Culturing and Collecting Micro-Organisms Safely

by Richard L. Howey, Wyoming, USA

 Part I

 The very concept of micro-organisms makes some people uneasy and they have a tendency to regard them as inherently dangerous.  Over the years a number of people have asked me whether or not my collecting pond samples is safe.  My reply is:  "Safer than crossing the street in New York or London."  Risks are an inherent part of living.  Eating the wrong batch of food can prove fatal, drinking tainted water can make you very ill, and sky-diving with a homemade parachute is generally not to be recommended.  The point, of course, is that there are all sorts of degrees of risk and one needs to take reasonable precautions in studying micro-organisms just as one takes precautions in the rest of one's day-to-day activities.

 These precautions should extend to the general environment in which you are collecting.  For example, if there are rattlesnakes, grizzly bears, wild boars (or wild bores) in a particular area, then either avoid the area or take very careful measures to minimize the possibility of any encounters.  These are not the sorts of encounters you normally expect to have, but this year already several tourists have been mauled by grizzly bears in Yellowstone Park.  Others have been severely burned in hot springs as a result of wandering off the paths despite repeated warnings.  Human beings often don't exercise common sense and, as a consequence, become a danger to themselves and others.  So, in collecting micro-organisms, simply be sensible and learn the potential risks in the environment where you are collecting.  Ticks, fleas, and poisonous plants are also things you should be aware of.  I strongly suspect that far more people have gotten ill or injured from ignoring the macro-environment than from collecting from the micro-environment.

 So, now, what about the ponds and streams and lakes and rivers?  Well, watch out for the currents, sink holes, quicksand, alligators, and hippos.  Then we're ready to collect.  I often collect in high mountain areas where there are streams with crystal clear water dashing over the rocks.  The sun is very intense at 9,000 feet and the water looks so pure and inviting that many people are tempted to take a deep drink out of such a clear stream or mountain spring.  Some regret it for weeks.  Elk and other ruminants are also attracted to clear water and may leave their feces in the water.  Such waste can contain a nasty little protozoan called GiardiaGiardia can produce a very severe recurrent type of diarrhea.  Getting rid of the organism requires some potent drugs, since the organisms can encyst in the walls of your intestine and then become active again and again.  So, don't drink the water, no matter how clear and fresh it looks.  Carry your own plastic bottle, but label it clearly so that you don't end up drinking one of your samples.  By the way, in some parts of the world, Giardia even shows up in city drink water supplies, so when traveling it's best to drink bottled water.

 In collecting in ponds in pastures and grazing areas, one can often encounter cow and\or horse manure in the water.  For this reason, as well as the possibility of stepping on rusty nails or puncturing one's finger on an old piece of barbed wire, keep your tetanus shots current.  They last for a number of years, are inexpensive, and not something to procrastinate about.  You might also want to carry a pair or two of sturdy, but light-weight rubber gloves with you on your expeditions for collecting in such ponds, especially if you have any nicks or cuts on your hands.  Another annoyance you might encounter is what's known as "swimmer's itch".  In areas where there are sheep, one frequently finds, at certain times of the year, the cercaria or larval stages of the sheep liver fluke in ponds.  These can produce a skin irritation and rash that is rather like being exposed to nettles.

 Usually it is best to go collecting with another person or with a small group, but if like Garbo, you vant to be alone, then at least let someone know where you are going and when you expect to return.
 Now, with all of these warnings, you have no doubt decided that the only sensible course is to put some sample jars with dead leaves out in the back yard and wait for the rain, thus avoiding all of these dangers.

 In forty-five years of tramping around in forests, meadows, plains, semi-desert, and high mountains areas, I have never encountered a bear, wolf, rattlesnake, or Tyrannosaurus.  I have never, to my knowledge, contracted any illnesses or infections other than a few head colds.  However, I have seen eagles, avocets, blue heron, pelicans, antelope, mule deer, chipmunks, voles, beaver, butterflies, dragonflies, iridescent blister beetles, wild orchids, snow lilies, red algae growing on the surface of snow banks, and mosquitos—lots of mosquitos.

 Part II

 Having braved the wilds, we can now triumphantly return to the laboratory with  bottles of samples filled with micro-wonders.

 Here I want to introduce a distinction.  These bottles which we have just brought back to the lab, I will continue to refer to as samples.  Dishes into which one or several types of organisms are isolated and placed into special media, I shall refer to as cultures.

