Microscopy

• Magnification is how many times bigger the image is than the actual object/specimen.
• Resolution is the ability t distinguish between 2 points on an image.
• Cell Theory


  1665: Cell first observed by Robert Hooke. He observed the structure of thinly sliced cork using an early light microscope. He named the compartments he saw as 'cells' (we now know they were cell walls)
• Cell Theory


  1674-1683: First living cells observed by Anton Van Leeunwenhoek. He developed powerful glass lenses to make his own microscope to study pondwater samples.
• Cell Theory


  1832: Evidence for origin of new plant cells from Barthelemy Dumortier. He was the first to observe cell division in plants.
• Cell theory


  1833: First observed nucleus by Robert Brown.
• Cell theory


  1837-1838: 'Birth of universal cell theory' by Mattias Schledien. He proposed that all plant tissues are composed of cells.
• Cell theory


  1837-1838: Jan Purkyne was the first to use a microtome to make thin slices of tissue for examination. He said that both animal and plant tissues were composed of cells.
• Cell theory


  1837-1838: Theodor Schwann made a similar observation: "all living things are composed of cells and cell products". He is the credited scientist for the BUCT.
• Cell theory


  1844 (1855): Robert Remak is the first to observe cell division in animal cells. Rudolf Virchow published these findings as his own in 1855.
• Cell Theory


  1860: Spontaneous generation disproved by Louis Pasteur by demonstrating bacteria would only grow after sterile broth was exposed to air.
• How do light microscopes work?

  1. Mirror or lamp projects light through the condenser to the stage and specimen.
  2. Specimen is illuminated and the light refracts so the image can be viewed.
  3. Objective and eyepiece lens magnify the image for examination.
• Dry mount


  Specimen viewed whole or sectioned. Placed in the centre of the slide and the coverslip is placed over.
• Wet mount


  Specimen suspended in liquid and the coverslip is placed on from an angle.
• Smear slide

  Edge of the slide is used to smear the sample, the coverslip is placed over the sample.
• Squash slide

  Wet mount but lens tissue is used to gently press down the coverslip.
• How does a TEM differ from a SEM?

  TEM has a higher resolution of 0.5nm and a higher magnification but produces a 2D image.

  SEM has a lower resolution of 3-10nm an a lower magnification but it produces a 3D image.
• Why do we stain?


  Stains increase contrast and different components of the cell will take up the stain to different degrees of intensity.
• What is heat-fixing?


  Passing a specimen through a flame which allows the specimen to adhere to the microscope slide and take up the stains.
• Why are dyes attracted to different materials?

  Negatively charged dyes like nigrosine or congo red are repelled by the negatively charged cytosol so stay outside the cells.

  Crystal violet and methylene blue are positively charged though so it is attracted to the cytosol and stains it.
• What is differential staining?


  Can distinguish between 2 types of organisms that would otherwise be hard to identify. Can also differentiate between different organelles in a tissue sample.
• Gram Staining


  Used to separate bacteria into gram-positive and gram-negative.

  1. Crystal violet is first applied to a bacterial specimen.
  2. Then iodine, which fixes the dye.
  3. Slide is washed with alcohol.
• Gram positive bacteria


  Retain crystal violet stain and appear blue/purple under the microscope.
• Gram negative bacteria


  Have thinner cell walls so lose the crystal violet stain, they are stained with safranin dye (counterstain)
• Acid-fast technique


  Used to differentiate species of mycobacterium from other bacteria.

  A lipid solvent is used to carry the carbol fuchsin dye into the cells being studied. The cells are then washed with a dilute acid-alcohol solution. Mycobacterium are not affected by the solution so retain the dye which is bright red.
• Fixing


  Chemicals like formaldehyde are used to preserve specimens in as near-natural state as possible.
• Sectioning


  Specimens are dehydrated with alcohols and then placed in a mould with a wax or resin to form a hard block which can be sliced with a microtome.
• Staining


  Specimens are treated with multiple stains to show different structures.
• Mounting


  Specimens are secured to a microscope slide then a coverslip is placed on top.
• Managing risks in staining


  Most stains are toxic or irritants s a risk assessment must be completed. CLEAPSS provides student safety sheets that identify specific risks, and advice to be taken to reduce risks or in an emergency. Microscope slides are often pre-prepared for schools.
• Calibrating an eyepiece graticule

  1. Put the stage micrometre in place and eyepiece graticule in the eyepiece.
  2. Get the scale on the micrometre slide in focus.
  3. Align the micrometre slide with the eyepiece graticule and take a reading from both scales.
• 1 graticule division = number of micrometers ÷ number of graticule divisions
• Magnification = image size ÷ actual size