by Richard L Howey, USA
Part II Part IIIMy students hate my bad puns and some of them have started collecting money for a fund to have me lobotomized; if you would like to contribute, you can just send the money directly to me.
I want to talk about two things in this essay: 1) a variety of techniques for observing Paramecia and 2) the larger issue regarding the ways in which we gather information, interpret it, and synthesize it in an attempt to understand a tiny part of the natural world. Since I also want to show you how these two aspects are inextricably bound up with one another, I shall be considering both facets throughout the discussion.
Paramecia are one of the most common and most familiar micro-organisms on the planet and almost everyone with any formal education has at least heard of them. Because they are so common and have been widely researched, there is sometimes a tendency to regard them as rather ordinary and not very jazzy. Oh, thereís another Parameciumóho hum! However, Paramecia are anything but ordinary and, in a very important sense, do not really serve very well as an example of a prototypical ciliate. Iím not sure that there is such a thing except as a construct which we concoct for utilitarian and pedagogical reasons, along with the human passion to order and classify. Consider, for example, the remarks of R.S.J. Hawes in his preface to An Introduction to the Study of Protozoa by Professor Doris L. Mackinnon, Oxford, 1962.At this point we already encounter an important problem, namely, which species of Paramecium is going to be our reference type. Traditionally P. caudatum or P. multimicronucleatum have been the models, but the extensive research of T.M. Sonneborn has also made P. aurelia a candidate. These organisms and a fair number of others all belong to the genus Paramecium, but the species, subspecies, and variants can and do show considerable diversity. Consider P. bursaria which is one of the smaller members of the group and has the distinctive characteristic of possessing symbiotic algae of the genus Chlorella."Perhaps some historian of our subject will one day explain by what mischance this genus was adopted as a type for elementary study, a role for which it is ill adapted. Paramecium is an extremely specialized member of a peculiar and imperfectly investigated suborder. Its structure is difficult to observe and almost impossible to demonstrate to the inexperienced. Very few people understand how it feeds. Only recently has it found a tenable taxonomic position...We use it to illustrate to elementary classes the general features of the Protozoa (or the Ciliata) with about as much sense of proportion as if we introduced the Phylum Arthropoda with compulsory dissections of Drosophila. Is it too much to hope that one day it will be abandoned as a morphological type in favour of some more generalized ciliate such as Tetrahymena?" (p. vii)
So, here we get to a fascinatingly difficult set of questions. If some species are quite small, only around 40 microns and others are relative large (over 350 microns); if some have a single nucleus and two micronuclei, whereas others may typically have four or more, and one species can have from 3 to 8; if one species has symbiotic algae and the rest donít; if some strains of a given species contain a viroid particle, the so-called kappa particle, and other strains of the same species donít, so that when conjugation takes place and nuclear material is exchanged along with some kappa particles, the kappa strain flourishes and the other strains are killed by the viroid particlesóif all of these remarkable variations occur among these organisms, then what are the justifications for classifying them all in the genus Paramecium?
Human beings are inveterate classifiers as is evidenced by the fact that as early as Plato, we find in his Theory of Ideal Forms, what is essentially a theory of classification. Aristotle substantially modifies his teacherís conception and produces a much more practical classificatory system. The modern system of taxonomy appears centuries later and largely derives from the work of the great Swedish scientist, Linnaeus. Taxonomy has always been a controversial enterprise and increasingly in the 20th Century, as a consequence of technological advances, serious questions were raised about the adequacy of an approach based primarily on gross morphology and cytological criteria available from optical microscopy. With the advent of cellular physiology, biochemistry, electron microscopy, and the plethora of new high-tech imaging techniques, such as, laser confocal microscopy, scientists were provided with an array of detailed data in staggering quantities and more than a few thought that counting the bristles on the appendages of tiny aquatic crustacea in order to make species discriminations was no longer adequate. The utility of theories of classification depends upon varying degrees of generalization; if the categories are too general they lose their usefulness; if they are too strict, they also lose their usefulness in a different way, for even Aristotle wrestled with the problem of the uniqueness of each particular thing and Darwin understood that species do not occur in nature. Classification systems are tools to aid our understanding of nature, its processes and organisms. In that respect, taxonomic systems are rather like maps; they are useful precisely because of their distortions.