 The samples just collected should be immediately uncapped and placed in a cool, well-lit area, away from direct sunlight.  Your samples are artificial environments and are quite different from the pond, lake, or stream from which you collected them.  Because of the small volume of water, the temperature will rise significantly and rapidly and, as a consequence of the lack of currents or wave action, the oxygen will be depleted more rapidly and that corner of your lab may begin to smell like a malarial swamp.

 There are three main causes for rapid deterioration of samples:  1) too much vegetation in too small a volume of water, 2) a high concentration of alkaline salts, or 3) samples from muddy bottoms which are oxygen depleted.  If you find a sample that smells like the brimstone that the old-style preachers predict for sinners such as myself, then close the sample up immediately and dispose of it outdoors.  The rotten egg smell could be an indicator of hydrogen sulfide which is a highly toxic and dangerous gas, so it's the sulfurous-smelling ones to be wary of.  Fortunately this is rarely a problem and in having kept thousands of samples over the years, I have run into this problem only five or six times.

 However, I have had quite a few very smelly, but non-sulfurous samples.  This kind of investigating is not good training for working in a perfumery.   If you happen to be one of those few individuals whose olfactory nerves don't function, then no worry.  However, most of us may want to resort to some kind of solid air freshener that can be placed in the vicinity of the riper samples.  However, don't expect too much; the combination of your samples and the freshener may produce some exotic aromas, such as, Rotting Gardenia, or Floral Pond Scum.

 But what about nasty bacteria, protozoa, fungi and other pathogens and parasites?  Well, again, one has to exercise common sense and in order to do that there are some basic guidelines:

1) No beginner should work with pathogenic or parasitic organisms except under controlled laboratory conditions and under expert supervision by a specialist.  If you are curious about pathogens and parasites, then either obtain prepared sides or work with preserved material that has been well fixed.  I made a decision years ago to work almost exclusively on invertebrates and the few vertebrates I have worked on have always been preserved.  Even if you develop a passion to be a bacteriologist or parasitologist, you must begin with preserved materials until you can find access to a properly equipped laboratory under supervision.

 If you find a dead animal, the best course is to leave it lie, unless you have come prepared with rubber gloves, tongs, a proper fixative, such as formaldehyde1, and appropriate containers in which to preserve the creature.  As you move up the evolutionary chain from fish to amphibians to reptiles to birds to mammals, the risk increases.  If you find a small dead fish at the edge of a pond, it's very likely safe enough to pop it in a jar or plastic bag and examine at home.  But with mammals, the risks are too great.  You should never handle dead animals and should especially avoid contact with their blood, feces, and entrails. Small rodents are often objects of curiosity and interest, but some of them carry Hanta virus which can produce a deadly fever and others harbor fleas that can carry plague.  Some waterfowl can carry cholera; armadillos which are a natural magnet for one's curiosity, can carry the organism that causes leprosy.  Ticks, fleas, and mites that feed on mammals can all be vectors for transmitting diseases.

 Well, all of this sounds awfully grim.  The trick is to use common sense and keep things in perspective.  I have been collecting in Wyoming for over 30 years and there are ticks here that carry various sorts of fever, including Rocky Mountain Spotted Fever, which can be quite serious—and, in all that time, I've only ever found two ticks and I felt them crawling along my arm and simply flicked them off.  I take some care about dressing appropriately for collecting, taking sun screen and insect repellent and then I can relax and enjoy the experience.

2) When you return to your lab and are examining samples or setting up cultures, NEVER pipet by mouth.  ALWAYS use a rubber bulb on the pipet.

3) It is by far the best not to have food or drink in your laboratory area.  You might be in for an unpleasant surprise if you absentmindedly take a sip of your coffee and it turns out to be a sample instead.

4) Keep your lab, glassware and tools clean.  Be careful not to spill the samples on your hands and if you do, wash immediately with soap and water and rinse with rubbing alcohol.  If you have any nicks or cuts, put a little iodine on them.  If you are working with samples that you have reason to believe might have manure or other contaminants, then wear thin latex gloves (assuming that you're not allergic to latex).

