a host it is inert, an infectious particle that can be crystallised and stored, and it is that inertness which makes biologists argue about whether a virus counts as alive at all.

Named examples show how varied the group is. Tobacco mosaic virus has a helical capsid of protein wrapped round a single RNA molecule and no envelope; it was the first virus to be crystallised, by Wendell Stanley in 1935, and it stunts the leaves of tobacco and tomato plants. Bacteriophage T4 has an icosahedral head of DNA, a tail and tail fibres and no envelope, and it infects the bacterium Escherichia coli. HIV carries two copies of RNA inside a cone-shaped capsid, has a phospholipid envelope with glycoprotein spikes, and brings the enzyme reverse transcriptase with it to copy its RNA into DNA in the host cell. SARS-CoV-2 is an enveloped RNA virus whose glycoprotein spikes bind to the ACE2 receptor on human cells; because its envelope is a lipid bilayer, soap and alcohol break it apart, which is why hand-washing is effective against this virus but much less so against a non-enveloped virus such as norovirus.