Part II: Glassware and Related Accessories
by Richard L. Howey, Wyoming, US
Once again, I am assuming that you have both a compound microscope and a stereo-dissecting microscope since, in some instances, there are considerable differences in the types of glassware needed for the two instruments. In any case, you will want a good stock of standard 1" x 3" (or 25 x 75 mm.) slides. I keep two grades of slides on hand: 1) the cheapest I can find (these are usually Chinese). They are of reasonable quality for routine work and I regularly use them with the stereo microscope for creating small pools of glue when making dry mounts and for quick test mounts. 2) One also needs a high grade of slides made from optically clear glass and optically flat glass for any preparations which are to be carefully examined with the compound microscope. Such slides will cost 2 to 5 times more than the ones for routine use, but they are a necessity. Remember that slides and cover glasses are part of the optical system and so when you are doing critical microscopy, especially at high magnifications, it is important to have the best quality.
This brings us to cover glasses. There are 3 standard thicknesses (#1, #1½, #2) and they are available in a wide variety of sizes and three basic shapes: square, rectangular, and circular. For the older microscopists, the circles were standard and are still the choice for making fine permanent slides. For the majority of routine work, square cover glasses are used as they are considerably less expensive. Recently, I was looking for some circular ones and the cheapest high quality ones I could find were $42.50 per ounce! And even then they weren't the thickness I desired. An ounce of square cover classes can be purchased for one-fourth of the price. As mentioned before, the thickness of slides and cover glasses is important. The optimal thickness for slides is about 1 mm.; for cover glasses, it is more critical and here the optimal thickness is 0.17 mm (usually the #1½). On some of the first class objectives, you will find engraved "0.17" which is there to tell you that this is the ideal cover glass thickness for that lens and in some of the high power objectives of these manufacturers, you will find a correction collar to compensate for variations in the thickness of cover glasses. If you think this is all too fancy and fussy, consider this: it's rather like buying a Ferrari and never driving it over 40 miles per hour. The Ferrari must have just the right fuel mixture and everything must be properly tuned and then—the result is unexcelled performance and that's what you want from your microscope too.
The more expensive slides and the cover glasses are items which you may wish to save and reuse provided that they haven't been subjected to stains or chemicals. Rinse the slide and/or cover glass in water and then store in a jar of 70% isopropyl (rubbing) alcohol. When you are ready to use one, take it out and clean and dry it with a lint free towel or a lab tissue. The cover glasses must be handled carefully as they break easily. The selection of the size of a cover glass is in part determined by the specimen. For looking at protozoa and algae, I use an 18 mm. Square #1½. Remember that the bigger the cover glass, the larger the area which you have to scan and as you increase magnification, you will dramatically increase the time it takes to scan, since each increase in magnification gives you a smaller area of view.
You may also wish to purchase a few depression slides (these were invented before Prozac). Depression slides have a circular cavity in the center and can be used for larger and/or very delicate organisms which you don't want to risk crushing. You can obtain a dozen single concavity slides for $2.40 or a dozen double concavity slides for $3.00. I would also recommend investing in one or two well slides which are thicker and have a deeper concavity. These are excellent for making hanging drop preparations. They cost several dollars per slide, but are well worth it. Many protozoologists insist that using a hanging drop is the only adequate way of studying amoebae. You place a small drop of a rich amoeba culture on the center of a cover glass, then turn the cover glass over and carefully place it over the well in the slide so that the drop remains suspended and does not touch the slide. The drop must be small so that it does not come in contact with the slide, but also because the larger the drop, the more problem there will be with depth of field when trying to view the organisms.
Many companies sell plastic slides and cover glasses—avoid them unless you're under 7 years of age. You may wish to buy some microscope slide boxes to store permanent preparations which you have made or bought. Buy the ones that will hold 100 slides. You can get a perfectly adequate polystyrene 100 slide box for $5.00 and it's just the right size so that you can store it vertically in a bookcase. You always want to store your slides so that they are flat, otherwise, even after years, the mountant may gradually shift, moving the specimens and ruining the preparations.
