PAG 2.2 Flashcards
Aim
To use dissection tools safely and effectively to produce stained sections of celery (Apium graveolens var dulce) and to observe and draw these using a microscope.
Equipment
- Microscope
- Microscope slides
- Coverslips
- Fresh head of celery
- 0.5% Toluidine blue
- Scalpel or single edged razor blade
- White tile
- Forceps
- Dropping pipettes
- Tap water
- Eyepiece graticule
- Stage micrometer
- Watch glass
notes
Note: take care when using the sharp dissecting instruments.
Note: The table below shows the colours that you should expect to see in your preparation. Generally non-lignified tissue should be pink/purple and lignified tissue should be green/blue. Both colours tend towards dark blue when over stained.
Tissue Element or Structure Colour
Xylem Green or Blue-green
Phloem Red
Sclerenchyma Blue-green, sometimes Green
Collenchyma Red-Purple
Parenchyma Red-Purple
Transverse Sections procedure
- Obtain a stick of celery (Apium graveolens var dulce) about 5 cm long.
- Rest the stem horizontally on a white tile and use a blade to cut one end as perpendicular to the length of the stem as possible.
- Now use the blade to cut very thin perpendicular slices (transverse sections) of the celery from the edge you have just cut. Do not discard the remaining celery stem – you will take further sections later.
- Use forceps to gently lift the transverse sections into a small beaker containing tap water and leave to soak for 2 minutes.
- Use a stage micrometer to calibrate the eyepiece graticule for use with x4 and x10 objective lenses. Make a note of each calibration for later use.
- Use forceps to gently lift the transverse sections into a watch glass containing toluidine blue and leave them in the stain for 1 minute.
- Use forceps to gently lift the transverse sections back into tap water to rinse off excess stain.
- Place a transverse section on a microscope slide. Add a drop of tap water and a coverslip. Repeat this for your three thinnest transverse sections.
- View under the lowest magnification (x4 objective lens).
- Find the clearest view that shows a variety of structures within the stem and produce a scientific drawing of what you see. Use the graticule and your previously noted x4 objective calibration factor to add a scale bar and work out the magnification of your drawing.
- View under a higher magnification objective lens (x10 objective lens) and find the clearest view that shows one vascular bundle.
- Produce a scientific drawing of what you see. Use the graticule and the relevant calibration factor to add a scale bar and work out the magnification of your drawing.
Longitudinal Sections procedure
- Take the remaining celery stem. Just as in step 2 above cut a short piece off the end perpendicularly to remove the part that has dried out while you have been working on the transverse sections.
- Make another perpendicular cut to produce a piece of stem about 2 cm long.
- Carefully cut the piece of stem in half lengthways i.e. split it down the middle.
- Now use the blade to cut very thin lengthways slices (longitudinal sections) of one of the split halves, starting from the freshly cut inner surface.
- Use forceps to gently lift the longitudinal sections into a small beaker containing tap water and leave to soak for 2 minutes.
- Repeat steps 6 to 12 from the transverse sections method with the longitudinal sections.
- Why is it important to produce very thin slices of plant tissue?
The clearest images are obtained from preparations just one cell thick. In preparations several cells thick the light must travel through the other cell layers, reducing the quality of the image, and at low magnifications several cells could simultaneously lie within the focal plane giving a muddled overlayered image of several cells at once.
- Why is it important that the sections are truly transverse (or longitudinal) and not cut at an angle?
Cutting at an angle makes it much harder to interpret the image especially in vascular tissue where the appearance of longitudinal tubes is altered by oblique sectioning (e.g. where a truly tansverse section would give a clear view of the diameter of the tube, an oblique section of a cylindrical tube will appear as an elongated oval).
- What did you find was the best way to get sections as thin as possible?
Many valid suggestions are possible e.g. concentrating on getting thin sections rather than complete discs (transverse) or strips (longitudinal).
- Why are stains useful in microscopy?
they allow transparent structures to be visualised and by differential staining they allow different structures and cell contents to be distinguished.
- Why is Toluidine blue useful for this protocol?
It is a differential stain allowing us to distinguish different cells. It is also relatively safe and cheap.
