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What is Cell Fractionation? Cell fractionation is a laboratory process used to break open cells and then separate their organelles so that each can be studied in isolation. It is particularly useful because different organelles carry out different functions (e.g., mitochondria → aerobic respiration, chloroplasts → photosynthesis). The Three Key Stages
1. Homogenisation Cells are broken open using a homogeniser (blender). This produces a liquid called the homogenate , which contains all the organelles. To prevent organelle damage, the homogenate is prepared in a solution that is: o Cold → slows enzyme action, prevents organelle digestion. o Isotonic → prevents osmotic damage (no shrinkage or bursting of organelles). o Buffered → keeps pH stable, preventing denaturation of proteins/enzymes.
2. Filtration The homogenate is filtered through a gauze or mesh. This removes large debris, whole cells, and unbroken tissue. The filtrate that passes through contains the organelles. 3. Ultracentrifugation The filtrate is placed into tubes and spun in an ultracentrifuge. Spinning separates organelles by size and density : o Heavier organelles settle first (form the pellet at the bottom). o Lighter organelles remain in the supernatant. The supernatant is re-spun at faster speeds to collect smaller organelles. Order of organelle separation in animal cells: 1. Nuclei (largest, densest) 2. Mitochondria 3. Lysosomes 4. Endoplasmic reticulum 5. Ribosomes (smallest) (In plant cells, chloroplasts come after nuclei and before mitochondria.)
MICROSCOPES
Electron microscopes use a beam of electrons instead of light. Electrons have a much shorter wavelength than visible light. This gives a much higher resolution compared to light microscopes.
1. Transmission Electron Microscope (TEM) How it Works A beam of electrons is transmitted through a very thin specimen. Denser areas absorb more electrons → appear darker. Less dense areas allow electrons through → appear lighter. Produces 2D images of the internal structure of the cell. Resolution & Magnification Resolution: up to 0.1 nm (highest available). Magnification: up to ×500,.
Advantages Very high resolution → can see ultrastructure (e.g., cristae in mitochondria, membranes, ribosomes). Provides detailed view of internal organelle structure. Limitations Specimens must be dead (vacuum is required so electrons don’t scatter). Specimen must be extremely thin. Image is only in black and white (false colour may be added by computer). Complex preparation may create artefacts (structures seen that are not real). Produces only 2D images.
2. Scanning Electron Microscope (SEM) How it Works A beam of electrons is directed onto the surface of the specimen. Electrons scatter depending on surface contours. A detector collects these scattered electrons → builds up a 3D image of the specimen’s surface.
Comparison Table (TEM vs SEM vs Light Microscope) Feature Light Microscope
TEM SEM
Radiation used Light Electrons Electrons Resolution ~200 nm ~0.1 nm (highest) ~20 nm Magnification Up to ×1, Up to ×500, Up to ×100, Image type Colour, 2D Black & white, 2D Black & white, 3D Specimen thickness Thick allowed Very thin Thicker specimens allowed Living specimens? ✅ Yes ❌ No (vacuum needed) ❌ No (vacuum needed) Internal structure Limited detail Yes (ultrastructure visible) No (surface detail only) Artefacts risk Low High (complex preparation) High (complex preparation)
Why a Vacuum is Needed in TEM and SEM Prevents Electron Scattering Electron microscopes use a beam of electrons instead of light. Electrons can collide with air molecules as they travel. These collisions scatter the electrons , reducing resolution and blurring the image. A vacuum removes air , so electrons can travel in a straight line from source to specimen. Protects the Electron Source The electron gun produces electrons via thermionic emission or other methods. Presence of air would interfere with the electron beam and reduce efficiency. Prevents Oxidation of the Specimen Some specimens, especially metal-coated ones in SEM, could oxidize or react with air under the electron beam. A vacuum prevents unwanted chemical reactions.
o Reduces enzyme activity. o Prevents hydrolytic enzymes (e.g. lysosomal enzymes) from breaking down organelles.
- Isotonic o Has the same water potential as the cell’s cytoplasm. o Prevents osmosis, which could cause organelles to burst (lysis) or shrink.
- Buffered o Maintains a constant pH. o Prevents changes in pH that could denature enzymes or damage proteins in organelles. After Homogenisation The result is a mixture called the homogenate. The homogenate contains all the organelles from the broken cells, suspended in the protective solution. This homogenate is then filtered to remove large debris before moving on to ultracentrifugation. Why Homogenisation is Important If cells were not carefully homogenised, organelles would be damaged or digested, making it impossible to study them properly.