For $2.00, you can purchase a 1/4 ounce dropper bottle of immersion oil for use with high power immersion objectives. Only use oil that is specially designed for microscopes! It should have a refractive index of 1.515. If you have an oil immersion lens on your microscope, learn to use it. Once you have positioned the lens into the oil, focus slowly and carefully using fine focus only. If the lens meets resistance, stop immediately and raise the lens up out of the oil using the coarse focus and then begin again by lowering the objective while you are watching it, until it just touches the surface of the oil on the cover glass. At that point, return to the fine focus and look through the eyepieces. If you have a good condenser designed for critical high magnification work, then it too should have a drop of oil on the top lens, which you should raise until the oil drop touches the underside of the slide. The oil drop needs to be small enough so that it doesn't run down the side of the condenser, but large enough so that it spreads smoothly without forming air bubbles which can seriously distort the images. This is especially true if you are using a darkfield condenser which must be oiled to function properly.
Speaking of oil, never, Never, NEVER use oil of any kind on any of the mechanical parts of your microscope. There are special greases which are designed for microscope parts and it is generally best to let a good technician deal with those sorts of problems unless you have acquired expertise and confidence in the relevant areas.
Back to oil immersion. Sometimes amateurs get discouraged from using oil immersion because the procedures do involve some care and can, at times, get a bit messy, since you need to clean up the oil at the end of a session. Use a cotton swab to remove the majority of the oil. Take a clean swab and moisten it with any high quality, specially designed lens cleaner. In this country biological supply houses, Edmund Scientific, and the McCrone Institute all sell such a product. Do NOT use strong solvents and avoid the use of alcohols as they will eventually dissolve the cement that holds the lenses in place. Use the moistened swab to remove the final traces of oil and then use a second and a third dry swab to remove the remains of the cleaning fluid. Finally, a short blast of canned air to remove any vestiges of cotton lint from the swabs. If, on the next occasion when you use oil immersion, you notice that the image is not as sharp as it should be, stop and go through the cleaning procedure again. A good 100x objective can produce stunning results and make you wonder why you procrastinated so long about using it.
Next you will want an assortment of jars, bottles, culture dishes, vials, and tubes for collecting and storing specimens and for keeping solutions of stains and chemicals. To begin with, you can start saving jam jars, pickle jars, olive jars, and especially baby food jars which come in two very convenient sizes. Someone is always having a baby and you can sometimes find a box full of baby food jars at a yard sale for a dollar or two. If you hike very far when you are collecting, you will soon discover that glass is heavy, not to mention breakable, and so you may want to purchase a number of plastic bottles just for collecting. You can get ten 4 oz. ones for $4.80 or ten 8 oz. ones for $6.30 and you can wash them and reuse them. Buy a bottle brush and scrub them under the tap, but don't use soap or detergent. If you can't get them clean to your satisfaction with just water and scrubbing, then use some sodium bicarbonate (baking soda), rinse thoroughly in tap water, then rinse once in artesian water. Soap or detergents may leave residues which could be toxic to the micro-organisms you are trying to collect.
If you are going to isolate specimens and attempt to culture them, you will need some culture dishes and some tubes. Unfortunately, glass culture dishes are rather expensive; the least expensive I have found is $4.50 each for a 3½" x 1½" dish. If you try to keep many cultures going, at that rate you'll soon be taking out a second mortgage on your house. Check your local discount store and see if they have some small, inexpensive custard dishes. Buy a sleeve or two of disposable plastic Petri dishes at $2.25 per 20 and you're in business. You can take a top or a bottom of the Petri dish, whichever fits best and use it as a cover for your custard/culture dish. Another alternative is to use 1 oz. (approx. 30 ml.) or 2 oz. (approx. 60 ml.) jars with low sides and a clear flat bottom. Leave the covers lose, so that oxygen can get in and add water every few days to replace evaporation. The advantage of culture dishes is that they are stackable and that becomes important where space is a crucial consideration.
