Study guide: Cell Biology Techniques

📋 Course Outline

1. Cell Theory and Microscopy Resolution
2. Optical Microscopy and Tissue Preparation
3. Fluorescence Microscopy
4. Electron Microscopy Principles
5. Transmission Electron Microscopy
6. Surface Imaging and Cryofracture
7. Cell Culture and Animal Cell Lines
8. Stem Cell Potency and Plant Cultures
9. Differential Centrifugation
10. Density Gradient Centrifugation
11. Viruses as Cellular Tools and Agents

📖 1. Cell Theory and Microscopy Resolution

🔑 Key Concepts & Definitions

• Microscopy resolution: The minimum distance between two points at which they can still be distinguished as separate.

★ Must-know

• The unaided human eye has a resolving limit of approximately 100–200 µm, so most cells cannot be seen without a microscope.

• The first two principles of cell theory were defined in 1839 by Théodore Schwann: the cell is the structural and functional unit of every living organism, and every living organism consists of at least one cell.

• The third principle of cell theory states that every cell arises from the division of another cell.

Further detail

• In Pasteur’s swan-neck flask experiment, boiled water remained clear when the curved neck prevented bacteria from entering, supporting the conclusion that bacteria came from outside rather than arising spontaneously. - Louis Pasteur

📖 2. Optical Microscopy and Tissue Preparation

★ Must-know

• The optical microscope uses focused light and has a best resolution of about 200 nm (0.2 µm), sufficient to observe cells and large organelles but not fine cytoplasmic details.

Further detail

• For bright-field microscopy of tissue, the sample is fixed, dehydrated through increasingly concentrated alcohol, embedded in resin or paraffin, cut with a microtome, deparaffinized, rehydrated, stained, mounted, and covered with a coverslip.

• Bright-field microscopes combine objective magnification (×4, ×10, ×40, or ×100) with eyepiece magnification (×10 or ×20), reaching a maximum total magnification of ×2,000.

• Most fixation, sectioning, and staining procedures kill cells, while vital stains such as neutral red allow cells to remain alive.

• Phase-contrast and interference optical microscopes are used to observe living cells.

📖 3. Fluorescence Microscopy

★ Must-know

• Fluorescence microscopy can locate a target molecule by binding a target-specific antibody to it and associating the antibody with a fluorochrome that emits visible light when excited by ultraviolet light.

Further detail

• Fluorescein emits green light, whereas rhodamine emits red light.

💡 Memory Hook

Antibody binds target → fluorochrome emits visible light

📖 4. Electron Microscopy Principles

★ Must-know

• Electron microscopes use a high-speed electron beam in an absolute vacuum instead of light; the vacuum prevents collisions between air molecules and electrons.

• Electron microscopy cannot image living cells because the specimen must be in the vacuum column and is necessarily dead.

Further detail

• The course gives practical electron-microscope resolution as 0.5–1 nm and theoretical resolution as 0.1–0.2 nm.

💡 Memory Hook

Electrons, not light; vacuum, not living cells

📖 5. Transmission Electron Microscopy

★ Must-know

• A transmission electron microscope (TEM) transmits electrons through an ultrathin specimen to form an image and can resolve approximately 0.2 nm, or about 0.5 nm in practice.

• TEM heavy-metal salts accumulate in regions rich in proteins, lipids, or nucleic acids; these regions scatter electrons and appear dark, while electron-transmitting regions appear light.

Further detail

• Positive staining for TEM uses an ultrathin section of about 100 nm, collected on a grid and contrasted with heavy-metal salts that accumulate in cellular material.

• Negative staining is used mainly for intact isolated organelles, viral particles, or macromolecules; heavy-metal salts surround the specimen, leaving it light against a dark background and emphasizing its outline.

💡 Memory Hook

Positive staining darkens internal material; negative staining outlines the whole element

📖 6. Surface Imaging and Cryofracture

★ Must-know

• Metal shadowing coats an isolated structure from one side with heavy metal, producing a three-dimensional view of its surface relief rather than its interior.

• Cryofracture freezes tissue in liquid nitrogen at −196°C, fractures it—often through the middle of membranes—removes any surface ice, applies metal shadowing and a perpendicular carbon layer, and dissolves the biological material to leave a metal replica for TEM.

• A scanning electron microscope (SEM) scans an intact, fixed, dehydrated specimen’s surface without sectioning; reflected electrons are detected to form a relief image, with resolution of about 5–10 nm.

Further detail

• Cryofracture showed that proteins are inserted within membranes.

