Using a Microscope
On the one hand it is frustrating to discover, when trying to identify a mushroom you have found in the woods, that microscopic analysis is required in order to separate it from other mushrooms. On the other hand, microscope work can be fun and rewarding. Many of the techniques required are not that difficult to learn, and a decent used microscope can often be picked up for under $400. The best part is, if you are obsessed with mushrooms and live in an area where they do not come up year-round, you can spend quality time with your little friends in the off season.
Studying mushrooms with a microscope takes practice, but new frontiers open up at each level. Beginners will find that simply looking at a mushroom's spores is fascinating, and often helps substantially in the identification process. More advanced microscope skills lead not only to facilitated identification but, sometimes, to stunning views and gorgeous microstructures (would you believe cup fungi, species of Inocybe, and species of Pluteus, for example, are often beautiful?).
Plus, how cool will you be? "What are you doing?" someone will ask. "Mushroom microscopy," you'll reply with cavalier confidence. "Mushroom. Microscopy."
What kind of microscope do I need, and where do I get it?
You'll need a pretty good microscope. The many microscopes in people's basements and closets—forgotten gifts to 12-year-olds whose enthusiasm dwindled a few weeks after Christmas—are usually toys, great for looking at hair follicles and the like, but usually not powerful enough to get you very far with mushrooms. Some of the larger microscopic structures of mushrooms can sometimes be seen with these "garage-sale microscopes," however, and if you'd like to whet your appetite I recommend trying to view the asci and spores of morels, which have particularly large microscopic features, by slicing a thin section of the spore-bearing surface and using a tap-water mount (equipment required: garage-sale microscope; slide; cover slip; sharp razor blade; tap water; morel).
But if you want to go beyond having a little fun just seeing some or the larger microscopic features of mushrooms, you will need a microscope with an oil immersion lens, capable of magnifying things about 1,000 times—and the eyepiece of the microscope will need to have an ocular micrometer in it so that you can measure things. You will want an electric light source, controls to move the stage (the platform that holds the slide) mechanically, and a fine-focus knob (not just a single, coarse-focus knob).
You could buy a new microscope, of course. You can probably find new microscopes online that meet the requirements above for under $400 at the time of this writing (2019). However, and this is a big however, you cannot find a good new microscope for that price. With microscopes it is all about the lenses, and there are many knock-off, substandard lenses and microscopes out there. If you're going to make this investment, I recommend you get good lenses. Nikon. Olympus. Zeiss. Leica. We're not talking about handbags, where the reasons for getting the real Luis Vuitton as opposed to the "Luis Vuitton" the guy in the parking lot at the Leaning Tower of Pisa wanted to sell you are mostly social and legal. We're talking about the quality of your views of mushrooms.
. . . which probably means, if you're like me, that you can't afford a new microscope, since the good ones are much more than $400. It's a good thing, then, that a used microscope will work perfectly well, assuming it's in good condition. Good used microscopes can be found online, but I suspect that your best bet is to contact someone in one of the life science departments at a local university or community college. Former Biology 101 microscopes are not too hard to get hold of, and are often relatively cheap.
Calibrating a Microscope
The little ruler in the eyepiece of your microscope is divided up evenly into units—but those units do not necessarily correspond to anything in particular. You will need to compare the units on your microscope to the units on a special slide (called a "stage micrometer") that has known values on it. Most microscopic mushroom measurements are expressed in "micrometers," also called "microns." One micrometer is equal to 0.001 millimeter; the symbol for a micrometer is µm.
"Calibrating" your microscope is simply the process of comparing your yard-stick's units to the predetermined units on a special slide. Borrow the stage micrometer slide, if possible; you probably won't need to calibrate your microscope more than once. Once you have a conversion basis, you may need to do a little math every time you measure something. For example, if each unit on your yardstick equals 1.07 µm when you're using the highest magnification, a structure that measures 10 units long is 10.7 µm (roughly 11 µm) long. Or, if you're lucky, you may find that your highest magnification conversion is 1:1, in which case the math may not be needed (however, you will still need to calibrate your other lenses).
What other equipment will I need?
The obvious microscope things: slides, coverslips, extra bulbs, lens paper, and immersion oil. You will also need some very sharp razor blades, and some chemicals (see below).
Slides and coverslips are available from many sources, online and in brick-and-mortar stores. Your experience and preferences will dictate what kind of slides and cover slips you need. Glass cover slips are wonderful, but more expensive and easily broken. Plastic, disposable coverslips are much cheaper, but the view through them is inferior.
You will need lens paper to clean your oil immersion lens after each use. There are many online suppliers; also try your local camera shop, or your optometrist. Immersion oil can be purchased online, as well.
