by Richard L. Howey, Wyoming, USA
Here I assume that you are already proficient at observing a wide variety of organisms at lower magnifications and are comfortable finding your way around in the world of protozoa, algae, and bacteria. You have, no doubt, already discovered for yourself the delights of Paramecium, Amoeba, Stentor, diatoms, desmids, filamentous algae, and the larger forms of bacteria. However, hidden away in clumps of decaying bits of plant and animal detritus is yet another world of marvels and monsters.
When in the field, I often tend to collect samples into which I put a considerable quantity of pond vegetation—too much, many would say. I bring these samples back to my lab and let them simmer and stew until they look like some black witch's brew (sorry about that, I didn't mean to get literary) and then with a pipet, I place on a slide a drop of the rich culture fluid which most definitely does not smell like eau de cologne. To tell the truth, some of these samples smell perfectly vile—not at all the sorts of effluvia that nice people want in their houses, but microscopists are a strange and hardy breed and seek out mysteries in some remarkably malodorous places.
Naturally, one needs to take sensible precautions in handling this kind of sample, and indeed every kind of biological sample. I use an 18x18 cover glass and start at the lower left hand corner and begin to scan at a minimum of 200x and sometimes I will begin directly with 400x. Now, it may well be that large organisms, Paramecia, hypotrichs, rotifers, etc.-will go zipping across the field. Ignore them. We're out for small game.
Many of these creatures are very difficult to identify, but whenever possible continue to make careful drawings and fully descriptive notes. Then weeks or months later you may be browsing in a book such as Kudo's Protozoology and come across a drawing and description that fits one of yours. Kudo's book, by the way, although dated, is a classic and if you work much at all with protozoa, you should try to find a used copy of the fifth edition.
In old cultures made with a wheat grain, one is very likely to find an abundance of tiny amoebas. Talk about a taxonomist's nightmare! But don't worry too much about classification—observe, keep records, and enjoy. If you want to get a general sense of the freshwater amoeba, there are several good basic references. The small amoebas can provide you with some amazing surprises. One day I was looking at an old wheat grain/Giese salt culture of the ciliate Lacrymaria olor and the bottom was dense with bacterial growth. However, within these bacterial forests were tiny glades like miniature hemispheric domes that one might imagine humans constructing on some densely forested alien world. Naturally, this had to be investigated. Within each tiny dome is an extraordinary little amoeba of the genus Nuclearia, and they actually do construct a gelatinous dome in which they house themselves. Remarkably, they have very thin thread-like (filose) pseudopodia which can extend through the dome to feed on the bacteria. How's that for an engineering marvel!
Another intriguing small amoeba that is found in the alkaline lakes of the high plains is Radiophyrs. I had taken some samples of vegetation which had dried around the edge of the lake as the water receded and stored this material in plastic bags. Several years later when I was sorting through some miscellanea, I came across these plastic bags. Almost from my first encounter with protozoa fifty years ago, when I read about encystment and excystment, I have been fascinated with these two processes. Many micro-organisms form highly resistant cysts which can survive heat, cold, drying, and a remarkable range of conditions and contaminants. Then when conditions are right again and appropriate food is available, the organisms stir themselves from their state of suspended animation, come out of the cysts and start feeding and reproducing again. As a consequence, I often keep samples of dried material and, from time to time, I put some of this material in a nutrient culture to see what emerges. Some day I hope to have time to systematically study the processes of encystment and excystment. But back to Radiophyrs. I took some of the dried material from the alkaline lake and placed it in culture dishes and added nutrient solution. Several days later I checked it, but there wasn't much of interest. After a couple of weeks, I decided to take another look and found something quite surprising. On the bottom were clumps of amoebas with shared pseudopodia which looked like miniature protoplasmic bridges and I could observe the movement within them. I later read that apparently Radiophyrs forms these temporary pseudo-colonies to increase the efficacy of feeding.
Everyone who has looked at pond samples has observed the wonderful glass shells of the larger diatoms. However, what often goes unnoticed by the microscopist who is not a specialist in diatoms, is a stunning variety of very small diatoms. When scanning at 200x, one suddenly becomes aware of tiny kayaks, circular and rectangular etuis, miniature crescents, and triangles—an abbreviated textbook in 3-dimensional geometry.
In addition to diatoms, there are many small elegant algae which you will encounter. Some of them are housed in a gelatinous matrix, almost invisible in brightfield and just discernible with phase contrast. However, there is a simple effective technique for clearly demonstrating this matrix—just add a drop of India ink to your preparation. The ink particles will run up against this invisible boundary, thus outlining it quite distinctly.
There are, it seems, countless numbers of small flagellates which one will encounter. Some have a fairly rigid body, a long flagellum projecting forward, held stiffly and proudly like the standard of a flag and only the tip seems to be moving, pulling the organism along sedately. They will almost certainly have a second flagellum, often called a trailing flagellum, but frequently it is short and kept close to the body, thus ordinarily making it indiscernible. Other flagellates will have four, eight, or even more flagella. Some move with great speed, rushing about attending to the business of the microworld. Here again, phase contrast is a great advantage, but if you don't have access to that, the India ink trick sometimes works quite well.
If you are lucky, you might come across a rather ordinary looking little flagellate and then discover that it has broken off from a colony which has broken off from a reddish-brown "stem". In fact, you might first come across a tangle of the "stems" which looks like a random mass of brown fibers. At the tips of these stalks are clusters of flagellates, a hemispheric colony arranged like a bouquet. Each flagellate has two flagella. Often when one places the cover glass on the sample, some of the colonies break off from their "stems" and go swimming off around the slide giving no hint that they were once attached. These lovely creatures have the unlovely name of Anthophysis vegetans.
A small ciliate common in putrid samples is Cyclidium glaucoma. One can often get quite a good look at them as they seem to be of a rather manic-depressive temperament. Sometimes they will dart about in a frenetic fashion and then suddenly stop and sit meditatively for a minute for more. Then, they will dart off again with alacrity. They have a caudal cilium which is about half the length of the body and so once you've learned to recognize them, they're quite easy to pick out.
Perhaps my most exciting encounter while scanning at high magnification came from another small amoeba. When I first noticed it, it didn't seem particularly noteworthy, but then I thought I saw something very bizarre. I increased the magnification from 400x to 630x and, sure enough, this amoeba had a flagellum! Nature's always playing this kind of trick on us. We get everything neatly sorted out into orderly pigeonholes and then something appears that doesn't fit into any of our categories. How inconsiderate! An amoeba with a flagellum! There are in fact, two common genera of flagellate amoebae, Mastigella and Mastigamoeba and there are also some that have a flagellum some of the time, but not all of the time, including the nasty little Naegleria which can cause meningo-encephalitis in humans, when it gets into the brain through the nasal passages.
But these little creatures which I found are benign and are a striking reminder of the arbitrariness of our attempts to order the world. If you would like to see an extensive catalog of creatures which challenge our taxonomic ingenuity, then let me suggest that you browse in the Handbook of Protoctista, edited by Margulis, et al. Here you will find a feast of some of nature's most incredible creations.
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