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Using a Microscope: Viewing and Measuring Spores

by Michael Kuo

Observing spores, and measuring them, is the easiest of the various microscope routines involved in mycology. 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. If you are studying a Basidiomycete (for example, a Gilled Mushroom or a Bolete), place a drop of Melzer's Reagent on the dust, add a cover slip, and take a moment to note in your journal whether the spores are amyloid, dextrinoid, or inamyloid. Melzer's Reagent has iodine in it, and a color reaction will be visible if you hold the slide up to the light. If the spores turn bluish black in Melzer's, they are amyloid; if they turn reddish brown, they are dextrinoid; if there is no noticeable change in color, the spores are inamyloid. In some cases you will need to wait until you are viewing the spores through your microscope before you can see dextrinoid and amyloid reactions (see the graphic at the bottom of the page), but strong reactions, at least, can often be seen with the naked eye. (If you are studying an Ascomycete--say, a Morel, a False Morel, or a Cup Fungus--see this page.)

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).

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, brace the camera by holding the tripod against my chest, 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 single gill and mounting it in a Melzer's "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.


Spore Shapes

Sometimes simply knowing the shape of a mushroom's spores can eliminate hours of sorting through keys. The spores illustrated to the right belong to Agrocybe erebia and have a snout-nosed end, which helps to separate them many other Agrocybe spores. The spores of Inocybe and Entoloma are notorious for their funky shapes, and a quick peek at these spores can often separate the mushrooms from thousands of other LBM's (Little Brown Mushrooms).

 

Agrocybe erebia spores

The graphic below is meant to give you a broad sense of spore shapes, but it only represents the tip of the iceberg, including some of the shapes most commonly encountered under the microscope. For comprehensive treatment, see Largent et al. (1973; citation here).


Spore Shapes




Measuring Spores

Measure spores with the ruler in your eyepiece, and convert the values to microns using the conversion multiplier you established when you calibrated your microscope. Be sure your spores are completely in focus; in the illustration top-right, for example, the spore is slightly out of focus and would probably look a little thinner if it were brought into focus with the fine focus knob. Also note, in the bottom illustration, that spores with ornamentation can create a little confusion with measurements; it's usually best to measure the width or length of the spore without the ornamentation, and measure the ornamentation separately (see below). Officially, you should "measure the length of at least 10 mature spores and record them" (Smith, Smith & Weber, 1981; 13). You may find yourself eyeballing spores after measuring one or two, however, and to this I can only say that I do it too. But I have looked at a lot of spores through the microscope, and my eye is pretty good, after a few measurements. Be sure to 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. You will want to express the dimensions of the spores as two ranges of possibilities--for example, 7-9.5 x 4-5.5 µ, which means that the thinnest spore you found was 4 µ wide, the fattest was 5.5 µ, the shortest was 7 µ, and the longest was 9.5 µ. Tip: Only do the math once. Write everything down in terms of your microscope's units, ignoring the fact that your units do not equal microns until you convert the final numbers in the format above. Eventually, you will be so used to the conversion that you will be able to do it in your head with enough accuracy for all but the most technical mycological purposes.

 

Measuring spores
Clavariadelphus lignicola


Measuring spores
Lactarius deceptivus



Spore Surfaces

Many spores have smooth surfaces--but other spores feature spines, warts, ridges, lines, and other distinctive surface structures. (Some spores that look smooth actually have amazingly ornamented surfaces if you look at them with a Scanning Electron Microscope--but since virtually no one has an SEM on hand, the matter is pretty much moot.) You will need to view spore surfaces the same way mycologists do if you want to be able to compare your results to theirs. In the case of most Basidiomycetes, this means using Melzer's Reagent. The chemicals in the reagent actually affect the way the spore looks, and since the Smith & Thiers monograph of Lactarius (for example) is based in great part on observations of Lactarius spores with Melzer's mounts, you will have to use Melzer's if you want to be sure you're comparing apples to apples.

Some of the most commonly encountered spore surfaces are featured in the graphic below (for comprehensive treatment, see Largent et al., 1973), as well as examples of dextrinoid, amyloid, and inamyloid spores.


Spore Surfaces



Spores with subtle ornamentation, like the Lacrymaria velutina spores in the graphic above, do not require measurements beyond an assessment of basic dimensions (discussed in "Measuring Spores," above). But prominent ornamentation, as in the Scleroderma spores in the graphic, should be measured separately. The distance that spines stick out from the spore's surface should be measured--on several to many spores, to get a sense of the range of possibilities. One particularly frustrating distinction occurs fairly often with Russula and Lactarius spores, which can be merely spiny, or feature connecting lines and ridges between the spines. When present, the connecting lines can be merely "scattered"--or they can form "partial" or "complete" reticulation (see the illustrations in the graphic).



Cite this page as:

Kuo, M. (2006, February). Using a microscope: Viewing and measuring spores. Retrieved from the MushroomExpert.Com Web site: http://www.mushroomexpert.com/microscope_spores.html

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