Light microscopy required practical- B1

optical microscopes

an invaluable tool for scientists- they allow tissues, cells and larger organelles to be seen and studied

what happens in light microscopy

• 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 lense of the microscope

ideal tissue of observing

onion epidermis- found between layers of onions- one cell thick

• being a non photosynthetic tissue, onion epidermis is not green as it does not contain any chloroplasts

key apparatus of an optical microscope

• eyepiece lens

• objective lenses

• the stage

• light source

• coarse and fine focuses

other apparatus used

• forceps

• scissors

• scapel

• coverslip

• slides

• pipette

• iodine

what needs to be prepared first before microscopy?

specimens must be prepared on a microscope slide to be observed uner a light microscope- should be done carefully to avoid damaging the biological specimen and the structures within it

preparing a liquid specimen

1. add a few drops of the sample to the slide using a pipette

2. cover the liquid/smear with a coverslip and gently press down to remove air bubbles

3. wear gloves to ensure there is no cross-contamination of foreign cells

preparing a solid specimen

1. use scissors to cut a small sample of the tissue

2. peel away or cut a very thin layer of cells from the tissue sample to be placed on the slide (using scalpel or forceps)

3. some tissue samples need to be treated with chemicals to kill/ make tissue rigid

4. gently place a coverslip on top and press down to remove any air bubble

5. a stain may be required to make the structures visible

take care using sharp objects and wear gloves to prevent the stain from dying your skin

improving technique 1

1. 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 prevent damage to lens or coverslip incase stage has been raised too high

improving technique 2

to prevent dehydration of tissue:

• add a drop of water to the specimen to prevent cell from being damaged by dehydration

improving technique 3

unclear or blurry images:

• switch to 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

common specimens

onion epidermal tissue

• stained with iodine

cheek cells

• stained with methylene blue

practical using microscope to view specimen

1. clip the slide carefully onto the stage

2. ensure the lowest powered objective lens is over the slide

3. use the coarse adjustment knob to bring the stage up just below the lens

4. look down the eyepiece and gradually move the stage downwards using coarse adjustment knob- stop when image is rougly in focus

5. to bring image into focus, adjust the fine-adjustment knob until a clear image is obtained

how to observe the image with a higher magnification

change the objective lens to a higher power and readjust the stage using the coarse and fine adjustment knobs

viewing an animal cell

human cheek cells are good because..

• plentiful

• easy to obtain safely

• can be obtained without an overly intrusive process

• relatively undifferentiated and so will display main cell structures

viewing cheek cell safety considerations

• don’t perform the sampling on a person with a cold, cough or throat infection

  • to avoid spreading infections to others

• concentrated methylene blue is toxic if ingested

  • wear gloves and do not allow children access to handle the solution

apparatus used in cheek cell observation

• glass microscope slides

• cover slips

• paper towels or tissue

• staining solution

  • methylene blue, 0.5% to 1%.

  • dilute accodring to concentration of stock solution

• plastic pipette or dropper

• sterile, individually packed cotton wool buds or swabs

method

1. brush teeth

2. sterile cotton swab inner cheek surface for 5-10 seconds

3. smear cotton swab on the centre of the microscope slide

4. add drop of methylene blue solution and place the coverslip on top

5. absorb any excess solution using a paper towel

6. place on microscope- either 4x or 10 objective in position and find cell

7. view at higher magnification

8. cells seen are squamous epithelial cells from outer epithelial layer from mouth

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

why methylene blue?

it stains negatively charged molecules in the cell, including DNA and RNA

• this causes the nucleus and mitochondria to appear darker than their surrounding

using a graticule to measure cells, cell structures and organelles

to take measurements of cells, use a calibrated graticule

• an eyepiece graticule and stage micrometer are used to measure the size of the object when viewed under a microscope

eyepiece graticule and stage micrometer

rules for biological drawing

• always draw what you see with a sharp pencil, using clear, unbroken lines

• all structures drawn in proportion

• label all features using straight, uncrossed lines

what maginifaction is animal cells viewed under:

x150 magnification

calculaing size of a single cell

1. clip a ruler or eyepiece graticule on top of the slide

2. view the ruler and slide under 100x objective lens and adjust focus to obtain clearer image

3. line the cells along 1mm andd count the number of cells that fit across that length

4. 1mm = 1000um- divide 1000um by the number of cells.

limitation 1

• size of cells may appear inconsistent in different specimen slides

  • cell structures are 3D- different samples = cut at different planes

  • results in inconsistencies on a 2D slide

limitation 2

• optical microscopes do not have the same magnification power as other types of microscopes and so some structure cannot be seen

limitation 3

treatment of specimens when preparing slides could alter the structure of cells