Topic 1

• Use of microscopy

  To observe and investigate different types of cell and cell structure in a range of eukaryotic organisms
• Types of microscopy

  Light, transmission electron, scanning electron, laser scanning confocal
• Types of mount

  Wet, dry, squash, smear
• How to do a wet mount

  Suspend the specimen in liquid, place cover slip on from an angle
• Examples of liquids used in wet mounts

  Water, immersion oil
• Organisms that can be viewed in wet mounts

  Aquatic organisms
• How to do a dry mount

  Section the sample if it is too large, place specimen on centre of the slide, put cover slip on top
• Things that can be viewed in dry mounts

  Hair, pollen, dust, insect parts, parts of muscle tissue, parts of plant
• How to do a smear slide

  Use the edge of a slide to smear out the sample on another slide, put cover slip over the sample
• How to do a squash slide

  Prepare a wet mount, use a lens tissue or two microscope slides to press down on the cover slip
• What can you view with a squash slide?

  Root tips during cell division
• What can you view with a smear slide?

  Blood
• Why must a sample be thin for light microscopy?

  So the light can shine through it and details can be seen.
• How a light microscope works

  Objective lens produces a magnified image, image magnified again by the eyepiece lens, illumination provided by a light underneath the sample
• How to calibrate a microscope

  Stage micrometer on the stage and the eyepiece graticule in the eyepiece, get in focus, align micrometer with the eyepiece graticule, take readings from both the micrometer and the graticule, use ratios to find how much one graticule division is worth, find the magnification factor, measure stuff
• Why must the liquid medium used in wet mounts have a similar refractive index to glass?

  To prevent diffraction between the liquid and the glass and thus preventing image distortion.
• Why are cover slips placed on wet mounts at an angle?

  To prevent the trapping of air bubbles.
• Purpose of differential staining

  To identify different cellular components and cell types
• Examples of differential staining

  Gram stain technique, Acid-fast technique
• Purpose of the Gram stain technique

  To differentiate between Gram positive and Gram negative bacteria.
• How to do the Gram stain technique

  Apply crystal violet, add iodine to fix the dye, wash the slide with alcohol, Gram positive bacteria retain the dye so will seem blue or purple, stained with safranin in a counterstain which makes Gram negative bacteria appear red
• Purpose of the Acid Fast technique

  To differentiate between species of Mycobacterium and other bacteria
• How to do the Acid Fast technique

  Carbolfuchsin dye is carried into the cell in a lipid solvent, cells are washed with an acid-alcohol solution, Mycobacteria retain the stain which turns them bright red, methylene blue is applied in a counterstain to turn other bacteria blue
• Stages in pre-preparation of slides

  Fixing, sectioning, staining, mounting
• Fixing


  Using chemicals like formaldehyde to preserve specimens in a near-natural state.
• Sectioning

  Dehydrating a sample with alcohol and then placing it in a mould of resin or wax to form a hard block before slicing it with a microtome into thin slices.
• Staining

  Treating specimens with multiple stains to show different structures.
• Mounting

  Securing a specimen onto a microscope slide under a cover slip.
• Magnification Formula

  Magnification = Image size / Object Size
• Difference between magnification and resolution

  Magnification is how many times larger the image is than the actual size of the object whereas resolution is the ability to see individual objects as separate entities.
• Resolution of light microscopes

  200nm
• Magnification of light microscopes

  1500x
• Resolution of a transmission electron microscope

  0.5nm
• Magnification of a transmission electron microscope

  500000x
• Resolution of a scanning electron microscope

  3-10nm
• Magnification of a scanning electron microscope

  100000x
• Parts of the ultrastructure of eukaryotic cells

  Nucleus, nucleolus, nuclear envelope, SER, RER, Golgi Apparatus, ribosomes, mitochondria, lysosomes, chloroplasts, cell surface plasma membrane, centrioles, cell wall, flagella, cilia
• Function of the nucleus

  Contains genetic information in the form of DNA molecules which directs the synthesis of proteins for the cell, thus making it indirectly responsible for the metabolic activity in the cell.
• How is the nucleus indirectly responsible for the metabolic activity of a cell?

  Some of the proteins produced will be enzymes necessary for metabolism.
• Function of the nucleolus

  To produce ribosomes when the ribosomal RNA in the nucleolus combines with proteins