Wild Cultures and Subcultures: Part I
by Richard L. Howey, Wyoming, USA
Well, there are the Yanomamo in the Amazon Basin, the Ik in Africa, punk rockers, and teenaged Americans into body piercing. However, the wild cultures I want to talk about here are ones made from freshwater samples collected from ponds, rivers, and streams. In Part II, I’ll discuss some techniques regarding subculturing.
When we go out into the field with our bottles and nets, we are often under the impression that we are going to bring a bit of nature back into the lab–raw, wild, and untouched. But, think about what we have done in the process of collecting.
1) We use nets to “concentrate” organisms for our jar–a perfectly justifiable and pragmatic procedure, but one which has already altered the context in which these organisms live “in the wild”.
2) It is often desirable to include bits of aquatic plants from the edge of the pond, stream or river. The concept of a “bit” is not a scientific measure and I find myself including such vegetation in a sample ranging from none at all to “chock-full” (a much more scientific measure than a “bit”). Clearly the amount of plant material in each sample will affect the growth rate of bacteria, the rate of decay of the plant material, the depletion of oxygen, the excystment of other organisms not actively present when the sample was collected, and the reproduction rate of dominant species.
3) The chances are very high that the water temperature in your sample will go up when you place it in your lab environment and this also will affect the bacterial growth rate and the plant decay rate. Samples which I collect in the early spring just as the ice is melting or even midsummer samples from high alpine lakes are in very cold water indeed. Many micro-organisms are quite sensitive to temperature and some tolerate only a rather narrow range. When I am collecting in cold water environments and want to insure that temperature remains in an acceptable range, I have a couple of old thermos bottles which do an effective job. But then, what do you do when you get them home? My wife drew a hard line–no cultures in the refrigerator! In college and university towns, one can often find, for a reasonable price, a used shelf-top refrigerator of the sort that students have in dormitories and apartments and such an appliance is ideal for storing heat-sensitive cultures and certain chemicals which require refrigeration.
4) If you divide samples and dilute them, the kind of water you use to do so becomes another factor.
4a) For diluting samples and setting up cultures, distilled water should generally be avoided. It contains no salts and is, as a consequence, toxic to some organisms.
4b) Tap water usually contains an acceptable balance of salts, but also has chlorine. If you are going to use tap water, draw it off and let it sit in an open container for at least 24 hours.
4c) If you have a “water softening” system in your house, avoid using such water for these purposes; it often contains excess of salts for micro-organisms.
4d) Filtered pond water from the collecting site is often very desirable. You must filter it, of course, to remove larger organisms which might be potential predators on the organisms you want to study. Ostracods, cyclops, Daphnia, and small cladocerans can be special nuisances, since they have voracious appetites. If these are the organisms which you wish to culture, then you don’t have to worry, unless you have certain insect larvae or fingerling fish in your sample. However, if your goal is to culture some protozoan and algal forms, then you need to exclude micro-crustaceans. Filtering with cheesecloth won’t do it, since the nauplii of cyclops are small enough to make it past the filter. There are, of course, alternatives. You can use a coffee filter which micro-wise is fairly coarse, but also reasonably rapid or you can go to commercial filter papers specially designed for the purpose. There are grades of such paper ranging from coarse to microfine. If you use the microfine grade, you will need the patience of Job. It make take you a month to filter an ounce of pond water–a slight exaggeration perhaps, but you get the point–this process makes snails seem like speed demons!
4e) Another option is to coarse filter the pond water and then boil it to kill any unwanted organisms that have escaped the filtering process. There are advantages and disadvantages here. Pond water is a very complex material –and water itself is one of the stranger substances on this planet. In the process of boiling the pond water, we not only purge it of potential contaminating organisms, but we also subtly alter the chemistry and composition of that water by providing additional material to decompose in the water. Another very pragmatic disadvantage regarding the use of pond water is the issue of transport. Water is heavy and awkward to carry for any significant distance.
4f) My lazy solution: I go to the supermarket and buy several plastic gallon jugs filled with artesian water and for most of my uses, it has proved quite acceptable.
4g) Some organisms, such as, Paramecium, will grow in almost any medium that has plenty of bacteria for them to feed on and they tolerate a wide variety of conditions. Other organisms are quite fussy and have a narrow range of parameters for thriving. There are some general purpose salt solutions, such as, Giese’s and Chalkey’s which some investigators prefer. Cerophyll or other cereal grass solutions are also popular. There are also specific media which have been devised for amoebae, others for flagellates, and yet others for specific groups of ciliates. An excellent reference for such recipes is Culture Methods for Invertebrate Animals by Needam, Galtsoff, Lutz, and Welch.
