This lesson is called observing the structure and distribution of stomata practical, and it's from the unit, transport and exchange surfaces in plants.

Hi there, my name's Mrs. McCready and I'm here to guide you through today's lesson.

So thank you very much for joining me today.

In our lesson today, we're going to use a light microscope to observe the imprints of stomata in leaves and investigate the distribution of stomata on leaves.

So we've got a partial practical lesson today.

I hope you're looking forward to it.

Now, we're gonna come across a number of key words today and they're listed up here on the screen for you.

Now, you may wish to pause the video and make a note of them, but I will introduce them to you as we come across them.

Now in our lesson today, we're going to first of all look at how a light microscope works before we use a light microscope to observe stomata and their distribution in the leaf.

And then we'll move on to comparing the distribution of stomata in different leaves and explaining it.

So are you ready to go? I certainly am.

Let's get started.

Now, most cells are too small to be seen with the unaided eye.

That's not true of all cells, but most of them are.

And in order to be able to see them, we will need to use a light microscope to sufficiently magnify it and be able to see the cells themselves and maybe some of the detail inside them.

Now a light microscope has various parts to it.

It has an eyepiece lens, objective lenses, a stage, a light source, a coarse focus wheel, and a fine focus wheel.

And each of these parts have different roles.

So the eyepiece lens contains a times 10 magnification and allows us to view the specimen.

The objective lenses usually include three different magnification powers of times four, times 10, and times 40.

And these allow us to view the specimen at different levels of magnification.

The coarse focus wheel allows us to adjust the focus in large increments and the fine focus wheel allows us to adjust the focus in small increments.

The light source may be an internal lamp or a mirror to reflect an external lamp, but either way it shines light up through the specimen and up through the objective in the eyepiece lenses so that we can see the specimen.

And finally, the stage is where the specimen is placed.

So which label or labels indicate a part of the light microscope used to illuminate the specimen? I'll give you five seconds to think about it.

Okay, you should have chosen part b, well done.

Now, when you are using a light microscope, there is a very specific method that you need to follow.

Firstly, you need to turn the objective lenses, that's part b in the diagram to the one with the lowest magnification.

That's the shortest lens.

Then you need to put your specimen onto the stage, part c in diagram and secure it under the clips.

After that, you can turn on the light source.

If your microscope has a mirror, you'll need to angle it so that it best reflects light up through the specimen.

And be careful because lamps get hot and therefore you need to avoid touching the lamp either within the microscope or the external lamp when you are using it, when it's turned on.

And you must also be careful, especially if you are using a mirror to reflect light from an external source.

You must never use sunlight for this purpose because it may blind you.

Once you have that part of the microscope set up, you can then look from the side and turn the coarse focus wheel so that the stage moves up towards the objective lens to as close as it possibly can be without them touching.

Now observe from the side so you can see when to stop.

This is so that you can stop the slide from being smashed into the objective lens, which may well damage the lens or the specimen itself.

And once it's set up, you can then look down the eyepiece lens and turn the coarse focus wheel so that the stage moves away from the objective lens, bringing it into focus.

And if you need to use the fine focus wheel to bring it into sharp, clear focus, do that once you have got the large amount of focusing done with the coarse wheel.

So watch the video to see how to safely and correctly use the light microscope.

Okay, let's quickly check our understanding.

So you should look through the eyepiece lens when moving the stage towards the objective lens.

True or false? So you should have said that that is false, but can you explain why? So you might have explained that by saying that you should look from the side when moving the stage towards the objective lens.

And this is because this allows you to be able to see when to stop moving the stage upwards so that you don't smash it into the objective lens and damage either the lens or the specimen itself.

Well done if you got both of those right.

So what I'd like you to do now is to firstly write a method for setting up the light microscope.

Make sure you use all of the keywords from the diagram in your method and then I'd like you to write two pieces of safety advice for anybody following this method.

So pause the video and come back to me when you are ready.

Okay, let's check our work.