 From the human perspective there are some incredibly nasty organisms out there, seemingly just waiting for us to drop our guard for a moment—the Ebola viruses, HIV, Plasmodium vivax (the malarial parasite—a protozoan), trypanosomes (the cause of sleeping sickness), Naegleria (a tiny organism that has both flagellate and amoeboid stages and can enter the brain through the nasal passages and produce a fatal type of meningitis—in crude terms they are brain-eaters).  There have been outbreaks in swimming holes and even swimming pools, in rare instances where there was no chlorination, and the pollution  brought the Naegleria into the upper layers of water.  They are basically anaerobes and as the oxygen depleted level rises, so do Naegleria.

 However, unless you are extremely careless, you are much more likely to be hit by lightning or a car, than you are to expire as a consequence of collecting and studying micro-organisms in ponds, lakes, streams, and rivers.

 Life is incredibly tenacious and that is one of the most fascinating lessons one can lean from studying micro-organisms.  Every organism is looking for niches in which it can survive.  As it turns out, given the enormous numbers of bacteria, fungi, viruses, protozoa, round worms, and flatworms, only very few, relatively speaking, find us a desirable target and, of those that do, many are beneficial to us.

 Life has adapted to incredible environments, from a red algae that lives on snow banks to bacteria that thrive in hot springs. But this is a subject for another essay.

 Let us now turn to the issue of cultures. Sometimes you may develop a special interest in a particular organism and want to examine it in a variety of ways using a range of different techniques.  This, of course, means that you need large numbers of the specific organism.  There are several different types of cultures.  Professional protozoologists have developed, for a few species, axenic cultures; that is, cultures which contain only the specific organism being studied.  Elaborate procedures are employed, such as, special washing techniques for the organism, sometimes involving the use of detergents and antibiotics to remove any bacteria clinging to the surface of the organism. This type of culture also involves figuring out the nutritional needs of the organism in question.  If not even bacteria are permitted in the culture as food, then the specialist must have a detailed and precise knowledge of the essential metabolic requirements.  These artificial media consist largely of inorganic salts and the formulas sometimes run for over a page.  Clearly this is not for the beginner nor for most amateurs.  However, on reflection, two important points emerge:  1) if you want relatively "clean" cultures, then it is necessary to use an appropriate medium and 2) all cultures are artificial environments.

 Let's examine the second point first.  Organisms generally behave quite differently when their environment is radically altered and most protozoa are no exception.  A culture dish is no substitute for a pond.  In the small contained world of a culture dish, the balance of salts, the oxygen levels, the pH, and the temperature can all change significantly in a short time.  In alpine lakes, the water temperature can be 45 degrees Fahrenheit or less and I sometimes take along a couple of small thermos flasks to keep a sample or two in a near natural state until I can get them into the lab for immediate examination.  Interestingly these are protozoa and algae that thrive in the cold and nutrient rich water and many of them disappear from the sample within 24 to 48 hours to be replaced by quite a different range of species.

 Also here there are alkaline lakes and ponds in the high plains which as the summer progresses, begin to dry up thus increasing the concentration of salts.  Some of the lakes are surrounded at the edges by hardy sagebrush and during the spring runoff from the melting snow, the shrubs are covered by water.  As the summer progresses and the water recedes, the alkaline salts are deposited on the sagebrush sometimes to a thickness of half an inch, thus creating a miniature sculpture garden of strange and wonderful forms.  Now, imagine what's happening in the lake or pond!  As the salt concentration increases, the number of species decreases dramatically, but there remain some fascinating ciliates, especially hypotrichs, rotifers, and tiny flagellates which again demonstrate the ability of life forms to adapt to extreme conditions.

 It can be rewarding to investigate a wide variety of environments.  Just this afternoon, I was checking some samples that I collected earlier in the morning at a lake about 20 miles southwest of Laramie.  The lake was warm and relatively still and almost no vegetation was to be found at the edges in the water.  However, I did notice small schools of fingerling trout, no more than two inches long.  I thought no more about them and collected my samples and headed home to examine them.  Often, in midsummer, freshly collected samples are initially rather disappointing, especially protozoa-wise.  However, sometimes after sitting for a day to two, all sorts of interesting things will appear.  Well, these samples were rather uninteresting and then I noticed a gelatinous blob filled with elongate, cigar-shaped eggs.  Ordinarily I don't much bother looking at eggs, except snail eggs which are fascinating, but I decided to take a closer look at these which I am fairly certain were trout eggs, but since I am so ignorant in this area, they could be coelocanth eggs for all I know, except they're marine, so I guess not.  The egg, under higher magnification immediately brought to mind the organic patterns and designs of Giger in the huge space ship at the beginning of the film Alien.  But what really caught my attention was the fact that within this mass of gelatin, in which the eggs were imbedded, were diatoms and some very active protozoa.  In fact, so far, I've found five species of ciliates and two flagellates and one of these ciliates, a fairly large fellow of 150 to 200 microns, is a diatom eater and every specimen was stuffed full of diatoms, the soft parts of which were slowly being digested.