Cultures As Distinguished From Samples
I tend to regard samples as those collections which one makes pondside and which one tends to leave largely in the state collected, perhaps divided and diluted or added to an aquarium, but with no attempt to isolate out individual species or groups of organisms. Cultures, on the other hand, are designed to provide quantities of a specific organism or group of organisms, so that they may be studied at length, at leisure, and in detail. One of the most frustrating experiences for the microscopist is to come across a beautiful, bizarre, or bewildering organism and have it lyse (come apart) on the slide just after you have started examining it. You go back to your sample with the optimism of the hungry man who just a few days ago killed the last Dodo. Where there was one, there must be more. All too often that’s not the case or you may find 5 or 6 more and plan to study them the next day only to find that the next day, they have vanished. To avoid this frustration is one of the primary reasons for trying to culture organisms. Whenever I find a really intriguing organism and there seems to be only a few specimens of it in a sample, I try either to photograph or video-tape it, the latter being preferred, since even though the image quality is not as good as photograph, video provides a chronicle of behavior of the organism along with a series of different perspectives. There are many different approaches to trying to understand an organism and minute cytological detail is often not the first priority. I sometimes initially sacrifice detail and resolution in order to get a larger overview of an organism’s morphology and behavior. I sometimes break one of the fundamental Commandments of Microscopy and don’t use a cover glass.
Here is another way in which we, often without thinking about it, create a rigorously artificial environment. We place a drop of pond sample on a slide and then place a cover glass on top of the drop. Think about it; at the micro-level, this is an enormous object with great weight, exerting an enormous pressure. For bacteria, very small flagellates and ciliates, such an environmental change usually makes little observable difference. However, the situation is quite different when we consider large, elegant organisms, such as, Stentor coeruleus, Amoeba proteus, Bursaria truncatella, Spirostomum ambiguum, Volvox globator, Nassula ornata, Frontonia, and Actinosphaerium. A cover glass may very well crush them and, at the very least, produce significant distortions. It is easy to forget that these are 3-dimensional organisms and a vivid way of reminding ourselves of that fact is to watch Amoeba proteus through a stereo microscope as it extends its pseudopodia not only on the X-Y axes, but on a Z axis as well–moving up toward us as we are observing it!
So, first of all, I video-tape at low magnification without a cover glass. This way I at least have a rough visual record to help me identify it if it’s an unknown or possibly such a record may provide me with additional information if it is an organism know to me. The classic case for me was when I found a Paradileptus conicus dividing. This striking organism is rather uncommon where I live and it looks rather like a squat ice cream cone with an adder’s neck and head projecting up from the rim and curving above the cone. The “neck” is heavily ciliated and flexible giving Paradileptus a highly distinctive manner of swimming. The dividing specimen was still joined along the cone and each part had a “neck” and their struggle to separate made their motion quite erratic. I managed to capture their strange dance for only about 10 seconds on videotape and then the pressure of the cover glass crushed them. I keep a small dropper bottle of artesian water next to the microscope, but the problem was that if I added water to the slide, they might very well go rapidly swimming off out of the field of view and by the time I found them, they might very well have separated looking like two “ordinary” Paradileptus. I took a risk and didn’t add water and, as a consequence, I both won and lost. I do, at least, have 10 seconds of videotape showing Paradileptus conicus dividing and who knows, it might be the only visual record of this phenomenon in existence. I am sure that amateurs all over the world have treasure troves of private observations and images which new technologies have just begun to let us share with one another.
If you don’t have access to such equipment, make drawing. If you do have such equipment, make drawings after you photograph or videotape. Drawing can get you into the habit of noticing detail you might otherwise very well miss. Technology, as wonderful as it is, can make us a bit lazy and not as observant as we could and should be.
If we could produce stable cultures of any organisms we wished to study, life would, in some respects, be much easier. I spent almost two years, along with the help of my friend and former student, Mike Shappell, figuring out how to get stable cultures of Lacrymaria olor. But some organisms are utterly intractable; they are like rebellious teenagers and vigorously resist any attempt to “tame” and cultivate them. However, whenever you find an interesting organism which you would like to study in detail, then it’s always worth the effort to try to culture them.
A few simple suggestions may be helpful:
Use a culture dish of the appropriate size. I use 3 sizes: 1 inch, 2 inch, and 4 inch in diameter. If you have only one specimen or even 4 or 5, it generally makes best sense to try to start them in a 1 inch dish with very limited food. If you toss them into a 4 inch dish, it’s likely that you’ll never find them again unless they are hardy, adapt quickly, and reproduce relatively rapidly and, if they meet these conditions, they won’t be much of a problem to culture anyway.