Chemicals, Reagents, and Stains
While water can be used to mount mycological specimens, features are often difficult to see without using stains and/or reagents that mycologists use. The bad news is, some of these chemicals can be very difficult to obtain (even for mycologists). At a minimum, you will probably need the chemicals below.
KOH (potassium hydroxide) is a strong base often used to study mushrooms. Although it is sometimes difficult to obtain, KOH can usually be purchased without too much difficulty. Several major online vendors have it available. KOH is used in a 2 percent aqueous solution as a mounting medium for microscopic examination of mushrooms. As a medium it often does a good job of clarifying mounts and making tissues and structures visible. It has its drawbacks (for example it tends to swell some structures) but it has been used for so long by mycologists that using it is necessary if one wants to compare data with their work.
Melzer's reagent is an iodine-based stain regularly used in mycological microscope work to better see tissues and to determine whether spores and tissues are amyloid, inamyloid, or dextrinoid. It is unfortunately extremely difficult to obtain. Melzer's contains water, iodine, and potassium iodide, all of which are fairly easy to get hold of—but it also contains chloral hydrate, which is a controlled substance. Thus, you won't be able to buy it easily. Virtually your only option is to beg it from a professional mycologist. Even mycologists have difficulty obtaining Melzer's, however, and if the mycologist you know can't afford to provide you with some of her precious supply (or if you cannot find a mycologist), your last resort is to try explaining your situation to your doctor and getting a prescription for chloral hydrate (not likely; it's a date-rape drug) or for Melzer's reagent itself, which a compounding pharmacist could mix according to the formula below, and which your doctor would need to write on the prescription (still not very likely, but not unheard of).
Water: 20.0 gm
Viewing and Measuring Spores
Observing spores, and measuring them, is probably the easiest of the various microscope routines involved in mycology, and it's a good place to start. You will be surprised at how useful it can be in the identification process to know whether a mushroom's spores are smooth, "ornamented," "ridged," or "pitted" (etc.), and to know their dimensions.
You want to measure mature spores since, like other parts of a mushroom, spores are little before they are big. The spore sizes quoted in field guides and in technical mycological literature represent mature spores—which, by definition, have fallen off the mushroom. This means that a spore print should be the source of your material. Take a clean, dry razor blade and scrape lightly on the spore print, collecting spore dust on the blade's edge. If you have made a spore print on paper, don't scrape too hard; you will be scraping paper particles as well, which will confuse things under the microscope. I place a piece of glass underneath the paper before I scrape spores from a paper print, to give myself an even and hard surface to work on.
Tap the spore dust off the razor blade, onto a clean slide. Place a drop of KOH or Melzer's reagent on the spore dust, add a coverslip, and tap gently on the coverslip with a pencil eraser to coax air bubbles away. Now put the slide on your microscope's stage. Start at low magnification, bringing the spores into focus (they may be very tiny), and move progressively through your magnifications, bringing the spores into focus each time. Carefully add a drop of immersion oil to the top of the cover slip before moving to the highest magnification (your oil-immersion lens), and then turn the coarse focus knob very gently and carefully until your spores slide almost into focus. Use the fine focus knob to make them completely visible. Going from low to high magnification is the only way to first locate the spores and then put them in focus; you probably won't have much luck if you start the process with your oil immersion lens (plus, you make easily break the slide or cover slip by forcing the oil immersion lens too far down).
At least one mount of spores should be made in Melzer's reagent. This will allow you to determine whether the spores are amyloid, inamyloid, or dextrinoid, which can be very important information in the identification process.
Sketch and describe the appearance of spores in your journal. You can also use a digital camera to take pictures through your eyepiece—and this works better than one might expect, after a little practice. You will have to experiment with your camera's settings. I have pretty good luck when I turn off the automatic flash, zoom my lens just a little bit, and take a whole lot of pictures, pausing to re-adjust the microscope's fine focus frequently. With this method I typically get one or two good shots for every ten I attempt—so you may well discover a more reliable method.
If you can't get a spore print out of your mushroom, you may still be able to see spores by taking a small portion of a mature gill and mounting it in a "crush mount" (press on the cover slip with a pencil eraser, gently crushing and stretching out the gill). Under the microscope, search through all of the tissues for spores—but remember that you may be viewing immature spores if you find them. If you can't find spores with this method, odds are high that your mushroom is simply immature, and has not yet developed spores.