A topographical map is useless if you are trying to drive from Laramie to Denver, but very helpful if you are looking for certain kinds of geological formations. The scale of a map is also a factor in its utility for a given purpose.
Suppose we wished to develop a classification scheme based on color. Oranges, limes, tangerines, tangelos, and lemons would be easy, if they were ripe. But what about apples? A ripe Granny Smith is green and there are Golden Delicious apples, as well as lots of red apples. Well, thatís O.K.; we put the Granny Smiths in the category of green things, the Golden Delicious in the category of yellow things, and all those red ones in the red things category. But, hold on, itís not quite that easy. Some apples, like the Gala, are multi-colored; some of the red ones are more rose-colored than red and so now we start to need subcategories. We canít just have red things; we have to have rose things, crimson, pink, burgundy, and vermillion things. And in the red category, we have not only apples, but rubber balls, pomegranates, roses, holly berries, strawberries, Christmas tree ornaments, and, of course, Red Square and red pens for grading student papers. (Some of my colleagues have, however, informed me that I am not being politically correct by using the harsh red pen and that I should convert to using a more student-friendly color, such as, soft green, light brown, or purple. Their comments had a profound effect upon me and I went to the bookstore and ordered a gross of red pens.)
Where are we? What have we learned? Well, it seems to me that color is probably not a very good basis for trying to organize objects in the world. Consider what would happen if we tried to do this with protists. There are a few organisms that do have distinctive colors and it would be quite helpful if someone were to make up an extensive list of these while retaining the present classification system. Stentor coeruleus possesses a distinctive blue pigment, with a tint of green, called stentorin and, interestingly, this pigment is dichroic; that is, if you shift the angle of light S. coeruleus turns a light rose color. There is, however another species of Stentor which has a light pink pigment which is not dichroic. Many species of Blepharisma possess a photoactive pink pigment called blepharismin. Just from these two examples, you can see a number of difficulties about classifying according to color.
There are other problems. Euglena rubra is, in certain angles of light, the typical deep green of chlorophyll, but, in other angles, it is a brilliant red and this is not a consequence of dichroism. Rather E. rubra possess two kinds of chromatophores, one kind containing chlorophyll, the other a carotene pigment. Thus, the conditions under which an object or organism is viewed can affect the color judgment.
Here in the mountains, if you go for a hike in the early spring, you have a good chance of
encountering "red snow" which is the growth of special algae on the surface of the snow and
usually it appears pink (bright red when concentrated) and, oddly enough, it smells and tastes like watermelon. If you don't believe me and must taste it to satisfy your curiosity, taste only a tiny it and then spit it outóif you ingest very much, it can be toxic."
There are some fairly large groups of protists that have chlorophyll pigments and as a result appear green; others which possess xanthophylls and appear yellowish or brown, and those that possess carotenes and have a reddish or sometimes orange color. However, the vast majority of protists are colorless and thus a color classification system would create a giant class of organisms which would be unrelated except for the lack of pigmentóa totally unworkable system.
However, even though pigment is not a viable classificatory tool, it is certainly very much worth noting distinctive cases such as Blepharisma and Stentor coeruleus. Letís return to our problem about Paramecia. Clearly general appearance is, in itself, not a sufficient criterion for including an organism in a particular genus. Earlier I mentioned P. Bursaria which, you will remember, has green symbiotic algae, is relatively small compared to P. caudatum and P. multimicronucleatum which were long regarded as the paradigm for Paramecia, and P. bursaria has a slight curve to its body at the anterior end, all of which makes P. bursariaójust on the criteria of general appearanceóa less likely candidate for inclusion in the genus Paramecium than a number of other ciliates.