If you place them in a 2 inch dish and you intend to add a rich food source to produce bacterial growth, you must adjust that carefully. If, for example, you plan to use a rice grain or a boiled wheat grain in such a small dish, you need to cut or mash it, so that you can add just 1/4 to 1/3 of a grain. Mashing or cutting (be careful not to cut your fingers!) has the advantage of opening the grain so that more of the inside food materials that produce rapid bacterial growth are available sooner. In a small dish, such bacterial growth can rapidly alter the chemistry of the environment and become toxic to the organisms you are trying to culture, so the small dishes should be monitored daily.
If the organisms begin to reproduce steadily, then transfer them to a 2 inch dish with an appropriate amount of food and, if they become abundant, you may wish to transfer them to 4 inch dishes which generally allow you to a create a somewhat more stable environment requiring less maintenance.
Food and Salt Media
I have already mentioned a few aspects regarding this issue, but some additional remarks may prove helpful.
a) By boiling wheat grains for 5 to 10 minutes, you can destroy most of the spores and cysts that have lodged on their surface. Use wheat that hasn’t been treated with an antifungal agent, since that would add an additional chemical complication in terms of your cultures. I believe the practice has been banned (but that doesn’t mean that it’s not still being practiced) of using mercury compounds as an antifungal agent.
Unless you plan to dry and store some of the wheat grains, prepare only what you need for the batch of cultures you are making up at the moment. I usually cover the bottom of a beaker with the grains and then add 2 to 3 inches of water. I find it can also be convenient to microwave them, but only for 2 to 3 minutes!
You will need to experiment to find the right ratio between food and water, but initially, it’s best to be conservative with the food. Ideally, the culture dishes should be prepared 36 to 48 hours in advance of inoculating them with the organisms. For me, that rarely happens. I often tend to go collecting rather on the spur of the moment and I sometimes bring back enough material to occupy any sane person for a month. However, if the collecting has been particularly good or exceptionally poor, I might be back out collecting the very next week. When working through samples, it is not uncommon to come across organisms which are new or unusual to you and for which you want to start a culture, but have made no previous preparations. However, by no means is all lost.
Divide your sample with the interesting beasties into at least 2 dishes. Make one (or more) your reference sample which you keep in as nearly a natural condition as is feasible. Take the other portion and pipet out the desired organisms and place them in a culture dish, preferably in a salt medium, such as Giese’s or Chalkey’s. Add a modest amount of boiled wheat (or unboiled rice–it tends to get sticky when boiled) and then see if you can determine what sort of organisms in the sample may be a good food supply. For many protozoans, Chilomonas is apparently a tasty staple and for others, bacteria, and for yet others, organisms as large as Colpidium or even Paramecium.
Add a supply of what seem to be like food organisms while excluding potential predators. This may help your organisms survive until the culture has had time to achieve some sort of balance.
If you wish to have a supply of boiled, dried wheat grains on hand, take the ones you don’t immediately use, spread them on a paper towel and leave them to dry, then place them in a vial for future use. They will accumulate some fungal spores and some bacterial and tiny protozoan cysts from the air. This is generally not a particular problem and is virtually unavoidable unless you take exceptional precautions and have elaborate facilities. Some professionals have gone so far as to develop axenic cultures, that is, ones containing only the organisms being cultured. This requires mapping out the metabolic needs of the organism, creating a salt solution containing the proper balance of nutrients, sterilizing everything, and even using multiple washings to remove bacteria from the surface of the protozoan. Clearly these techniques are suitable only for a very limited number of species. Also such cultures are virtually impossible for large predatory ciliates.
Cultures of just one species of organisms along with only food organisms in a salt solution are somewhat easier, but still involve elaborate preparation. Generally, the best sort of cultures for the amateur aquatic biologist to strive for are ones that don’t require a great deal of maintenance and ones in which the desired organism retains dominance and reproduces in sufficient number to allow the application of numerous techniques of examination and experimentation.
Naturally, there are exceptions as you have no doubt come to suspect in dealing with complex biological systems. Sometimes a chaotic, unmanageable culture or an old apparently depleted one will produce surprising and pleasing results. Let me give just one example. Lacrymaria cultures can lose viability very quickly and I am not always prompt about eliminating old cultures and tidying up. In any case, on examining some old former Lacrymaria cultures, I noticed some grayish-brown clouds of dense bacterial growth on the bottom. Nothing surprising in that. However, I noticed something else that was quite surprising–tiny, clear, circular spaces scattered here and there in the bacterial cloud. Looking more carefully, I noticed that in the center of the little dome, there appeared to be something inside. I took a sample and put it on the slide, hoping that some of this material would remain intact and I was lucky. A couple of little domes were intact and what I found was a tiny amoeba with filose pseudopodia enclosed in a thin membrane (the dome) of mucous through which apparently the pseudopodia can extend to obtain food. I later got a much better view of these amazing little creatures when I cultured them in a special chamber and observed them with an inverted microscope. So, don’t always automatically toss out old cultures.