But as always, I digress, so back to cultures!

 Another type of culture is the so-called pure culture, which means that the culture contains the organism you're trying to raise and one or two other food organisms, sometimes bacteria, sometimes bacteria and a small protozoan.  The small protozoa eat the bacteria, the bigger organisms you're trying to grow eat the small protozoa and you get lots and lots of beasties to study and everybody's happy.

 Yet another type of culture is the single strain culture. Say you want a few thousand specimens of Spirostomum and you want them all to be genetically the same.  You know that Spirostomum are bacterial feeders, so you take a small culture dish, put in a grain of boiled wheat and add one, yes only one—Spirostomum.  It's best to let the bacteria build up by allowing the culture to sit for a day before adding the Spirostomum.  Why boil the wheat?—which by the way, you should boil for about five minutes.  To kill off most of the fungal spores, protozoan cysts, bacteria spores, rotifer eggs, gastrotrich eggs, etc.  Set up a number of cultures, because, unless you are much luckier than I am, some of them won't take off and you'll just be left with some dishes of smelly water.

 Usually what happens to me is that I'll be sorting through some new samples and come across an interesting beastie and I decide to try to culture it.  So, I get out a culture dish or two, add some diluted Giese salts—I'll tell you about salt solutions in a minute—, add a boiled wheat grain, add some Chilomonas culture—I'll tell you about Chilomonas in two minutes—, then using a micropipet—I'll tell you about micropipets in three minutes—I isolate the specimens I want and put them in the culture dishes.  Again if you are taking the trouble to try to culture this organism, start at least two cultures if possible; however, it's not always possible.  This afternoon I found a lovely large amoeba—small ones are common, but the big ones are rare in the wild.  I spent an hour sorting through the sample, convinced as always, that if there's one, then there has to be four or seven more.
But, no—just the one.

 O.K., so let's clean up the details I mentioned above and then see if I can get to the point.

1) Boiled wheat or rice grains are excellent for producing rapid bacterial growth and work well for culturing many ciliates, flagellates, and amoebas.

2) Dried lettuce leaves or dried cereal grasses (commercially available from biological supply houses under the name of Cerophyll) work well for many kinds of organisms that require metabolites from green plant material.

3) Water is important.  Tap water is often chlorinated and can be toxic to the organisms, in fact, that's the whole idea of adding chlorine to drinking water.  If no other source is readily available, then draw off a gallon or two of tap water and allow it to sit uncovered for 24 hours to permit the chlorine to dissipate. If this still does not prove suitable, it may be that there are traces of metals in the water that adversely effect the organisms.  In that case, try artesian water as a substitute or one can, of course, drag home several gallons of pond water—not so convenient, if you are hiking or cycling—and then filter and boil it.

 Interestingly, distilled water is toxic to some micro-organisms.  The complete lack of salts messes up the osmo-regulatory system.  You can use distilled water, if you add inorganic salts.—Will this man never get to the point?  Well, I mentioned Giese salts above.  There are many salt solutions that protozoologists have concocted over the last couple of centuries and there are two that I keep stock solutions of:

1) Giese salts (diluted with artesian water, which you can buy in plastic gallon containers at the local market)

Giese Salt Solution

Concentrate (dilute 1 part solution to 9 parts water)

NaCl  1.04 gm.
KCl  0.23 gm.
MgCl2 0.85 gm.
MgSO4 0.01 gm.
CaCl2 0.02 gm.

Superconcentrate—(dilute 10cc. to 990cc. water)

NaCl      10.40 gm.
KCl  2.23 gm.
MgCl2 8.50 gm.
MgSO4 0.04 gm.
CaCl2 0.15 gm.

2) Chalkey's solution

NaCl  1.0 gm.
KCl  0.04 gm.
CaCl2 0.06 gm.
Distilled Water 10 liters

These seem like such tiny amounts of salts per volume of water that one might get the impression that they can't make any difference, but, as a matter of fact, they do make a very real difference and these are very simple solutions compared to some.