To measure spores, use the ruler in your eyepiece (converting the values, if necessary, to microns using the multiplier you established when you calibrated your microscope). Be sure your spores are completely in focus; roll the fine-focus knob until the dimensions are as small as they can be. In order to get a good sense of the size of the spores, you will need to measure a bunch of them. How many? You will find different answers to this question, depending on the mycologist. Most will tell you that 10–20 measurements is probably enough. Others suggest more. Others, even more. I doubt my friend Rod Tulloss, the reigning North American Amanita expert, thinks one has ever measured enough spores to be confident about spore sizes. Whatever you decide, record the spore sizes and shapes in your journal, with the other data you recorded about the mushroom. Record the dimensions of the smallest and largest spores you can find—but if these big and little spores seem aberrant (like, there's only one little one amongst hundreds of others), exclude their dimensions from your accounting. If the spores have significant, measurable ornamentation (for example spines), it's best to measure the width or length of the spore without the ornamentation, and measure the ornamentation separately (the spines, for example, may vary in their length). You will want to express the dimensions of the spores as two ranges of possibilities, length and width: for example, 7–9.5 x 4–5.5 µm, which means that the thinnest spore you found was 4 µm wide, the widest was 5.5 µm, the shortest was 7 µm, and the longest was 9.5 µm.
Creating a Section to Study
Many of the microscopic structures you will want to observe can be seen by creating a cross-section of the mushroom's cap, with the gills included. Thus, "sectioning a pileus" is one of the most essential and basic routines for studying mushrooms. The concept is illustrated in the graphic below. You are creating a cross-section that contains a bit of the cap surface, the flesh, and several gills—represented by the thing in the middle of the illustration. I call this a "Roman Aqueduct Section," since the section looks a bit like an aqueduct. On the left is the section in relation to the mushroom and its cap; on the right is a schematized diagram of part of the section, under your microscope.
Use good razor blades!
Prepare to budget some money for razor blades, because successful sectioning requires super sharp edges. We're talking three or four uses, and it may be time for a new one. You'll have to search around at your drug store to find the old-fashioned, single-edged blades in the little safety boxes. Avoid double-edged blades; they are flimsy and, more importantly, dangerous, since you don't have a nice, safe edge to handle.
Step by step
The illustrations to the left take you through the process of creating a section like the one schematized above. You will notice that things don't work out so perfectly for me in the photo series--for example, the Agaricus bisporus specimen I'm using has so much flesh in its cap that creating a paper-thin section all the way from the cap surface to tips of the gills would be impossible (for me, anyway), so I have given up and simply used a small piece of the cap's flesh to hold the gill sections together. Also, the section I have cut is too thick to be ideal--but I wanted something that would show up well in the photos.
For boletes, a similar method should be used if you want to study the pileipellis (the cap surface)—but for studying the hymenium, a small sliver of cross-section sliced from the pore surface will usually suffice.
Polypores, especially the tough-fleshed, woody-textured ones, are notoriously difficult to section. Since a
For other types of mushrooms, you will usually want to create a section from the hymenium. In the jelly fungi this means pretty much any surface of the mushroom; slice a very thin, cross-sliced section (your razor blade must be exceptionally sharp with jelly fungi) and create a "squash mount" by pressing gently on the coverslip with a pencil eraser. The concept is more or less the same for cup fungi, morels, false morels, species of Helvella, and so on; find the hymenium (with the morels, by the way, that means the pits rather than the ridges), slice a very thin section from the surface, and make a squash mount. Puffballs and similar mushrooms usually require only grabbing a tweezers-pinch-full of the mature gleba and mounting the dusty tissue in a crush mount. Crust fungi can be approached more or less like polypores, above.
Structures in the Hymenium
Basidia are occasionally distinctive in shape, but they are usually clavate (shaped more or less like inverted clubs). Their length and width (at the widest point) should be measured—but assessing the precise length often involves a little bit of guesswork, since the bases of the basidia are often obscured within the palisade that makes up the hymenium (a crush mount will separate basidia, if you need precise measurements). Also measure the length of the sterigmata (the prongs at the end of the basidium), and count the number of prongs on as many basidia as you can. This can be a little difficult, since basidia are often large enough that their entire depth cannot be brought into focus at once; you will usually need to roll the fine focus knob back and forth to be able to see all the prongs.
Between the basidia there may be basidium-like cells that lack prongs and do not hold spores. These are called "basidioles," and they probably represent immature (or aborted) basidia. Analysis of basidioles is not usually relevant to mushroom identification, but I mention them in case they cause confusion. True, spore-bearing basidia have prongs that are almost always large enough to be seen easily with an oil-immersion lens. See also the glossary entry for brachybasidioles, also known as "pavement cells."
Cystidia are sterile cells that are found, in some mushrooms, popping up between the basidia. Unlike the basidia, cystidia do not produce spores. Their shapes and sizes vary widely between mushrooms—and many mushrooms do not have cystidia at all. Some mushrooms have boring, club-shaped cystidia that are hardly different from the basidia, except for the absence of spore-holding prongs. Others have elaborately ornamented cystidia, thick-walled and enormous cystidia, long and pointy cystidia, and so on. In fact mycologists have given names to many types of cystidia (and, to be accurate, cystidia can occur elsewhere on a mushroom—not just on the hymenium).