As I mentioned earlier, boiled rice can get sticky and this can be a disadvantage, since some organisms will get trapped in the “glue”. An unboiled rice grain will very quickly develop fungal hyphae and these will look like minute rootlets which soon develop small spheres at the tips which are bundles of spores and give the rice grain the appearance of an elongated, fuzzy, white football. In a number of instances, this can be a benefit and sometimes large numbers of organisms will take refuge in the miniature forest of hyphae.
c) Timothy Hay
Why Timothy hay in particular? I have no idea, but again and again, especially in older literature, this specific type of hay is recommended. Other kinds will certain work and only recently did I manage to find some Timothy hay–at a large discount store in the pet section, recommended as bedding for guinea pigs.
Whatever sort you use, cut it into 1 inch sections and boil them for about 10 minutes. For certain organisms, hay infusions are a better culture medium than either wheat or rice, although sometimes combinations are used. Hay is also sometimes added to soil media.
d) Cereal Grass, Dried
The commercial product called Cerophyll is often recommended and consists, I gather, largely of dried shots, oops, that’s shoots of rye. Also some biological supply houses provide protozoan pellets, which seem to consists largely of plant material. That such material serves as good culture media shouldn’t surprise us, since you have undoubtedly noticed that ponds will have grasses and leaves floating or submerged in them and such plants are often covered with a rich assortment of micro fauna and flora. If it’s midwinter (and I wish it were–I start to melt when it get much above 70 degrees F.) and you’ve run out of leaves or your supply of cereal grass, you can spread out some lettuce leaves on a cookie sheet and bake them until they are dry and crisp, but still green. These can then be crumbled up and stored in a small jar or a plastic sandwich bag until needed.
e) Salt Media
As I have already mentioned I find Giese’s solution and Chalkely’s solution to be valuable general purpose salt media especially when combined with wheat, rice, or hay. For media for specific groups of protozoa consult a standard reference on biological techniques. Algae often require somewhat more specific types of salt media and Carolina Biological Supply Company has a booklet titled Culturing Algae which, at the back, lists recipes for 12 different media and also advertizes 2 of their own proprietary media. They describe the preparation of soil-water medium and this is a medium well worth experimenting with for certain forms of algae and protozoa.
If you use salt water (or the version made up from marine salts) and distilled water, you can grow certain marine and brackish algae and protozoa. The issue of the culture of marine micro-organisms is, however, a topic for another essay.
Direct sunlight is rarely desirable for cultures with the exception of certain forms of algae which reproduce rampantly and become micro-versions of the notorious kudzu vine. Moderate, indirect light is the usual standard and some protozoa and algae have special “eyespots” or contain special pigments which provide biochemical signals to help them maintain an appropriate spot in the water column with optimal light and temperature gradients. Large commercial and research laboratories sometimes have rooms with special lighting controlled by timing mechanisms. Some interesting studies on photoperiodism in both protozoa and algae have been conducted and one intriguing result was the discovery that with certain protozoa, an extended period of darkness can induce conjugation.
The accepted ideal temperature for protozoan and algal cultures is 20 degree C. or 68 degree F. However, there are organisms which survive at remarkable extremes–algae that thrive in hot springs in Yellowstone Park and small algae and protozoa which grow on the surface of snowbanks. Clearly organisms from either of those environments are not going to do well at room temperature and require special techniques and facilities for culture and examination. In general, it’s better to have cultures a bit too cool than too warm. As temperature increases the rate of bacterial growth increases enormously and fairly rapidly creates difficult or even toxic environments for many organisms.
The generally recommended pH for cultures is 7.0 for the majority of larger aquatic micro-organisms. As always, there are exceptions. There are some which appear only in somewhat acid bogs and others that seem to prefer alkaline pools and lakes. Here in the high plains, the water is quite alkaline and as the summer progresses and evaporation takes place, the alkalinity increases significantly. For 7 years now, we have been experiencing drought and this year the spring levels of runoff from the mountain snow peaks was very low, so already in June, white deposits of alkaline salts could be seen around the edges of most lakes and ponds. Usually this doesn’t occur until August. For some reason, certain hypotrichs seem to thrive in pools where I would not have expected to find any mid-sized or large protozoans.
In Part II, we’ll take a look at issues related to subculturing.
All comments to the author Richard Howey are welcomed.
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