So firstly, with your method, you should have started by saying that you need to turn to the objective lens with the lowest magnification.

Then you need to put the slide onto the stage and fasten it with the clips.

Then turn on the light and look from the side.

Use the coarse focus wheel and move the stage up so that it is as close to the objective lens as possible.

Then you can look through the eyepiece lens, turn the coarse focus wheel and move the stage away from the objective lens, bringing the specimen into focus.

Then finally, if you need to focus it further, use the fine focus wheel to make the image nice and sharp and clear.

So check over your method, make sure you've got all of those salient points well done.

Then I asked you to write two pieces of safety advice.

So you might have included that lamps get hot.

So you mustn't touch the microscope's light whilst it is switched on, or an external lamp for that matter as well, that you must look from the side while you are moving the stage upwards so that it doesn't smash into the objective lens.

Or you might have included that if the microscope has a mirror, you mustn't use it to reflect direct sunlight because this can damage the eye or that you need to carry the microscope with both hands and place it on a firm flat surface.

So again, check over your work.

Have you got two of those key points? Well done if you have.

Okay, let's move on to the practical part now by observing stomata and how they are distributed across a leaf.

Now, leaves have holes, pores, in them called stomata.

So one stoma, many stomata, and you can see them in the pictures there.

Now each stoma, each hole, each pore is surrounded by two guard cells that change shape and open or close the pore.

So you can see the guard cells in that electron micrograph.

They are sausage shaped cells and they form either side of the hole which sits between them.

Now when it is light and sunny, the stomata open and this allows gases to diffuse in and outta the leaf.

So it allows carbon dioxide to enter the leaf and it allows waste oxygen gas to diffuse outta the leaf.

And this allows photosynthesis to take place within the leaf.

Now the structure of stomata can be observed using a microscope.

So if we look at this electron micrograph of the stomata in a leaf at two and half thousand times larger than real life, we can see the stoma in the middle, the hole in the middle, nice and big surrounded by guard cells on either side, which are making the hole.

But this is observed with an electron microscope at very high magnification and this is not something that we can do within the school laboratory.

So quick check.

Which piece of apparatus was used to produce this image of a stomata? Was it a, an atomic microscope? B, an electric microscope, c, an electron microscope or d, a light microscope? I'll give you five seconds to think about it.

Okay, so you should have selected the electron microscope.

Well done.

Now it's really difficult to observe stomata using a light microscope in school because the leaf is rather thick.

And what that means is the light can't really easily penetrate through all the layers of the leaf and we can't really easily focus on the lowest side of the leaf in order to be able to see it nice and clearly and to see the stomata and the guard cells there.

Instead though we can circumvent some of these problems if we paint the surface of the leaf using clear nail varnish.

And then if we let that dry we can apply a piece of sticky tape to the nail varnish, carefully peel it off, the nail varnish will come off with it.

And in the nail varnish, the dried nail varnish will be the imprints of the underside of the leaf, including the stomata.

Then we can use the piece of sticky tape, attach it to a slide, put it onto the stage of a light microscope and observe that and we'll be able to see the imprints of the stomata from the leaf rather than seeing the stomata themselves directly.

So we're only seeing the imprints, but we get a rough idea of what they look like and we get to see how many of them there are as well.

Now this is a light micrograph of stomata viewed in that method.

So this is the imprint of the stomata from the underside of a leaf that have been retrieved by painting nail varnish onto the leaf and then peeling that nail varnish off of the leaf using sticky tape.

Then if we put that onto the light microscope slide and view it using the light microscope, that's the kind of thing that we see.

And this is at times 40 magnification, so it's not a massively magnified, it's just the lowest power magnification objective lens with the eyepiece lens to give us this magnification.

So we can use these imprints to see how many stomata are present in this section of the leaf.

So we can count 'em up, we'll do a tally, so whenever we see them we'll tally them up.

So that's the first five.

Then the next five, there's the next five, another five, five more, another five, five more and two remaining.