Proper homogenisation ensures organelles are intact and functional when separated later.
- Second spin – faster speed o The supernatant is spun at a higher speed. o The next heaviest organelles sediment and form a pellet. In animal cells: mitochondria (and in plants, chloroplasts).
- Repeated at increasing speeds o Each time the centrifuge speed is increased, progressively lighter organelles are separated out. o This continues until even the smallest organelles are isolated. Order of Organelle Separation In Animal Cells (AQA spec):
- Nuclei (largest, densest).
- Mitochondria.
- Lysosomes.
- Endoplasmic reticulum.
- Ribosomes (smallest). In Plant Cells:
- Nuclei.
- Chloroplasts (large and dense).
- Mitochondria.
- Lysosomes / ER.
- Ribosomes. Key Terms to Remember Pellet : solid layer of organelles that collects at the bottom of the tube. Supernatant : liquid above the pellet containing remaining organelles. Differential centrifugation : technique of separating organelles by spinning at different speeds. Why Ultracentrifugation is Important It allows scientists to isolate and study individual organelles. Example: Studying mitochondria to confirm their role in aerobic respiration. Provides organelles intact and functional for biochemical experiments and microscopy. Eukaryotic Cell Structure Notes
Euchromatin (light, active for transcription). Heterochromatin (dark, inactive). Function : o Controls cell activities by directing protein synthesis. o Retains genetic material in the form of DNA. o Nucleolus manufactures ribosomal RNA and assembles ribosomes. Exam link : In microscopy, nucleus stains dark. Look for nuclear pores and nucleolus.
2. Mitochondria Structure : o Double membrane: inner membrane folded into cristae → increases surface area for attachment of enzymes and electron carriers. o Inside is the matrix , which contains: Enzymes for respiration, Mitochondrial DNA, Ribosomes (70S). Function : o Site of aerobic respiration → produces ATP (energy currency of the cell). o Self-replicating (can increase in number when energy demand rises). Exam link : Describe cristae as “large surface area for enzyme attachment” not just “folds”.
3. Ribosomes Structure : o Small, dense organelles made of protein + rRNA. o Two subunits (large + small). o Found free in cytoplasm or attached to RER. o Eukaryotes = 80S type (larger). Function : o Free ribosomes : make proteins used in cytoplasm. o RER ribosomes : make proteins for export/packaging. Exam link : Remember mitochondria and chloroplasts have 70S ribosomes → evidence for endosymbiotic theory. 4. Endoplasmic Reticulum (ER) Rough ER (RER) : o Membranes studded with ribosomes. o Proteins made on ribosomes enter the cisternae (sacs) → folded + modified. o Proteins are packaged into vesicles → sent to Golgi. Smooth ER (SER) : o No ribosomes.
o Break down pathogens engulfed by phagocytosis. o Autolysis → enzymes released to destroy the whole cell when necessary. Exam link : AQA often tests lysosome role in immune system → macrophages.
7. Cell-Surface Membrane (Plasma Membrane) Structure : o Phospholipid bilayer with embedded proteins (fluid mosaic model). Function : o Controls entry/exit of substances. o Contains receptors → allows cell signalling and recognition. Exam link : Mention selectively permeable, not completely impermeable. 8. Cell Wall (plants, fungi, algae) Structure : o Plants: cellulose microfibrils. o Fungi: chitin. o Algae: cellulose or glycoproteins. Function : o Provides strength to prevent osmotic bursting. o Provides shape and mechanical support.
Exam link : Do not confuse with plasma membrane (cell wall is non-living).
9. Chloroplasts (plants + algae only) Structure : o Double membrane (chloroplast envelope). o Thylakoids stacked into grana ; contain chlorophyll for light absorption. o Stroma = fluid with enzymes, sugars, organic molecules, DNA, and ribosomes. o Can reproduce themselves. Function : o Photosynthesis: Light-dependent reaction in thylakoid membranes. Light-independent reaction in stroma. Exam link : Chloroplasts contain their own DNA and ribosomes → can make proteins for photosynthesis. 10. Vacuole (plants only) Structure : o Large central vacuole, surrounded by tonoplast (single membrane). o Contains cell sap (salts, sugars, amino acids, waste, pigments).
