Cell culture & Microscopy

• G0 phase


  Cells that are not actively dividing
• Cell cycle progression


  1. Cells leave G0 phase

  2. Enter G1 phase

  3. Start first phase of mitosis
• Senescent cells


  Cells that are permanently stuck in G0 phase and cannot enter the cell cycle
• Telomere shortening following cell division limits the number of times a cell divides
• Tumour cells are effectively immortal and can rebuild their telomeres using the enzyme telomerase
• HeLa cells were isolated from Henrietta Lacks in 1951 from a cervical tumour
• Some of the tumour cells that were isolated were immortal and could be cultured indefinitely
• HeLa cells were the first human cell line and are still one of the most widely used cell models today
• HeLa cells were used to test the first polio vaccine in the 1950s
• HeLa cells can contaminate other cell cultures
• Culture media


  Provides all of the nutrients cells need to grow
• Different media compositions can be used for different cells depending on their requirements
• Typical culture media contains

  • Salts
  • Amino acids
  • Vitamins
  • Glucose
  • Phenol red dye
• Adherent cells


  Most cells in the body need to attach to a surface
• When cells run out of space in the dish they need to be split into new dishes
• Trypsin


  An enzyme used to detach cells
• Passaging


  The process of splitting cells into new dishes, needs to happen 1-2 times per week
• Some cells (e.g. blood cells and leukaemia cells) grow in suspension
• Growth conditions for cells in culture are also ideal for growth of bacterial cells
• Aseptic conditions


  Conditions needed to maintain sterile environments in cell culture
• Use of antibiotics in culture media and laminar flow cabinets help maintain sterile conditions
• Incubators maintain temperature at 37 degrees
• Common contamination issues in cell culture

  • Bacteria
  • Yeast
  • Fungi
• Biosafety levels (BSL)


  • BSL-1 lab
  • BSL-2 lab
  • BSL-3 lab
  • BSL-4 lab
• Microscopes work by using lenses to magnify light coming through a biological sample
• Modern microscopes have a built-in light source that is focused using a collector lens onto a mirror
• The mirror bounces the light through a condenser lens which focuses it on the specimen, located on the microscope stage
• The light from the specimen is magnified by the objective and projection lenses until it arrives at the detector
• Types of standard light microscopy

  • Bright-field microscopy
  • Phase contrast microscopy
  • Differential Interference Contrast (DIC) microscopy
• Azan trichrome stain stains nuclei bright red, collagen, basement membrane and mucin blue, muscle and red blood cells orange to red
• Hematoxylin & Eosin stain is the most commonly used stain in histology
• Hematoxylin stains acidic structures such as the nucleus, eosin stains basic structures such as the cytoplasm and cell walls
• Electron microscopes work using the same basic principles as light microscopes but use electrons instead of light
• Electrons have smaller wavelengths than photons, allowing visualization of smaller objects
• Electron microscopes can’t produce colour images and can’t be used to view live cells due to the need for a vacuum
• Fluorescence microscopy is a type of light microscopy that relies on the ability of some molecules to emit light when stimulated
• Uses of fluorescent proteins

  • Cell dynamics
  • Sub-cellular localisation of proteins
  • Transgenic animals
• Photobleaching is the destruction of the fluorescent protein by high intensity laser light
• Phototoxicity is toxicity caused by high-intensity laser light (mainly due to free radical formation)
• Overexpression occurs when tagged genes are expressed at very high levels to produce a good signal