7.1.1 Practical - Using Light Microscopes to View Cells

Viewing plant cells

• 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 larger organelles to be seen and studied
• Light is directed through a 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
• Getting a visible image requires a very thin sample of biological tissue because light has to pass through the sample and into the lenses of the microscope
• An ideal tissue is the onion epidermis (found between the layers of onions) because it forms a layer just one cell thick
  • Being a non-photosynthetic tissue, onion epidermis is not green as it does not contain any chloroplasts

Apparatus

• The key components of an optical microscope you will need to use are:
  • The eyepiece lens
  • The objective lenses
  • The stage
  • The light source
  • The coarse and fine focus

• Other apparatus used:
  • Forceps
  • Scissors
  • Scalpel
  • Coverslip
  • Slides
  • Pipette
  • Iodine

Method

• Specimens must be prepared on a microscope slide to be observed under a light microscope
• This must be done carefully to avoid damaging the biological specimen and the structures within it
• The most common specimens to observe under a light microscope are cheek cells (animal cells) and onion cells (plant cells)
• 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 to 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
    • Commonly used stains include methylene blue to stain cheek cells and iodine to stain onion cells
  • 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 lowest 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 in case 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

Care must be taken to avoid smudging the glass slide or trapping air bubbles under the coverslip

Light microscopes have a lens in the eyepiece which is fixed and two or three objective lenses of different powers

Viewing an animal cell

• Human cheek cells are a good choice for examination under the light microscope because they are:
  • Plentiful
  • Easy to obtain safely
  • Can be obtained without an overly intrusive process
  • Relatively undifferentiated and so will display the main cell structures

Safety considerations

• Do not perform the sampling on a person who has a cold, cough, throat infection etc.
  • To avoid spreading the infection to others
• Concentrated methylene blue is toxic if ingested
  • Wear gloves and do NOT allow children to handle methylene blue solution or have access to the bottle of solution
    

Apparatus

• Glass microscope slides
  
• Cover slips
  
• Paper towels or tissue
  
• Staining solution
  • Methylene blue solution
    • 0.5% to 1% 
      
    • Dilute according to concentration of the stock solution
• Plastic pipette or dropper
  
• Sterile, individually packed cotton wool buds or swabs

Method

• Brush teeth thoroughly with normal toothbrush and toothpaste
  • This removes bacteria from teeth so they don't obscure the view of the cheek cell
• Take a clean, sterile cotton swab and gently scrape the inside cheek surface of the mouth for 5-10 seconds
• Smear the cotton swab on the centre of the microscope slide for 2 to 3 seconds
• Add a drop of methylene blue solution
• Place a coverslip on top
  • Lay the coverslip down at one edge and then tilt it down flat
    • This reduces bubble formation under the coverslip
• Absorb any excess solution by allowing a paper towel to touch one side of the coverslip.
• Place the slide on the microscope, with 4 x or 10 objective in position and find a cell
• Then view at higher magnification to reveal more detail
• Methylene blue stains negatively charged molecules in the cell, including DNA and RNA
  • This causes the nucleus and mitochondria to appear darker than their surroundings
• The cells seen are squamous epithelial cells from the outer epithelial layer of the mouth

Method for sampling cheek cells from inside the mouth for microscopy

Parts of the cell that can be seen

• Nucleus
• Mitochondria
• Cell membrane
• Cytoplasm

Parts of the cell that cannot be seen with a light microscope

• Ribosomes
• Endoplasmic reticulum
• Golgi
• Details of the nucleus, mitochondria and cell membrane

Using a graticule to measure cells, cell structures and organelles

• In order to take measurements of cells, you need to use a calibrated graticule
• An eyepiece graticule and stage micrometer are used to measure the size of the object when viewed under a microscope

The three lines of a stage micrometer and the 100 division-markings of the eyepiece graticule, as seen if looking down the lens of a light microscope

Producing labelled scientific drawings from observations

• Producing biological drawings of what you see under the microscope is a key skill
• The key is not to try to be too artistic with your drawings – they are supposed to be scientific so make sure you follow the rules

Biological drawings should be as large as possible – aim to take up at least half of the space available on the page with your drawings.

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 cannot be seen
• The treatment of specimens when preparing slides could alter the structure of cells