Microscope 101: How To Properly Prepare a Microscope Slide

Often times, new microscope users are puzzled by how to properly prepare slides. A slide incorrectly prepared can often lead the novice microscopist to believe their new microscope is defective (as we’re more prone to think our equipment is wrong with lower priced purchases than we are to question our techniques–it’s natural to the human psychology).

To help you avoid this situation, and help you get started even faster with your first microscope, here’s my guide to proper preparation of a compound microscope slide.

1. Purchase the correct equipment for your microscope.

Equipment Checklist:

• Blank Glass Slides
• Glass Cover Slips
• Tools (Dropper or Pipette, Tweezers, Scalpel, Vials, etc)
• Stains/Dyes
• Paper Towels

Slides & Coverslips

Microscope slides and coverslips come in a variety of shapes, sizes, and thicknesses. You want to make absolutely sure that the ones you purchase fit the specifications of your new microscope’s objectives.

The objective lenses are the lenses that point down to your sample, and determine what magnification you are at. They have a series of numbers on them, and knowing what each of them mean is important. Below we’ll go left to right, then down to the second row, explaining what each  means in regards to compatibility with equipment.

First Number = 10

This number is the magnification value of the objective. So, this objective magnifies an image by 10x, or 10 times.

Second Number = 0.25

This is the numerical aperture of the objective (commonly called NA rating). It measures the resolution of the objective. So the condenser of your microscope must have at least a 0.25 NA itself in order for this objective to work properly (more on NA in the Glossary).

Third Number = 160

This number is a bit fancy, but it refers to the distance between the nosepiece (where the eyepieces meet the head of the microscope’s ocular tubes) to the top of the observation tube (top of the objective where it meets the objective turret). You want to be sure that the microscope has a tube length that matches your objective, or you will have focusing problems. This is usually ensured by the company that sells the microscope with the objective beforehand.

Fourth Number =  0.17

In this discussion, this is the important number we need to pay attention to. The final number is the thickness of coverslip glass that this objective is designed to use. If you are using a coverslip that is too thick for the objective, then you will not be able to achieve proper focus with the objective. Not enough light will be passed through the coverslip, or the objective lens itself won’t be able to be placed in proper position (if you’re using two slides with the sample in between them, for example–which is not good at all.)

So make sure you have the correct sized glass slides and coverslips for your objectives. Standard microscope slides are 75mm x 75mm x 1mm (length x width x height). Glass cover slips are dependent on your objective, but 0.017mm is fairly common also.

Preparation Tools

Beyond microscope slides, you also may need tools to move or collect your sample.

These can vary from a dropper, to a pair of tweezers, to a scalpel (be careful with sharp edges, especially if learning with children).

What tools to purchase are mostly dependent on what you plan on viewing or collecting to place on the microscope. I personally recommend at least a dropper, tweezers, and a scalpel, as you can prepare just about any simple sample with these three tools.

If you need a child safe kit of microscope preparing equipment, Elenco makes a cost effective kit including everything you need in a child safe package here:

 

2. Pick a sample to make that is translucent.

Because a compound microscope has light from the bottom of the sample, it must pass through the sample in order for the item in question to be visible in the eyepieces. Without any light to pass through the sample, you will simply see a silhouette of the sample, if anything at all.

Great examples of easy to access samples are tap water, perhaps pond water (or puddle water). All of these are translucent, easy to put on a slide, and generally not dangerous or toxic.

Coming soon will be some great example and tutorials on projects that can be done at home for little or no cost, so be sure to subscribe to the blog to get notified of it on the right side of this page!

3. Mount the sample to the slide.

Mounting the Sample

This is best performed with a dropper, if your sample is a liquid, or the tweezers if your sample is a solid.

For liquid samples, take the dropper and place a drop on the center of the slide. From then, place the edge of the coverslip on to the slide perpendicular to the slide (so upright at a right angle), then lower the coverslip down onto the drop carefully to avoid any air bubbles between the liquid and the cover slip. This is called making a wet mount.

You may need to press down on the slip slightly to even it out–to do so, make sure you use a small piece of paper towel and not your finger! Fingerprints can ruin your image.

For solid samples, place a drop of water on the slide as above, and then put the sample inside the liquid. From there, follow the steps as normal.

 

You can also use a pair of tweezers or a needle for this whole process.

Staining or Dying the Sample

Some samples are unfortunately too translucent to be seen even with a microscope. If they have no color to them, then light simply passes through them entirely, making it very difficult to see, if not impossible.

There are advanced microscopes out there still capable of seeing these things (phase contrast microscopes in particular),  however for a novice, these types are typically both out of the budget range and too technical to use. So, we instead use a simple technique to add color to the sample–we stain or dye it!

For a bacterial cell, for example, methylene blue is a commonly used stain. This stain penetrates the cell, staining it a different color than translucent. This stain is what allows us to see the cell and some cell structures without using a phase contrast microscope. The downside in using a stain or a dye is that the sample will be killed by it, so live viewing cannot be achieved with a basic microscope if the sample is translucent.

To stain or dye a microscopic sample, we use a special technique to draw the stain into the sample while mounting it (to both avoid mess and ensure that the stain permeates through the whole sample). This is where the paper towel comes into play (it’s not just for cleaning up!)

So when you have your sample on the slide with a drop of water, and you need to get the stain into the sample, here’s what you want to do.

First, drop a drop of the stain near the edge of the glass cover slip.

On the opposite end of the drop of stain, take a small piece of paper towel and place it up against the edge of the glass slide.

The paper towel will draw in the water from under the cover slip, and in doing so, will pull the stain under the cover slip and into the sample. This also helps keep the stain from being too diluted to be effective as well, so it serves two purposes in one!

4. Place the sample on the microscope stage and center on it with your lowest power objective, and view!

That’s it–you’re all done preparing your first slide!

The process remains the same for any home made sample slides of any kind, so there’s no need to deviate from this guide unless you need a different stain/dye.

 

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