Biology Lab: Microscope slide preparation
Goal: Learn basic skills in slide preparation
Complete Illustrated Guide to Home Biology Experiments, Lab I-2, all four procedures for making wet mounts, smear mounts, hanging drop mounts, and sectional mounts. Instructions for making a microtome are given on p. 39 of IGHBE; you may also find this blog article a useful guide to making a very simple nut-and-bolt microtome you can use with a single-edge razor.
Also complete IGHBE Lab I-3, Staining. Carry out the procedures for simple staining and gram staining and compare your results. Be sure to wear old clothes and work on a covered table: stains really do stain, sometimes permanently. This is a good time to use food-service or exam gloves to prevent staining your fingers.
Alternative approach to slide preparation
Below is the slide preparation lab we used prior to IGHBE. You may find some of the activities useful in gaining further skill levels with your microscope. Doing these exercises is optional!
- Clean slides
- Cover slips
- Newspaper and scissors
- Nut and bolt, large (1/2 inch bolt is good)
- Parafin, beeswax, or candle wax (use parafin or beeswax if you have it; either gives better results than candle wax, which is much softer and so harder to work with).
- Single-edge razor blade (and safe place to store it when not in use)
- Plant sample (green onions work very well)
- Iodine (brown) and/or
- Staining kit
Procedure for basic microscope slide preparation
Be sure to follow the proper safety precautions for working around an open flame, and for working with sharp edges! Also, when using any staining medium, be careful to avoid staining anything but your samples. Always recap your staining bottles tightly after extracting the few drops of stain you will actually need. Spread newspapers out or work over an old cooky sheet to avoid damaging your work surface or the furniture.
- Cut two of the same single letter from the newspaper, magazine, or printer output, and trim down to the single letter. Small a and e are good because they have both left-right and up-down asymmetry.
- Try to view one of your letters through the microscope. What do you notice about how well it focusses? How well does it transmit light?
- Put the other letter on a clean slide, place a few drops of water on it, and view it through the microscope. Is it harder or easier to focus? Does it seem to transmit light better or worse than the dry letter? Be sure to look at the dry one again to confirm your impressions.
- Place a cover slip over the wet sample by placing one edge of the cover slip onto the drop of water and dropping the slip carefully down over the letter. If you get a bubble of air, you may need to remove the slip (carefully, they break very easily) and try dropping it again. Experiment with the amount of water necessary to get a bubble-less slide. Now view the result. Is it better or worse than the wet letter or dry letter without the slip?
Be sure to record your observations carefully. Make tables or take notes so that you can do comparisons later. You may use any format which will capture all the information you need, but here is a suggestion for this section:
|Letter a from magazine: focus
|Can't see anything with light from below; can focus if light above (reflection, not transmission). Can barely distinguish top and bar of e.
|Easier to see for a few seconds, then ink ran and blurred image. Sample destroyed. Need better sample or solution which won't dissolve ink.
|Had difficult with cover slip; broke two and couldn't get bubble out of third. Started over with new letter. Think result is better than wet mount alone, but hard to tell without earlier sample. Loop of e open.
|Letter a from magazine: light transmission
|See notes above on light.
|Light through magazine paper poorly. Second sample was from printer and light passage was dramatically better than dry mount. Can see fibers where ink spread and loop clearly defined (until ink ran).
|Getting better; didn't break coverslip and got all the bubbles out this time without tearing sample. Ink runs less; sample blurred by running ink but edges appear better defined.
You have just made a temporary wet mount. This is a good procedure for samples which you only want to view once.
Procedure for making a thin-slice cross section for observation
- Cut a small sample of onion or other plant material so that it will fit in the cavity made when you screw the bolt 1 turn into the nut.
- Light your candle, and put a drop of melted wax in the cavity. Let it cool and partially solidify.
- Put the sample into the cavity.
- Pour more wax over the sample and let it solidify. Your sample should now be encased in a cylinder of wax.
- Turn the bolt until a small portion of wax extrudes from the cavity.
- Use the razor blade to shave off a thin slice of wax and sample (you may have to shave several times to get down to the sample).
- Float the thin slice of wax-and-sample in hot water to melt the wax from the sample. Use your tweezers to retrieve the sample and place it on the microscope slide.
As with most lab techniques, this one takes practice to achieve a good result. Keep at it until you get several very thin slices of your sample.
Can you distinguish cells in the onion sample? What about membranes, nuclei, or other parts of the cell? Draw a picture of what you see, and identify as much as possible.
Procedure for staining specimens
- If you have a staining kit, follow the directions for staining your plant samples. You can also make a reasonable stain from iodine by diluting it in (distilled water). In fact, a standard stain (Lugol's solution) is a very weak solution of iodine and potassium iodide.
- Be sure to create stains of different concentrations. Record your concentrations and keep track of your samples so that you can compare results.
- Use an eyedropper or glass rod to place a drop of staining solution on several of your samples. Leave some unstained for comparison (this is your control group!).
- View your stained samples after 10 minutes, several hours, and several days. Let your stains dry in a protected place away from direct sunlight when you are not viewing them. Some stains take 72-100 hours to reach maximum absorption.
- If you have tuolene or another adhesive, you can make a permanent slide by using the adhesive instead of water and dropping a cover slide over your sample. Don't be impatient to do this, however! Wait several days until your slides are completely dry.
- Use several different types of samples. Plant samples are generally readily available; you may be able to find wings of dead insects when you visit your field area. Not all stains work equally well.
Please send a description of the staining technique you used and your observations of at least two different samples at different stain concentrations. Take notes while you are working, so that you can accurately reconstruct your procedure when you write up your lab.
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