💡 Memory Hook

MEB scans a surface; cryofracture exposes membrane planes

📖 7. Cell Culture and Animal Cell Lines

🔑 Key Concepts & Definitions

• Cell culture: A controlled in vitro system that grows many cells of one type from an initial sample to study cell behavior and function.

★ Must-know

📌 A primary culture comes directly from a tissue sample, whereas a secondary culture is produced by collecting and reculturing cells from an earlier culture.

• Animal cells can divide about 50–100 times before becoming senescent, while genetically modified cells or tumor-derived cells can divide indefinitely.

Further detail

• Animal cells generally stop dividing after covering the culture surface, but cells can be transferred to new dishes to establish secondary cultures.

• HeLa is a tumor-derived cell line sampled in 1951 and still used in laboratories; it has helped investigate cellular processes, including cancer-cell behavior.

• A cell-line culture comprises the primary culture and all cultures derived from it directly or indirectly, and its cells can be frozen for years before reculturing.

📖 8. Stem Cell Potency and Plant Cultures

🔑 Key Concepts & Definitions

• Stem cell: An undifferentiated cell that can give rise to other cells.

★ Must-know

📌 Totipotent cells can form all organismal cell types, including embryonic annexes such as the placenta and umbilical cord; pluripotent cells can form any cell type except embryonic annexes; multipotent cells can form a limited range of cell types.

• Plant tissue is treated with cellulase to digest cell walls and produce wall-less protoplasts, which are cultured until walls reform and cells divide.

• Cultured plant cells form an undifferentiated callus, continue growing rather than stopping at dish coverage, and are totipotent, so a single cell can generate an entire plant.

Further detail

• In animals, the zygote and cells of the earliest divisions are totipotent, while embryonic stem cells at an early stage can give rise to many cell types and fetal stem cells have a more limited range.

• Human bone marrow contains stem cells that give rise to blood cells, and small percentages of stem cells occur in various tissues.

💡 Memory Hook

Animal cultures stop at confluence; plant cultures form a callus

📖 9. Differential Centrifugation

🔑 Key Concepts & Definitions

• Cell fractionation: A method that isolates cellular components so they can be studied individually, including the functions of organelles.

★ Must-know

• Differential centrifugation separates cell components through successive centrifugations of increasing speed and duration: denser components sediment into a pellet, while less dense components remain in the supernatant.

• Successive pellets and the final supernatant contain:

  • First low-speed pellet: nuclei and intact cells.
  • Second, faster pellet: mitochondria, lysosomes, and peroxisomes.
  • Third pellet: microsomes.
  • Ultracentrifugation above 100,000 G: ribosomes.
  • Final supernatant: cytosol.

Further detail

• Cell homogenization in buffer breaks the plasma membrane and also fragments the endoplasmic reticulum and Golgi membranes into vesicles called microsomes.

💡 Memory Hook

Nuclei → mitochondria and related organelles → microsomes → ribosomes

📖 10. Density Gradient Centrifugation

★ Must-know

• Density-gradient centrifugation places a sample above a sucrose solution whose concentration increases toward the tube bottom; during centrifugation, each component sediments to the level where its density matches the sucrose, forming separable bands.

📌 Differential centrifugation separates components with relatively large density differences, whereas density-gradient centrifugation separates components with very similar densities and yields purer fractions.

Further detail

• A density gradient can separate mitochondria, lysosomes, and peroxisomes from a mixed pellet; filling lysosomes with detergent can reduce their density, and plant-cell pellet 2 contains chloroplasts but no lysosomes.

💡 Memory Hook

Particles settle into bands where their density matches the rising sucrose gradient

📖 11. Viruses as Cellular Tools and Agents

🔑 Key Concepts & Definitions

• Virus: A noncellular particle of macromolecules that contains DNA or RNA and is not considered living in the course’s account.

★ Must-know

• A simple virus consists of a nucleic acid enclosed in a protective protein coat called a capsid, and some viruses also have a lipid envelope.

• Viruses replicate only inside a host cell by using cellular machinery to replicate their genetic material and express viral genes; some RNA viruses first use reverse transcription to make DNA.

Further detail

• Viral genes can encode capsid proteins, and viral studies have helped investigate DNA replication, transcription, and translation.

• Genetically modified viruses can act as vectors that deliver a therapeutic gene after disease-causing or cell-destroying viral genes have been removed.

• Oncogenic viruses can transform normal cells into cancer cells; examples given are papillomavirus associated with uterine cancer and hepatitis B virus associated with liver cancer, and the course states that at least 20% of human cancers are caused by viruses.

💡 Memory Hook

Host-cell machinery enables viral replication and gene expression