Skip to 0 minutes and 11 secondsThis staining technique shows the location of the different tissues present in a cross section of a stem. Toluidine blue stains lignified and non-lignified cell walls a different colour. Once prepared this slide can be used by students for the development of their scientific drawing skills and their use of the eyepiece graticule to estimate the size of the cell. Take a stick of celery about 5cm in length. Use a sharp blade to cut one end of the stem as perpendicular to the length of the stem as possible. Cut very thin slices of the celery from the edge that you have just cut.
Skip to 0 minutes and 55 secondsUsing forceps gently put the slices of celery into a small beaker of tap water and leave to soak for 2 minutes.
Skip to 1 minute and 7 secondsUse forceps to lift the celery out of the beaker and place in a watch glass containing toluidine blue and leave for 1 minute.
Skip to 1 minute and 21 secondsUse forceps to put the celery into tap water again to rinse off the excess stain.
Skip to 1 minute and 30 secondsThen place the celery onto a microscope slide. Carefully add a drop of tap water and lower the cover slip onto the celery.
Skip to 1 minute and 50 secondsWhen looking through the microscope you will be able to identify different tissues more clearly with the different colours.
Skip to 2 minutes and 6 secondsYou should always carry a microscope with two hands, one holding the arm of the microscope, and the other under the base – never just by the arm. Put the microscope on a clean, flat surface. Plug it in and switch it on. Pick up the slide using only the edges, so that you don't press fingerprints onto your clean slide, and place it on the stage. Make sure you’ve got the slide in the centre and, if you can see it, line the specimen up with the hole in the middle of the stage. Then secure it in place with the 2 stage clips. Turn the microscope on. A small circle of light should appear in the centre of the slide.
Skip to 2 minutes and 51 secondsIf not it may be because the diaphragm is closed. The diaphragm is underneath the stage and allows you to adjust the amount of light coming through. You’ll need to have the maximum amount of light coming through when you start focusing, so turn the lever or disc on the diaphragm so that it is open all the way.
Skip to 3 minutes and 19 secondsMost microscopes have three rotating objective lenses which magnify the specimen. Each one is colour coded with the power written on the side. The lens with the lowest magnification is the smallest, and this is usually x4. This one is x10, and this one x40. Be careful never to touch the lenses with your fingers as the oils on your skin can damage them. If you need to clean them, use a lens tissue with ethanol. Always start focusing with the lowest power lens - so rotate the objective lenses round until the lowest power lens is above the middle of the slide, and you feel it click into place.
Skip to 4 minutes and 5 secondsThis lens gives you the widest view of the specimen and makes it easier to bring it into focus. For this reason it is sometimes called the scanning objective. The eyepiece here at the top of the microscope also has a 10x magnification. So this means the specimen will first be magnified by the objective lens by x4, then by the eyepiece by x10, giving a total magnification of x40. The x10 objective would give you a total magnification of x100 and the x40 objective, a x400 magnification.
Skip to 4 minutes and 52 secondsThere are two focusing knobs on a microscope. The large one is for positioning the stage and for course focusing. The small one is for fine focusing. Use the course focus knob to carefully raise the stage. So that the slide is near the objective lens, but not touching it. Look into the eyepiece and use the coarse focus knob to slowly move the objective away from the stage while you look into the eyepiece. You should see the image come into focus.
Skip to 5 minutes and 28 secondsNow you can use the fine focus to make the image sharp.
Skip to 5 minutes and 35 secondsYou might want to adjust the diaphragm below the stage now – reducing the light can make the image look less washed out. Now you can switch to a higher magnification to see more detail in your specimen, but be careful switching the lenses as the next one up is longer and could crack your slide if it’s too close.
Skip to 5 minutes and 59 secondsYou may need to use the course focus knob to move the stage lower and then bring it back up to the lens before you view the slide. Always use the fine adjustment knob when working with the higher objectives, such as the 100x, because the coarse knob moves the stage and you can damage the lens if it hits the slide.
Developing microscope skills
This video covers slide preparation and microscope operation. Feel free to download and use this video to support your students.
Sometimes microscopes are parfocal, which means that when you switch to the next objective lens there is no need to move the stage and re-focus as the specimen will remain in focus. In the previous step you will have broken down an example practical into the skills needed.
Toluidine blue stain
The table below show 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|
|Sclerenchyma||Blue-green, sometimes green|
Source: Table from Parker et al. (1982).
If you don’t have a full class set of microscopes, what approaches could you use to support students to learn to use this equipment and skills such as scientific drawing and measurement that microscope work develops?