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Making your first slide

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The Theory
To make a slide, we need to create an extremely thin well or cavity on a glass slide in which to place our specimen and our Fructose mounting solution. There are many ways of doing this. We could, for example, use pieces of a broken cover slip and stick these onto the glass surface of a slide. Ultimately, the cover slip would sit-down onto these pieces - which prevent the specimen from being crushed.

I'd like to show you a better and safer way. First of all, take a look at Fig. 2 which shows a sketch I made for you. Does this give you an idea of how we will build a well on the slide?

We will use PVA glue to create a thin wall that will run just inside the edges of the cover slip. PVA glue is ideal for this. It can be painted onto glass and dries very quickly. Some specimens, like my flea - for example, will require a higher wall to ensure the cover glass does NOT sit on the specimen itself. We can build our well to be as deep as we like simply by adding layers of PVA glue after the previous one has had time to dry.

Let's do our PVA glue wall now together. Lay your slide on top of a cover slip, ensuring that the slip itself is centrally placed under the glass slide. By doing this, we can see see where the edges of the cover slip will be when - in a later stage - we fix the slip on top of the slide.

Take a fine paintprush and moisten it. Maybe you should put some water in an eggcup and keep it close to hand for this purpose. Now open up your PVA glue, or lightly squeeze some out and gently dip the tip of the brush into the milky white liquid. You only need a tiny amount of glue. Do not overload the brush. With a steady hand, paint a fine line onto the top of the glass slide just inside where you can see the edges of the cover slip beneath it.

It's important to try and maintain a continuous line of equal thickness and depth all the way round. You can remoisten the brush and reload it with glue as you go, but remember to shake off excess water from the brush before loading it with glue. If you have problems holding the glass slide still and in position on top of the slippery surface of the cover slip, try this instead:-

Put a glass slide on a sheet of paper and draw a line around it with a pen or pencil. Place a cover slip centrally in the rectangle drawn and trace around this with your pen. Now you can use your drawing as a template by laying the glass slide on top of it instead of the cover slip when painting on the PVA glue.

After you have completed the PVA line, put the slide to one side for the glue to dry. It will be ready quicker if you put it somewhere warm. I sit mine on top or near a central heating radiater and they dry in a few minutes. You'll know when the glue is dry because it changes colour from white to nearly transparent.

You may wish to make several slides in your first batch. If so, preapare a few more and put these aside to dry.

Take a look at the specimen you are going to use for your slide. Is it as big as a flea? In my first batch, I made slides of a flea, pollen grains, hairs from a leaf, and a mould found on bramble leaves. The first two required me to build up a PVA wall in several layers to ensure the specimen would 'sit' down properly inside the well. If you fail to put on enough layers, you won't be able to get the cover slip to settle down on the dried glue properly!

Preparing the specimen

There are several ways you can prepare subjects for immersion in the Fructose. One way is to put a tiny amount of the solution into a clean small dish or egg cup. Put your specimen into this, being careful not to introduce air bubbles, and leave it for a few days for some of the water to evaporate - leaving the solution much thicker! If you wish to try this method, stand the eggcup or small dish between two pencils and rest an up-turned tumbler or glass over the eggcup but resting on the pencils. This will allow air to flow through but prevent dust from falling into the solution! It is a good idea to at least soak your specimen in Fructose for a night so that the solution can seep into its structure.

When you have been an amateur microscopist for a while, you will learn different ways of using chemicals to help clean and fix the specimen prior to mounting it in the fructose solution. I am deliberately skipping this important step here because it involves using chemicals which are normally highly inflammable or slightly toxic. As this is your first slide-making try, I wanted to keep the process as simple as possible.

Another method of preparing the specimen for the fructose is to place it into some clean water. For example, I plunged some leaves from a New Zealand bush into boiling water and left them in there overnight. This helps to force air out of the plant which, if not done, would prevent fructose mountant from seeping into the leaf's structure. I scraped a leaf's surface with a razor blade to remove the hairs into a saucer containing a few drops of fructose solution diluted heavily with water. Lets use this as my example for the best way of proceeding.

This is what you do:-

Using a smooth implement like a knitting needle (I use a slim glass rod), insert the tool into your jar of Fructose solution. Remove it and bring its point down into contact with your glass slide, ensuring the point of contact is centrally inside the 'well'. Let the Fructose drop slip down onto the slide.

You need to judge how big a droplet to leave on on the slide. You should be aiming to lay down a drop big enough that when you finally place a cover slip on top, the solution will spread out and fill up the PVA-glue formed well. In the long run, trial and error will enable you to judge this. You simply repeat the action to build up a bigger drop of solution in the well.