Disposable glass culture tubes are available in a variety of sizes and depending on size range from $7.00 to $12.50 for 250 tubes. [Caution: These are not test tubes and if you want to heat things in tubes, then you should buy special heat-resistant test tubes.] In the last year or so, I have been using small plastic sterile culture tubes which are polypropylene and are, therefore, chemically resistant. You can purchase 5 ml. tubes, which is what I use, for $36.00 per 500 or 16 ml. tubes at nearly twice the cost $65.00 for 500 which is about 7½ cents per tube for the small ones and 13 cents for the larger ones. For me, the great advantage of these tubes is that they have a 2 position cap, they are very light and convenient to carry in a jacket pocket or in the glove compartment of your car. You are always prepared to capture a sample of whatever catches your eye. When the cap is pushed all the way down, the tube is sealed and you can put the tube with the sample back in your jacket pocket without leakage. When you get back to your lab, put the tubes in a holder or rack and pull the cap up a fraction of an inch to its second position and now it has access to air. a very convenient arrangement!
There is yet a further advantage; I use these tubes to isolate spines, spicules, ossicles, and plates from sponges, soft, corals, starfish, sea cucumbers, etc. You can place a small sample of the material in the bottom of a tube and then add sodium hypochlorite (bleach) or potassium hydroxide or hydrogen peroxide to dissolve away the tissues, leaving only the spines, plates, ossicles, or spicules—ideally. Usually it doesn't go quite that smoothly, but I'll get to that in a minute. When you are using chemicals to dissolve the tissue, have the cap in the open (or up) position which will allow the generated oxygen to escape. If you have the tube closed during this process, the gas may build up enough pressure to blow the top off of the tube creating a mess, not to mention the risk of being spattered by the chemicals. The less than ideal part of this process is that rarely is one treatment enough, unless you use very small samples of tissue that have been very thinly cut and with some types of specimens that's simply not possible.
The good news is that after a day or two, if a second treatment is needed, you can simply use a Pasteur pipet and carefully remove and discard the chemicals and then add fresh ones. Continue this process until you have nice clear spines, spicules or plates. Sometimes I continue this process for several weeks. When your specimens are clean, you can begin the rinsing process by means of which you want to remove all traces of the chemicals so that they don't deposit crystals on your specimens. Use distilled water for rinsing, as you don't want any possibility of any kind of salt deposits or micro-crystals in your preparations.
Again use a Pasteur pipet to remove the fluid, being careful not to stir up the bottom deposit of specimens. Take a fresh pipet, fill it with distilled water and forcefully jet the water into the tube to get maximum agitation to dissolve out chemical deposits. Close the tube and press the cap all the way down. Let the specimens settle back to the bottom. This may take several hours, depending on their size. Repeat this process a number of times, always using a fresh pipet. Exactly how many repetitions are necessary must be determined by trial and error and will depend on the type of specimen. By this point, you will have invested a significant amount of time, so a couple of extra rinses may be well worthwhile. Nonetheless, even if I've done 10 rinses, I still conduct a little test before I go on to mount the specimens. I use a micro-pipet and take a tiny sample from the bottom of the tube, transfer it to a clean slide, and let the distilled water evaporate. I then examine it under the microscope and if I still find crystals, I renew the rinsing process.
Now comes the best part; suppose that you're not ready to make permanent mounts right now because you're off to climb Everest or swim the English Channel—well, all you have to do is put in fresh distilled water, or if you prefer, 90% isopropyl alcohol, push the cap all the way down and you can store your material indefinitely—that's a lot to get out of one kind of tube!
One of my favorite indulgences—completely justified, of course—is dropper bottles. There are Barnes dropping bottles, round dropper bottles, French square ones, micro dropping bottles, Army style dropping bottles (I guess the Air Force, Navy, and Marines don't need dropping bottles). This Army style has a pipet which is a ground glass stopper and when I was young and would see them listed in the catalogs, I would daydream about buying a number of them, but they were much too expensive. Now you can buy imported ones: 1 oz. for $2.60, 2 oz. ones for $2.80, and 4 oz. ones for $3.15. So, I bought a few of the 1 oz. and 2 oz. sizes. I needn't have wasted all that daydreaming as these type are no real advantage except perhaps for solutions of mountants and even then you need to use special silicone valve grease on the stopper or you'll never get it out after the first use or two.