So, whatís going on? How does one find out that P. bursaria does indeed belong in the genus Paramecium? This, of course, leads us directly into the issue of techniques of observation for gathering information. For the purposes of the discussion here, weíll limit ourselves to techniques available through optical microscopy, since I am profoundly ignorant of the new high-tech imaging equipment and procedures.
Part of what prompted me to think about this issue of various kinds of observation techniques and the sorts of information which we derive from them is a series of photomicrographs of P. bursaria in Professor David Pattersonís excellent book Free-Living Freshwater Protozoa: A Color Guide. This volume is now available in paperback for about $40 and is a marvelous addition to the reference library of any amateur microscopist interested in protozoa. If you canít purchase it yourself, then try to convince your local library to acquire a copy. So, for most of this discussion the reference species will be P. bursaria, although many of the observations will apply to other species in the genus as well.
Most amateurs begin with a compound microscope fitted for brightfield observation which immediately poses a challenge. Most protozoa are relative small and essentially transparent, thus providing relative little contrast and, to make matters worse, they are generally very active making observation at high magnifications difficult to say the least. As a consequence, one is tempted to abandon Köhler illumination [see endnote] by closing down the iris diaphragm or lowering the condenser, thus increasing contrast, but losing resolution. Over the last century, microscopists have demonstrated extraordinary ingenuity in inventing a variety of contrast techniques to allow the observer to see more and more detail. One of the most fascinating aspects of these techniques is that virtually each one provides information different from the others. Naturally, there is a certain amount of overlap, but no single technique can provide all the information we desire and, as a consequence, our understanding of a given organism rests finally on interpretation and synthesis of data from a whole series of techniques.
Letís go back to our problem of insufficient contrast with brightfield. It is always important to understand the rules for optical illumination for any given techniques, so that when special circumstances arise, you can break those rules in an intelligent and creative fashion. Clearly with brightfield illumination and low contrast specimens, one of the problems is too much light, so if you have a variable transformer, you might be tempted to reduce the light intensity. You will quickly discover, however, that this is not a desirable solution, as you will achieve a minimal gain in contrast and a significant loss of resolution plus an increase in chromatic aberrations.
Early microscopists began to experiment with various kinds of stops in the substage to redirect a portion of the light. These stops were often opaque metal or black heavy paper disks cut in various configurations to produce either darkfield or oblique illumination. Center stops of various sizes can be used to produce darkfield, different objectives requiring different-sized stops. These days there exists a variety of special darkfield condensers and, if you can afford one and learn how to use it properly, it can produce spectacular results. Many protozoa, diatoms, radiolaria, and a wide variety of other objects can be very advantageously viewed with darkfield. One can see detail not ordinarily visible with brightfield, such as, structural elements in diatoms and radiolaria and locomotive elements in protozoa, such as, flagella and cilia.
If one is fortunate enough to have a microscope fitted with a turret phase condenser, then you can produce yet another kind of contrast by breaking one of the rules. Position one of the phase rings so that it is in the optical path, even if you are using a brightfield lens. Then while looking through the microscope at the specimen, slowly rotate the phase turret ring until you get optimal side-lighting. By putting the condenser out of phase, you produce a pseudo-Nomarski image and can achieve some excellent contrast. You can experiment with using different phase rings in the turret with both brightfield and phase objectives. Keep records of your results so that you know which combinations work best with particular types of specimens.
The other classic response to the problem of low contrast specimens is the use of stains of which there is a very large number and, in addition, various investigators have devised methods of double and even triple staining. In the last part of the 19th Century and the early part of the 20th Century, the Germans produced many of the best quality stains as a product of their highly developed chemical industry. Many of these stains were coal tar dyes developed in a dazzling array for the textile industry.