Work carefully and precisely trying to avoid introducing air bubbles into the droplet. When you think you have enough solution in the 'well', position your specimen on the slide by pushing it gently into the frustose droplet. I used my glass rod to pick up a drop of watery solution (containing plant hairs) from the saucer and introduced this to the Fructose droplet on the slide.

Lowering the lid
Take the cover slip and, either using tweezers, or holding it gently between index finger and thumb, breathe two or three times rapidly onto its surface. This is to moisten it which will help the Fructose to run without trapping air when we lower the slip onto the slide in a moment!

Working quickly, lower the cover slip down onto the droplet, keeping the edges aligned with the PVA-glue on the slide. Just before the Fructose droplet comes into contact with the underside of the cover slip, release the slip - letting it float down onto the PVA glue walls. The Fructose should spread out across the underside of the slip and fill the 'well' without leaving any air gaps.

Check! If the slip is not resting squarely on the dried PVA glue walls, gently slide the cover slip sideways until it properly position. If you find you have used too much Fructose mountant - causing it to leak out onto the glass slide itself - use moistened cotton buds or tissue paper to gently clean and wipe the spilled fructose away. Ensure you leave the area around the cover glass as clean as possible.

Right, how did you do?

You may find you have air bubbles trapped under the cover glass. If these are very few and not all over the specimen itself, leave it be. If you have a lot of them, it might be best to recover the slide, cover glass, and specimen by placing the whole thing in a saucer of warm water. You can clean the glue from the slide by peeling it off. Dry and clean the glass slide and slip and reuse it again for another go.

This is what may be causing air bubbles to form at this stage:-

Before I move on to the next stage, here is a recap of what you need to do to get to this point:-

(a) Position the cover slip under the slide.

(b) Brush on layer/layers of PVA glue.

(c) Place Fructose Drop and specimen in the well.

(d) Lower the cover slip (after breathing on it).

(e) Ensure slip rests on the 'well' walls squarely.

If you are satisfied with your slide or slides up to this point, put them somewhere warm for a few hours or better still, leave them somewhere dust-free (remember the pencils and tumbler tip) for a day or two.

However, if you are in a rush - like me - just make sure the area around the cover slip is dry, free of water and spilled fructose, and carry on as below.

Sealing the slide

Nail Varnish
You now need to use the nail varnish to seal the cover slip to the slide. Nail Varnish comes with its own little brush. Use this by wiping off the excess when taking the brush from the bottle. Dab a drop of the varnish on the very edges of the cover slip in each of its 4 corners. Each drop should be half on the cover slip and half on the glass surface of the slide itself. The idea here is to fix the cover slip to the slide so that it doesn't move around in a moment when we paint right around the edge of the cover slip with the varnish.

Take care when applying the dabs so as not to move the cover slip or spill varnish onto the central area of the cover slip. Put the slide to one side for 5 minutes to let the varnish dry. Be sure to return the brush to the bottle right away so the varnish doesn't dry on the brush itself.

When the 4 spots have dried on the slide, carefully run the nail varnish brush all around the edge of the cover slip. The edge of the cover slip and the surface of the glass slide up to about 1/8 inch around the slip should be covered in one continuous line. The idea here is to completely seal the edge of the cover slip where it meets the glass slide.

Put the slide to one side and let the varnish dry. It will take about 5 minutes in a warm room, and then you can apply another layer on top of the first one. Work quickly so the solvent doesn't melt the first layer as you sweep the brush over it!

Here's one I made so you can see just how wide a strip of nail varnish to apply. Look at it carefully. Can you see all the elements we have spoken about so far? You should be able to see the PVA glue beneath the cover slip. To do a proper job, write out a tiny label and sellotape it to the slide as I have done here in Figure 5.

Enamel Paint
At this point, your first slide is finished but if you want to do it the right way, you should apply a layer of enamel paint once the varnish has dried. The paint must be painted on as you did the varnish.

Here's one I did! Notice that I didn't apply enough Fructose Mountant in this one which has left some air bubbles under the cover slip. They are well out of the way of the specimens in the middle, so I decided not to recover the slide but keep it instead.

After all that, I guess you want to see what my slides look like under the Microscope. Okay then... pick from these 3 I made. Alas, I can't show you the flea one cos I broke this accidently one night while I was looking at it under the scope: my first broken slide!

That's it for now. Drop in on me again and I'll show you a lot more things we can do together....

regards, Larry

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Note: The first use of Fructose as a Mountant is attributed by Langeron to Michaelis. It does not crystalise, preserves well, and works effectively with specimens stained with carmine or coal tar. Haematoxylin fades slightly.

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