But no one knows what they are. Don't you love mycology? Um, maybe cystidia hold the gill faces apart so the spores have room to fall? That theory crashes to earth right out of the gate, since plenty of gilled mushrooms lack cystidia and manage just fine. Maybe they hold gills together until the spores are mature? Yeah, and maybe your mail carrier is doing something unproductive on your lawn. I once spent nearly two full days concocting a theory that the little liquid-filled guys are sensors that expand or contract with temperature changes (or changes in humidity) and transmit the information to the basidia so that spores are produced in optimal conditions . . . then I ran out of coffee, thank God.
Pleurocystidia are found on the faces (the sides) of the gills, while cheilocystidia are found on the edges of the gills. The only way to be sure of which kind of cystidia you're looking at is to have created a section, like the Roman aqueduct section described above, that will orient your perspective. Cystidia can be seen (if a mushroom has them) by simply creating a crush mount of a small piece of a gill. But you will only be able to distinguish and compare pleuro- and cheilocystidia by creating orientation.
In boletes, the pleurocystidia are found on the inside walls of the tubes, while cheilocystidia appear on the edges of the tubes (where the pore is).
In order to observe the cap surface of a mushroom, you will need to have sectioned your material in such a way as to facilitate your view. The Roman aqueduct section described above often works to see the pileipellis clearly, but it can obscure your view in the case of a cutis (see below), since the hyphae are arranged so that they radiate away from the cap center, and the sectioning method slices them in cross-section. Thus a radial section is sometimes preferable; make your cuts parallel to the gills, rather than across them.
A cutis is a type of pileipellis in which hyphae are arranged more or less parallel to the cap's surface, radiating outward from the center of the cap. Occasional hyphal ends (terminal cells) protrude from the surface, usually at a very small angle, sticking up only a little bit; these are often referred to as "exserted ends." (Truly projecting, well-differentiated, sticking-up things constitute pileocystidia.) When observing a cutis, measure the width of the hyphae, note the color of the hyphae in your mounting medium and whether the surface of the hyphae is smooth or not (see for example the encrusted hyphal elements illustrated to the left); also describe the terminal cells, if they are differentiated—and, finally, search for clamp connections at the septa. An ixocutis is one in which the hyphae are partially gelatinized as a result of the mushroom being viscid; in an ixocutis one often sees poorly-defined hyphae that appear to be swimming in an amorphous, hard-to-define material. However, the extent of the gelatinization can vary considerably, and sometimes it can be difficult to determine whether you're looking at a cutis or an ixocutis. In such cases it may help to know that an ixocutis has often accumulated a lot of debris as a result of stuff adhering to the slimy cap surface; search for messy areas and look for signs of gelatinization in them.
Trichoderm, Epithelium, Palisadoderm, Ixotrichoderm
In a trichoderm the hyphae arise perpendicular to the cap surface. This type of pileipellis can be viewed with equal ease in a Roman aqueduct section or a radial section. When observing a trichoderm, measure the width of the hyphae, note the color of the hyphae in your mounting medium and whether the surface of the hyphae is smooth or not—and describe the terminal cells, which will definitely be differentiated in a trichoderm, since they are upright. The terminal cells may be merely cylindric (see the illustration accompanying the glossary entry for pileipellis), or they may become swollen into various shapes. If the terminal cells are consistently swollen and frequently septate, the pileipellis is sometimes called an epithelium or palisadoderm (see Xerocomus illudens). In an ixotrichoderm the hyphae are gelatinized (see Hygrocybe glutinipes for an example). As with an ixocutis (see above), the gelatinization is sometimes difficult to determine. In a hyphoepithelium, a trichoderm is covered by a very thin, cutis-like layer. In a lamprotrichoderm the upright elements are thick-walled, elongated, and pointed (see Lactarius subvellereus var. subdistans).
Hymeniform pileipellis, cellular pileipellis
A hymeniform pileipellis is one in which the hyphae are club-shaped and arise perpendicular to the cap surface; they are inflated and resemble immature basidia in a palisade. A hymeniform pileipellis can be viewed with either a Roman aqueduct section or a radial section. A pileipellis is sometimes called "cellular" when it appears to consist of swollen cells; a hymeniform pileipellis, for example, will appear "cellular" when viewed from above.
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Smith, A. H. & Thiers, H. D. (1971). The boletes of Michigan. Ann Arbor: U Michigan P. 428 pp.
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Kuo, Michael (2019). Using a microscope. Retrieved from the Mushroomexpert.Com website: www.mushroomexpert.com/microscope.html
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