And if we add all of those up, we can see there's five, 10, 15, 20, 25, 30, 35 plus two is 37.

So there are a total of 37 stomata in this field of view.

So how big is this field of view? Well, we can measure the radius at 0.

4 millimetres and then using pi r squared to calculate the area of the field of view.

If we use 3.

14 as our value for pi and times 0.

4 by 0.

4 to get r squared, then we've got 3.

14 times 0.

4 squared, which gives us a total area of 0.

5024 millimetres squared.

Now we know that within that area there are 37 stomata.

And so what is the distribution, we can estimate the distribution of the stomata by saying that there are 37 stomata in 0.

5024 millimetres squared.

And if we do that division, 37 divided by 0.

5024, we'll get 73.

6 stomata per millimetre squared.

That's our average, our estimation, and it's an estimation because we've not counted every single stomata on the leaf.

So let's quickly check.

Aisha is observing stomata and she observes 47 stomata in this section of the leaf, which has an area of 0.

785 millimetre squared.

So what is the density of these stomata? I'll give you five seconds to think about it.

Okay, so you should have remembered that we are dividing stomata by millimetre squared and that's evident in the units of the measurement, stomata divided by millimetre squared and therefore we need to put the number of stomata first divided by the area viewed.

So that means that c is correct.

47 is the number of stomata divided by 0.

785 millimetres squared, which is the area of view that gives 59.

9 stomata per millimetre squared.

Well done if you chose that option.

Now in order to view the imprints of stomata, you will need some specific equipment.

Firstly, you're going to need a leaf with which to do this investigation.

You'll need some clear nail varnish with its brush, of coarse.

You'll need some clear sticky tape, a slide onto which to stick that sticky tape, and of coarse your light microscope.

So the method for observing the imprints is as follows.

And firstly, before you do anything, you must put eye protection on to keep your eyes safe whilst using nail varnish.

So use the brush and paint the nail varnish onto one surface of the leaf, allow it to dry completely and when it is dry, then place clear sticky tape over that layer of varnish, make sure it's fastened and attached to the varnish properly, and then carefully peel the varnish away and that should come away from the leaf.

The leaf should remain, it shouldn't peel any of the leaf away and it should take the nail varnish off of the leaf as a clear layer and you'll need to stick that cellar tape with nail varnish onto a microscope slide.

Then once you've got that on the slide, you can use the microscope to observe your specimen.

So who summarises the method correctly? Jacob says, "Paint the leaf with nail varnish and observe using a light microscope." Sam says, "Stick clear tape onto a leaf, then varnish it, then transfer it to a microscope slide." And Sophia says, "Varnish a leaf, dry, stick clear tape on it, then transfer the tape to a microscope slide." But who is correct? I'll give you five seconds to think about it.

Okay, you should have said that Sophia is correct.

Well done if you did.

So what I would like you to do is to collect a leaf, then follow the method on your worksheet to create imprints of stomata using the nail varnish.

Now, whilst you are waiting for the nail varnish to dry, practise calculating the density of stomata per millimetre squared using the image and use 3.

14 as your value for pi when you are calculating the area of the view.

Once the nail varnish is dry, then you can follow the method on the worksheet to observe the imprints of the stomata and to count the number of stomata present within the field of view.

So pause a video and come back to me when you are ready.

Okay, let's see how you got on them.

So whilst you are waiting for the nail varnish to dry, you should have calculated the density of the stomata in the image.

So to calculate the area of the field of view, first you need to do pi r squared.

Well r is 0.

6 millimetres, so you are doing 3.

14, which is pi times 0.

6 squared, which gives a value of 1.

1304 millimetres squared.

So that's the area of the field of view.

The number of stomata include five, 10, 15, 20, 25, 30, 35, 36, 37, 38.

So there are 38 stomata in 1.

1304 millimetres squared.

And that means that the density is 38 divided by 1.

1304 making 33.

6 stomata per millimetre squared.

Well done if you got all of those correct and well done, particularly if you showed your workings.

