OCR AS Biology

Revision Notes

1.2.16 Practical: Using a Light Microscope

Practical: Using a Light Microscope

  • Many biological structures are too small to be seen by the naked eye
  • Optical microscopes are an invaluable tool for scientists as they allow for tissues, cells and organelles to be seen and studied
  • For example, the movement of chromosomes during mitosis can be observed using a microscope

How optical microscopes work

  • Light is directed through the thin layer of biological material that is supported on a glass slide
  • This light is focused through several lenses so that an image is visible through the eyepiece
  • The magnifying power of the microscope can be increased by rotating the higher power objective lens into place

Apparatus

  • The key components of an optical microscope are:
    • The eyepiece lens
    • The objective lenses
    • The stage
    • The light source
    • The coarse and fine focus
  • Other tools used:
    • Forceps
    • Scissors
    • Scalpel
    • Coverslip
    • Slides
    • Pipette

Method

  • Preparing a slide using a liquid specimen:
    • Add a few drops of the sample to the slide using a pipette
    • Cover the liquid/smear with a coverslip and gently press down to remove air bubbles
    • Wear gloves to ensure there is no cross-contamination of foreign cells
  • Preparing a slide using a solid specimen:
    • Use scissors to cut a small sample of the tissue
    • Peel away or cut a very thin layer of cells from the tissue sample to be placed on the slide (using a scalpel or forceps)
    • Some tissue samples need be treated with chemicals to kill/make the tissue rigid
    • Gently place a coverslip on top and press down to remove any air bubbles
    • A stain may be required to make the structures visible depending on the type of tissue being examined
    • Take care when using sharp objects and wear gloves to prevent the stain from dying your skin
  • When using an optical microscope always start with the low power objective lens:
    • It is easier to find what you are looking for in the field of view
    • This helps to prevent damage to the lens or coverslip incase the stage has been raised too high
  • Preventing the dehydration of tissue:
    • The thin layers of material placed on slides can dry up rapidly
    • Adding a drop of water to the specimen (beneath the coverslip) can prevent the cells from being damaged by dehydration
  • Unclear or blurry images:
    • Switch to the lower power objective lens and try using the coarse focus to get a clearer image
    • Consider whether the specimen sample is thin enough for light to pass through to see the structures clearly
    • There could be cross-contamination with foreign cells or bodies
  • Use a calibrated graticule to take measurements of cells

Using a graticule

  • An eyepiece graticule and stage micrometer are used to measure the size of the object when viewed under a microscope
  • Each microscope can vary slightly so needs to be calibrated when used
  • The calibration is done with a stage micrometer, this is a slide with a very accurate scale in micrometres (µm), it is usually in 10 µm divisions, so 1 mm divided into 100 divisions
  • The eyepiece graticule is a disc placed in the eyepiece with 100 divisions, this has no scale
  • To know what the divisions equal at each magnification the eyepiece graticule is calibrated to the stage micrometer at each magnification

Eyepiece graticule & Micrometer, AS & A Level Biology revision notes

  • In the diagram, the stage micrometer has three lines each 10 µm apart
  • Each 10 µm division has 40 eyepiece graticule divisions
  • 40 graticule divisions = 10 µm

1 graticule division = number of micrometres ÷ number of graticule division

  • 1 graticule division = 10 ÷ 40 = 0.25 µm this is the magnification factor
  • The specimen slide would be used to replace the stage micrometer and the eyepiece graticule at the same magnification would be used to measure the length of the object
  • The number of graticule divisions can then be multiplied by the magnification factor:

graticule divisions x magnification factor = measurement (µm)

Limitations

  • The size of cells or structures of tissues may appear inconsistent in different specimen slides
    • Cell structures are 3D and the different tissue samples will have been cut at different planes resulting in inconsistencies when viewed on a 2D slide
  • Optical microscopes do not have the same magnification power as other types of microscopes and so there are some structures that can not be seen
  • The treatment of specimens when preparing slides could alter the structure of cells

Exam Tip

The calculations involving stage micrometers and eyepiece graticules are often seen in exam questions, so make sure that you are comfortable with how to calibrate the graticule and calculate the length of an object on the slide.

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