Staining is an art and entire books have been devoted to stains and staining techniques. In this essay, I will limit my discussion to stains and techniques which I have found useful in my own investigations. Almost all stains are toxic in their concentrated forms and if preparing them from their powders or crystals, you must be careful to avoid skin contact and especially to avoid inhaling the "dust" from the dry stains. In solution, they are generally less problematic, but still need to be dealt with carefully as some are known to be carcinogenic and/or mutagenic. As far as the organisms that you are staining are concerned, some profound cytological changes will take place. These effects may be minimal and more difficult to trace with the so-called vital stains, but they are nonetheless present. Some stains, under certain conditions, will produce artifacts or pseudo-structures. The biochemical reactions involved between stains and organisms are enormous complex and are only partially understood. Sometimes scientists have been misled by these biochemical reactions into positing entities in cells which simply didnít exist or even conjecturing that a new and bizarre kind of organism had been discovered. Perhaps the paradigmatic case is Ernst Haeckelís Urschleim. While examining samples from the Challenger expedition, Haeckel noticed, that again and again, the samples which were collected and preserved contained a kind of gelatinous, undifferentiated "slime". He conjectured that this was the basic biological "stuff", the primordial ooze from which all other life had evolved and he built a theory around this remarkable primitive, homogenous ooze. Apparently, bottle after bottle appeared containing this Urschleim and it was not until after Haeckel had proposed his theory, that it was discovered that this substance was a precipitate formed by the fixatives reacting with the salts in the sea water.
Even the so-called ordinary biological stains that the amateur might use, can produce special problems both with regard to artifacts and also in the interpretation of the results of the staining. The purity of stains becomes a crucial consideration in certain types of techniques and some of the older microscopists used to specify a particular companyís stain, very frequently, Gruebler. Some stains can be consistently refined to remarkable high degrees of purity, whereas others seem to be intractable and, as a consequence of their impurities can become desirable, even though they are unpredictable. Methylene blue is a good example of a polychrome stain, which in this context really does amount to unpredictability. Methylene blue is one of a number of stains which produce "lakes" or a series of variations and gradations of colors as one moves from the edge of a dried drop toward the center, quite like what one sees in a computer-enhanced aerial photograph of a lake where the difference in depth show up as color variations.
If we apply this to protozoa, we discover something quite interesting. You can stain the same organism with a polychrome stain and get a variety of results and this variation may be the consequence of a range of factors from pH to salinity to temperature. (Hereís another great project for some enterprising amateur!) It is precisely the impurities in certain stains which can produce some striking contrasts and allow you to notice clearly, details which you might otherwise miss; the negative side is the problem of repeatability. For good results with stains in general, not only do you need high quality stains, but you need to use distilled water and/or high quality grades of alcohols for solutions, otherwise you may end up with undesirable precipitates which ruin your preparations (or lead you to think that you have discovered the primordial ooze.) Some stains have a long shelf life, indeed hematoxylin stains require months of aging or alternatively chemical "ripening", whereas other stains should be made up each time just before use. Accounts of staining techniques are, at best, rules of thumb. A stain that works well for Vorticella may be useless for Paramecium, so a great deal of trial and error is involved and when you have successes with a particular stain on a specific organism, write up your notes and send them to Micscape to share your work with other interested amateurs.
Fortunately, there are a significant number of staining and chemical techniques that do produce good results consistently. I added chemical techniques here, because there are some substances which are not stains which can nonetheless provide results that allow you to see structures you would otherwise likely not notice. Tannic acid, for example, when placed at the edge of a cover glass on a drop of Paramecium culture, produces an explosive discharge of trichocysts as it migrates under the cover glass. Trichocysts in Paramecia are something of a mystery; it would seem that they would provide some sort of defense, but there is not much evidence to support such a conjecture. I used to assume, very naively, that the type of trichocyst that Paramecia have was the definitive version; not, so, there are about 17 types of trichocysts (mostly with other names) and, no doubt, others waiting in the wings to be discovered.
Paramecia apparently discharge their trichocysts in batteries and with considerable force as one can often find "clouds" of them 3 or 4 body lengths away from the organism. The trichocysts of Paramecia seem to be like the spears in the old African adventure movies, where barrage after barrage would rain down upon The Great White Hunter and his safari party, but none of them would ever get hit. If you use a drop of blue ink or mix a bit of stain with the tannic acid, you can get the trichocysts to stand out even more vividly. Other ciliates, such as, Dileptus possess toxicysts which are used for prey gathering and possibly defense and which, when discharged, eject a drop of toxin.