 I mentioned artesian water above and usually this contains enough salts that one can use it without adding anything and it's less than a dollar a gallon—cheaper than petrol!  Why didn't you say so in the first place, I can hear you shouting?  Well, science is all about mysteries and we can't let people believe that science has any parts that are simple—that detracts from the mystery!

4)  Chilomonas cultures.  Chilomonas is a small colorless flagellate which is an excellent food organism for many larger protozoa and, best of all, it's exceptionally easy to culture.  You can find descriptions and drawings of it in Jahn's How To Know The Protozoa, Kudo's Protozoology or almost any basic book on protozoa.  They are common in pond samples containing abundant decaying vegetation.  Set up a culture dish with a salt solution, a grain of boiled wheat and inoculate it with Chilomonas.  In order to get only Chilomonas, take a small amount of the sample, dilute it in a watch glass and then using a micropipet, pick out the Chilomonas avoiding any other protozoa.  Once you have these single organism cultures established, it is very easy to subculture and insure that you always have a ready supply of Chilomonas on hand.

5)  Micropipets:  By far the best solution is to make your own using disposable Pasteur pipets.  You can buy a box of 250 for a quite reasonable price.  There are several styles.  Make sure the ones you get have a tip long enough for you to hold it in the flame of an alcohol lamp.  Use methanol (wood alcohol) and exercise caution—not only is it flammable, but it is poisonous and toxic, so don't try to drink it and if you spill any on your skin, wash it off immediately.  Some other types of alcohol will leave a black sooty deposit on the glass. Holding both ends, heat the tip, taking care not to burn your fingers while rotating it in the flame.  When it begins to glow red, then pull gently, but quickly, on the tip end.

 Be sure you carry out this procedure in an area away from all flammable materials and in a place where the alcohol lamp is not likely to get knocked over.  With a bit of practice, you will soon be able to produce pipets with very fine tips, so that you can pick out individual organisms from a sample or culture.  Use a small soft latex bulb as this will allow you the best control.  You can get them in packets of 10 or 12 from biological supply houses.  If you get some pipets that are drawn out too finely, don't worry.  Put a piece of paper on the stage of your stereo microscope and then, using top or side lighting, take a pair of fine forceps and break off the tip of the micropipet at the desirable point.  Dispose of the glass fragments in a wastebasket, since they can be most irritating if they get into your skin.

 There can be real delight in successfully culturing a variety of protozoa and once you find a good procedure, you can keep a supply of specimens available for your work for months or even years.  It is essential to keep careful records of your various attempts, so that when you succeed you can duplicate the procedure.  It took me over two years to find a successful means of culturing Lacrymaria olor, but once found, I was able to maintain cultures continuously for over ten years.  Now, when I come across some specimens in samples and decide I want to culture them again, I can do so with very little difficulty.

 What will work splendidly for one organism will be a complete failure with another, so it's important to experiment.  When you find a procedure that works, share it with other amateurs who are interested in protozoa.  Some organisms culture readily and others, despite the best efforts of professionals, have remained resistant to culturing.  Let me suggest a few that are interesting to examine and relatively easy to culture.  By the way, I use stackable 3 inch culture dishes which conveniently hold about 30 c.c. of fluid.

1) Paramecium—though common, in several species, this organism has some remarkable morphological and behavioral characteristics.

2) Amoeba—any large amoebas you come across, you should always try to culture.  They are relatively uncommon and wonderful to observe.

3) Heliozoa (or "sun animalcules")  There are several genera of these elegant organisms that culture fairly readily and they are always a delight to look at.

4)  Coleps—they look like armored cylinders and are the jackals of the protozoan world.

5)  Stentor, especially S. coeruleus, with its distinctive dichroic pigment (depending on the angle of light, it can appear its typical blue-green color or a delicate rose color).  This trumpet-shaped protist can be the subject of a wide range of experiments. (See Vance Tartar's excellent book The Biology of Stentor.)

6) Vorticella—the little bells on contractile stalks. This protist was first described by Leeuwenhoek.

7) Spirostomum—the ciliated giant of the microworld.

 Take commonsensical precautions and the risks are very small and you will be able to enjoy a steady supply of intriguing organisms.  Riding a bicycle is more likely to be a hazard to your health than sensibly studying pond organisms.

All comments to the author Richard Howey are welcomed.

1) Formaldehyde is a suspected carcinogen and should be treated with great care.
Avoid the fumes and any skin contact.  Use it in as dilute a solution as is still consistent with effectiveness. Return to article.

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