Now the measurements that you've taken for the leaf that you've been studying will be different and you'll be able to include that within your analysis going forward.

Okay, let's move on to comparing the distribution of stomata and trying to explain why this is the case.

So we can compare the distribution of stomata between different plants and between the top and the bottom surface of the same leaf.

So if we look at this data here, we can see that all of these four different plants have different densities of stomata in their leaves and they also have different densities of stomata on the top surface of the leaf compared to the bottom surface of the leaf where there are many fewer stomata on the top surface of the leaf compared to the bottom surface.

Now these are only estimates of the density of stomata on a leaf.

These are not accurate direct measurements, and this is because we haven't counted every single stomata across the entire leaf.

All we've done is take a sample sample and count those.

So this is not an absolute accurate measurement of every single stomata that is present on the leaf.

That is not what we have done.

Therefore, the density might be different in one part of the leaf compared to another.

And we don't know that because we haven't gone and checked, we haven't counted stomata across the whole leaf.

We've only looked at it from one specific area.

Furthermore, it's actually really difficult and perhaps you have found this, it's actually sometimes really difficult to work out whether it is a stomata that you are looking at or the imprint of something else, or maybe just a bit of a blob on the nail varnish instead of the stomata.

And therefore knowing that you've counted every single stomata not missed any or not counted as stoma where there wasn't one, you don't necessarily know that.

So actually counting the stomata on the leaf, all we have done is provide an estimate of the density of the stomata rather than an absolute accurate measurement.

So how could we improve our estimation? How could we make our results more accurate? Well, maybe you said we could count a larger area, absolutely, we could count the whole leaf for instance, or a larger part of the leaf, or maybe we could count several areas on the same leaf and then calculate a mean from that and see what that means, whether the density changes based on the mean that we've calculated instead.

So there are things that we can do to improve the accuracy of our estimate.

Now we've got this data, so we need to be able to describe it.

Now describing our data, because we've got lots of data from different leaves and from different parts of the same leaf, means that we can draw some comparisons.

This means that we're going to point out the similarities and the differences between parts of the same leaf and between the same parts of different leaves.

Now by doing this we can use words like more or less, or we can use comparative words that end in er for instance, larger or EST, such as largest.

And these are all words that we can use when we're drawing a comparison between different items. So for instance, we could say the bottom surface of each leaf has more stomata than the top surface of each leaf, or maybe we could say the density of stomata is approximately five times larger on the bottom of the leaf than it is on the top of the leaf.

Or perhaps we could compare plant to plant and say, well, plant D has fewer stomata than the other plants.

So these are all descriptions.

We are using the data and saying what we see.

And in this context we're comparing, we are saying what we see and how they are similar or different from each other.

Once we've described our data, we can then try and go on to explain it.

An explanation is suggesting reasons for the patterns and the trends, not just describing them, but actually saying why those patterns or trends may well exist.

And in order to do that, we need to use scientific knowledge and understanding to try to make sense of the data and the patterns and trends that we are seeing within it.

So how can we use our knowledge and understanding to explain why there are differences in our data, why there are more stomata on the lower surface of the leaf than there are on the top surface of the leaf, and why there are differences between different plants? Well, let's have a look.

So if we take a slice through a leaf, we can see that there are lots of different cells there and the stomata are present in the bottom of the leaf on the lower surface.

Now through stomata water is lost in this process called transpiration where water vapour diffuses out of the open holes.

Now the surface of the leaf, the top surface of the leaf, that is where the light is shining onto it.

And by that happening the leaf surface gets warm.

If you ever just go out into the garden or around the school field for instance, and touch leaves that have been sat in sunshine, you'll feel that they are warm.

So the light that's falling on the leaves is providing energy for photosynthesis, but it's also warming the leaf.

Now when water gets warmer, the rate of evaporation, the rate of diffusion, and therefore the rate of transpiration will increase.

So higher temperatures increase transpiration rate and that means that water loss from the plant will increase further.