Sometimes, as the water dries up in a preparation, the cover glass exerts pressure on the Paramecia and flattens them in such a way that if you focus along the edge of the organism, you can see the undischarged trichocysts in little capsules lined up like torpedoes. Furthermore, under these conditions, when the "cell" stars to lyse (break open), you can observe the process of the trichocysts being discharged, although strictly speaking the firing of the individual trichocysts happens so rapidly that we only observe the after-effects. To observe the actual discharge, one would have to use slow motion photography or videography.
With some large ciliates, the addition of dilute acetic or tannic acid, as well as certain stains, can produce a kind of "shedding" of what is apparently an entire membrane layer, complete with the outline of the spiral pathways of the kineties, which are the bands or lines that define the ciliary arrangements in the pellicle. It is rather like a snake shedding its outer layer of skin wherein the designs and patterns still remain evident.
I have already published a short article on vital stains on Micscape, but let me make a few additional comments here. Paramecia are very active organisms, so if you employ vital stains, you will also have to find a way to slow them down to observe much in the way of significant detail. To my mind, the best initial technique is to let evaporation and the weight of the cover glass do the work for you. This requires some patience, but also allows sufficient time to explore the slide and make some interesting progressive observations. Iíll discuss some other methods of slowing down organisms in a moment. A bit of powdered carmine mixed with a drop of Paramecium culture on a slide always produces interesting results. Carmine has a very low level of toxicity and, in its powdered form, it doesnít act as a dye. Rather, the carmine particles are ingested into the food vacuoles of the Paramecia and accumulate, showing the vacuoles very distinctly and one can trace their path through the organism.
Neutral Red will also produce interesting results in the food vacuoles when used sufficiently diluted to act as a vital stain. It is also worth noting that there are some quite small structures in Paramecia which are especially sensitive to Neutral Red and are thus called Neutral Red granules. Neutral red is not only a stain, but also a pH indicator. As food is digested in the vacuoles, the pH shifts and so does the color of the Neutral Red. (Iíll let you figure out the color shift/pH relation.)
But letís go back to the issue of other methods of slowing down active protozoa. It is obvious that no single method will work on such a group of diverse organisms. Nonetheless, over the past 100 years or so, microscopists have come up with some very helpful general suggestions. One very ingenious notion involved building lots of little cages by using cotton fibers placed in the drop of water containing the organisms. This is clever, but only modestly successful, since the result is a bunch of hyperactive Paramecia struggling to get away. An even cleverer method involved the introduction of a non-toxic substance of high viscosity to slow down the beasties. Mucilage from quince, various starches in solution, and other even more exotic substances were tried. Today the standard is methyl cellulose or some variation thereof. This works reasonably well, but for some active, large organisms, the need for a fairly high viscosity causes the specimen to contort and this can produce misleading images. The other problem with methyl cellulose solutions is that they produce distinct background noise with straight polarization or with Nomarski DIC, since the cellulose fibers in the solution are significantly birefringent.
I even remember reading the account of a Polish researcher who introduced powdered iron filings into a culture and then was able to direct the motion of the ciliates by the use of magnets. Apparently this technique didnít catch on.
The next obvious move is to try anesthetics and, to some devious minds, the first notion is to get the little critters drunk. Iím not being judgmental; in fact, I remember a remark which Dylan Thomas once made: "An alcoholic is someone you donít like who drinks as much as you do." (Iíll drink to that. Cheers!) For the Paramecia, you, of course, need a very small shot glass. Over the years, investigators have tried various concentrations of ethyl, methyl, isopropyl, butyl, and other types of alcohols. Solutions of about 10% ethyl or methyl alcohol added slowly to several drops of culture seem to work best.
The standard test for the utility of an anesthetic is whether or not the effects of the substance are reversible and the organism is able to resume full function including reproduction. Substances which significantly alter the biochemistry of an organism are very likely to have long-term effects. Alcohol, ether, menthol, chlorotone, chlorobutanol, benzocaine, hydroxylamine hydrochloride, and local anesthetics, such as novocaine have all been tried with varying degrees of success.