Two other types of dropper bottles which you may wish to consider are:
1) the sort that tincture of iodine used conveniently to come in; namely, a small amber bottle with a glass rod in the lid for applying the iodine in just the right spot. These are handy for applying adhesives, stains, Lugol's solution—an iodine/potassium iodide solution which is very good for demonstrating cilia, trichocysts, flagella, and starch bodies—and all kinds of other uses which will occur to you as you expand your explorations.
2) this is very like the first, except that in this case, instead of a glass rod, you have a small applicator brush and often the bottle is clear glass rather than amber—again, many possible uses.
So, my recommendation is to buy ordinary round dropper bottles or Barnes bottles. The former are inexpensive, serviceable, and can be purchased in 3 or 4 sizes and in clear glass, amber, or cobalt blue. They are very handy for stains, dilute reagents, and culture media. They are not really suitable for strong solvents, alkalis, or acids and even dilute acetic acid will eventually cause the latex stopper pipet bulb to swell.
Beakers and Flasks. These are both useful pieces of glassware, so much so, that some marketing "genius" came up with the idea of combining them and calling them—what else?—Fleakers! (I'm not making this up and personally I think they're rather silly.) There are two seeming antithetical rules for buying flasks and beakers:
1) if you buy a case (usually 6 or 12) of the most popular size (or sizes), you can usually save several dollars,
2) if you buy a case of an oddball size that most people seem to think is non-standard, then you can usually save several dollars. The psychology of marketing has always bewildered me.
Regarding point #1: If you buy a case of twelve 100 ml. beakers, they cost $28.00; but if you buy a case of twelve 250 ml. beakers, they only cost $25.00.
Regarding point #2: If you buy a case of twelve 250 ml. Erlenmeyer flasks, they cost $25.75; but if you buy a case of twelve 300 ml. Erlenmeyer flasks ( an "oddball" size), they only cost $19.80 as a "special buy", meaning "everyone thinks this is an oddball size, so we've reduced the price." Go figure!
If you decide to buy beakers or flasks, get ones that are heat resistant, so that you can boil up a batch of okra to make a slimy soup for your lunch (the protozoa won't eat it, they have better taste.)
You will probably want to acquire 2 or 3 graduated cylinders for making up various kinds of solutions. Here I recommend spending a bit extra and buying good quality glass cylinders. A good start is to get two 100 ml. ones ($9.50 each) and two 10 ml. ($5.50 each). Why two? Use one only for measuring chemical solutions and label it clearly and the other only for solutions for culture media. If you try to use one for both purposes, there is the risk of contamination. Even if you carefully rinse the cylinder, minute amounts of chemical may remain which could be transferred into a culture medium you are making up. The amounts may seem insignificant to us, but remember that we are dealing with micro-organisms which may be highly sensitive to certain substances. There are many toxic chemicals where the human tolerance levels are measured in parts per million and some which are designated as having no acceptable tolerance level in humans. So, it's better to take the precaution rather than end up with dead cultures or mutant microbes.
The variety of laboratory glassware is simply astonishing and there is always the temptation to buy items that look like they came out of an old Boris Karloff Frankenstein film, but start with the essentials and add the other more specialized items as you discover a need for them.
In the next article, I will discuss some of the more specialized kinds of glassware that you may wish to consider eventually and also some other basic sorts of laboratory apparatus. People from Glasgow are known as Glaswegians; those of us who are microscopists and have a passion for tiny glassware might be described as Wee-glassigians.
Prices are once again taken from the 2000-2001 Cynmar Scientific Equipment Catalog. For additional information see the endnote in Part I of this series.
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