So to reduce the amount of water loss that's occurring from the plant, plants locate their stomata in the lower side of the leaf rather than in the top part.

This is because the top part is warm and being warmed by the sun, whereas the lower side of the leaf is in the shade and is therefore much cooler.

And because it's much cooler, transpiration rates will be lower and plants will reduce the amount of water that they lose.

So we can explain the difference between the number of stomata found in the top surface of the leaf compared to the number of stomata found in the bottom surface of the leaf by explaining the fact that the bottom surface of the leaf is in shade, therefore it is cooler and therefore the rate of transpiration will be lower.

So by locating stomata in the lower side of the leaf, they lose less water.

So who has provided an explanation for the data? And Andeep says, "Plant D has fewer stomata than plants A, B, and C." Izzy says, "Plant D is to reduce water loss to very low levels." And Laura says, "The density of stomata in plant D is about eight times lower." But who has provided an explanation? I'll give you five seconds to think about it.

So you should have said that Izzy has provided an explanation.

She's trying to say why there are differences in the number of stomata, whereas both Andeep and Laura have only provided descriptions of the data and not an explanation.

I hope that makes sense.

So what I'd like you to do now is to firstly explain why plants have fewer stomata on the top surface of the leaf compared to the lower surface of the leaf.

And then I'd like you to consider these two plants.

Firstly, the Agave plant which lives in hot desert habitat where there is little water and strong intense sunlight.

And as a result, the Agave plant has a density of stomata of five stomata per millimetre squared.

I'd like you to compare that to the oak tree, which lives in the UK, which has a rather wet climate and plenty of water, therefore.

So the density of stomata in oak leaves is 500 stomata per millimetre squared.

So what I'd like you to do is to compare these two plants and describe and explain the differences in the density of the stomata between the agave plant and the oak tree.

So pause the video and come back to me when you are ready.

Okay, let's check our work then.

So I firstly asked you to explain why plants have few stomata on the top surface of their leaves.

So you might have said that the top surface of a leaf is warmed by direct sunlight and higher temperatures increase the rate of transpiration and therefore the rate of water loss through open stomata.

And because of the sunlight on the surface of the leaf increasing the temperature leaves are adapted to have fewer stomata on the top surface of the leaf.

And this reduces water loss by transpiration.

So check over your work.

Have you got that idea? Well done if you have.

Then I asked you to compare the Agave plant with the oak tree and describe and explain differences in density of stomata between these two plants.

So you might have described the differences along the lines that the density of stomata is 100 times lower in the Agave plant than in the oak tree, or maybe the density of stomata is 100 times higher in the oak tree than the Agave plant.

The explanation for this is the Agave plant is adapted to reduce water loss by having very few stomata, and what this means is that it loses less water, which helps it to survive in that hot dry desert.

Of coarse, you might have mentioned also the oak tree, which has lots of water present around it and therefore doesn't need to worry about conserving water.

Certainly not in the same way as the Agave plant does.

So again, just check over your work.

Have you got all of those salient points? And well done if you have.

Okay, we've reached the end of our lesson today and we've seen that with the light microscope, there are two lenses and two focus wheels, which enable us to view specimens at different magnifications and at clear focus.

And we've used the light microscope to observe the imprints of stomata by painting nail varnish onto the back of a leaf and then transferring that nail varnish onto a slide using sticky tape.

When we were using the light microscope to observe the stomata, we were able to count them to measure the density of the stomata and measure the distribution across the leaf.

And we also have been able to compare the distribution of stomata to different surfaces of the leaf and between different leaves.

Now we've been able to explain our observations of the density and distribution of tomato using ideas about water loss and transpiration rate, and minimising these things, especially for plants which are living in hot dry conditions.

So leaves are adapted to reduce water loss by having fewer stomata on the top surface of the leaf than on the bottom and therefore reducing the rate of transpiration.

So thank you very much for joining me today.

I hope you've enjoyed your practical lesson, and I hope to see you again soon.

Bye.