Before the advent of the "drug culture", researchers routinely had access to other more powerful and sometimes more effective substances, such as, cocaine hydrochloride, morphine, and chloral hydrate, but those days are long gone.
Interestingly, the metallic ions of potassium, magnesium, nickel, and copper can be quite useful in the investigation of protozoa. Potassium iodide, magnesium sulfate, and nickel sulfate all seem to act on elements that control ciliary activity and the effect is that the organisms slow down significantly and with minimal side-effect when using reversible concentrations. These metallic salts are easy to use and to prepare and are to be recommended as a first choice.
Copper salts are a different matter in that they are toxic to ciliates and, given the right concentrations of the right copper salts, can kill certain ciliates with relatively little distortion. Once that process is completed, one needs to follow with a fixative before maceration begins. The copper salts can act in a fairly delicate manner and produce very good results. I have found copper acetate to be particularly good in this regard. I use a 1-2% solution.
Magnesium sulfate, which is excellent for many aquatic invertebrates, especially marine ones, is unpredictable in its effect on ciliates. Lacrymaria olor, for example, has a very high tolerance for this salt. Potassium iodide (1-2%) and nickel sulfate (0.5-1.0%) are probably the best substances to try out to slow ciliary action. Several years ago, a researcher reported a more radical technique using dibucaine hydrochloride which cause Tetrahymena to shed their cilia, thus stopping motion. Surprisingly, the effect was reversible and when the organisms were returned to culture medium, they regenerated their cilia!
In order to acquire a wide range of information about micro-organisms, in this case Paramecium, you have to employ a whole battery of techniques which should always begin with extensive and careful observations of the living organism. For most living organisms, brightfield is of limited value as a consequence of the small differences in contrast. Darkfield and oblique illumination provide important means of obtaining additional pieces of information, but one has to remember that each technique has its limitations. Think of it this way: if you are photographing a person, a shift in the angle of the camera, a shift in lighting, a change of filter, a closeup of the face, a full length shotóeach "glimpse" provides different information and the appearance of that person can alter radically from photograph to photograph. Fortunately, with protozoa, we donít have to be concerned about facial expression, moods, or clothing, and can concentrate on the morphology and behavior of the organism, but these aspects are in themselves mind-bogglingly complex. Suppose you wish to observe the nucleus of a Paramecium. Sometimes, if you get just the right conditions with the right speciesósufficiently flattened, barely moving, if at all, good contrastóthen you may get a good look at the macronucleus and you wonít have to use a nuclear stain. However, often the conditions and species are not just right and there is the additional consideration that you will very likely want to try to observe the micronuclei as well to find out how many there are. Contrast techniques can be of some help, but in the end, you will probably resort to staining. A very good stain for this purpose is methyl green acetic and you can find the recipe in most traditional books on microtechnique or you can purchase it already made up from a biological supply house. To demonstrate nuclei, it must be used on living protozoa and the results are sometimes erratic, so try it on a number of organisms of the same species and observe them carefully and record your observations. There are a number of other good nuclear stains which you may want to experiment with, but I usually like to try methyl green acetic first. If you place a drop at the edge of the cover glass and let capillary action slowly draw it across the slide, you can frequently get a nice differentiation and insure that not all of the organisms overstain.
In a second part of this essay to be presented later, I will look at various contrast techniques in greater detail and discuss the types of information we can derive from them. Ultimately we are putting together a multi-dimensional image of great complexity and we need to know how and where the various kinds of information come together to give us a composite. Each particular "glimpse" that we get is limited and, like the maps which we spoke of earlier, produces a special and useful kind of distortion. If we understand in each case what those limitations and distortions are, then we are in a position to build up a progressively richer and more sophisticated conception of a particular organism.
In most every good book on basic microtechnique, you can find a discussion of Köhler illumination which will be better than what I could provide here in a brief summary.
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