15 October 2004

"The Amateur Scientist" Classics:

How to rear a plankton menagerie

Shawn Carlson

Although my formal training is almost entirely in physics and mathematics, I often wonder whether I wouldn't have made a better biologist. I have always reveled in the subject, sometimes even while enduring the consternation of professors who insisted that my many projects in the area meant that I wasn't a "serious" physics student.

They were quite wrong. In fact, biology frequently safeguarded my sanity during my graduate school days by providing the perfect respite from physics overload-a state of mind that I encountered all too often. Exploring the behavior of tangible things that lived and breathed let me idle my mental engines of high abstraction whenever my struggles with the vagaries of subjects like relativistic quantum field overheated them, while keeping the rest of my mind in gear.

In fact, it was none other than Nobelist Julian Schwinger who made me dive deeply into algae. Schwinger won his Nobel Prize, with Richard Feynman and Sin-Itiro Tomonaga, in 1965 for developing the theory of quantum electrodynamics, which theorists today almost universally regard as the best theory ever developed by man. As its name gives away, quantum electrodynamics describes electricity and magnetism at the quantum level, that is, where individual electrons absorb individual photons and spit them back out again.

But I didn't take quantum electrodynamics from Schwinger. Rather, I sat in his class on classical electrodynamics, which describes how electric and magnetic fields propagate through space and interact with matter on the macroscopic level. While the classical theory is supposed to be the easier of the two to grasp, Schwinger had a gift for using abstruse mathematics to make every subject impenetrable and mysterious. His non-stop two-hour lectures consisted of rapid-fire and unremitting onslaughts of high-level abstraction that were so incredibly taxing that I just had to put my mathematical mind in neutral after every class.

Schwinger was quite an odd looking fellow--short and large at the same time, as if he could have played halfback for the Munchkin football league. His great bulbous head, stuck on top of his short stocky frame, made him look like a walking caricature of an ivy-league egghead. He must have been teased mercilessly as a child. I once heard a gaggle of UCLA nymphets giggle out-loudly as he ambled by, “You follow that yellow-brick road, Poindexter!” How could any man so afflicted not have developed a powerful inferiority complex? And in modern physics lore, Schwinger's is the stuff of legend.

Schwinger's genius-level intellect provided him a ready way to compensate for his glaring physical inadequacies. In truth he was an extraordinarily brilliant man--a titanic intellect--and he needed every one of his students to fully appreciate that fact. If we happened to learn a little physics along the way, well, that was OK, too. But that never seemed to be his goal. I always felt that Schwinger's lectures were more exorcism than education, designed to banish his personal demons, not to bring enlightenment to his students. They were amazing. A tour de force of physics--supercharged, high volume, and death defying displays of mathematical legerdemain that he sometimes conducted without the safety of lecture notes.

The first time a student dared to stop his show with a question, Schwinger reacted as might a tightrope walker if stung by a peashooter. He turned from the chalkboard, glared at his interrupter and, with his voice rising in a steady crescendo responded "Infinitesimals. In-fin-i-tes-i-mals. Do you mean to say that I have to explain the concept of an infinitesimal TO A GRADUATE LEVEL CLASS?!" He scowled until the offending student broke eye contact and sank deeply into his chair. Then Schwinger turned back to the board and continued with what he was writing. Needless to say, it wasn't until near the end of the term that anyone dared to ask another question, and the only person who did was one of Schwinger's personal protégés.

Despite my love of theater, I was far too busy frantically scribbling notes to enjoy these mandatory matinees. Schwinger's was a required class, and UCLA physics professors were compelled by departmental policy to fail half of all their students in the required graduate-level courses. Since my entire future depended on beating out half my fellow students, I resorted to supercharging my attention by taking 200-milligram hits of caffeine before each lecture so I would have a fighting chance to keep apace. I then spent hours in the library afterward trying to decipher and digest the information that was hidden in what I had written down, until I was simply exhausted.

When I just couldn't bring myself to look at one more Langrangian or to integrate by parts to contemplate yet another set of boundary conditions at infinity, I walked home and enjoyed some quiet time amidst my algae and my rotifers. Without my biological diversions, I am quite certain that I never would have made the upper 50 percent in my required courses, and would, like so many of my classmates, have washed out on my academic behind.

Raising microscopic organisms is a wonderful and fulfilling pastime. What's more, it's the port of entry to many adventures in science. I highly recommend that all citizen scientists do this experiment if for no other reason than to see if you can find in it the same respite from the rat race that I have.

How to Rear a Plankton Menagerie

Shawn Carlson

Expanded from "The Amateur Scientist," Scientific American, August 2000.

The Monterey Bay Aquarium in California houses some of the finest marine exhibits in the world. So when the staff recently offered me a personal, behind-the-scenes tour, I couldn't refuse. Tim Cooke and Ed Seidel made the visit absolutely fascinating, and I am indebted to them for their hospitality. Tim, an aquarist extraordinaire, even let me in on a few secrets for raising plankton.

And he should know them: the Monterey Bay Aquarium grows a lot of plankton. Tim rears tons of the stuff each year to feed the thousands of voracious fish, crustaceans and jellies under its care. But these single-celled critters are not just fish food: they are quite intriguing in their own right and can provide amateur scientists with endless hours of delightful observation. When viewed under a microscope, the tiny phytoplankton (plants) and zooplankton (animals) are amazingly beautiful and complex.

These creatures can also be useful for many kinds of research. For example, phytoplankton such as green algae are great for investigating the fundamental biochemistry of photosynthesis. And members of a zooplankton group called rotifers, which measure a mere 400 microns across, serve as the microscopic equivalent of a miner's canary, because they are sensitive to toxins and therefore may be used to monitor the health of estuaries and streams.

Amateurs can easily rear both marine and freshwater plankton for examination, for feeding larger aquatic animals or for use in more advanced research projects. Ocean enthusiasts should go to their local aquarium store and purchase a kit to make 50 gallons of seawater (for about $15) as well as a simple salinity tester.

Citizen scientists who live near the sea can easily collect their own samples if they purchase a plankton net from a biological supply house. But if you do, you'll have to start out growing up diverse collections of organisms that may interact in non-reproducible ways. Colonies started with identical wide samples and grown up under identical conditions can sometimes turn out very differently, depending on random happenings inside the closed environment as various members of the plankton population vie for control of the limited resources inside the culture. With patients, and a technique I'll explain at the end, you can isolate a particular organism and grow up pure culture. However, for beginners it is far better to start off with a pure culture in the first place. There are a surprising number of suppliers in this field. I've used three in the past, Aquaculture Supply, www.aquaculture-supply.com, Aquatic Eco-Systems Inc. www.aquaticeco.com, and the famous Florida Aqua Farms (they have a strange Web address, so google them to find their home page.) Florida Aqua Farms are well-known innovators in this field. They manufacture some of their own products, and their staffers are all super-experts on this stuff, so I strongly suggest that you check them out first. But no matter what else you do, be sure you buy a good reference book. You'll find quite a few on any of these web sites. A few years ago I purchased Plankton Culture Manual, by Frank H. Hoff and Terry W. Snell (Florida Aqua Farms, 1999, $26.50) - and I believe it to be the Bible of plankton cultivation.

I've grown up batches of Nannochloropsis and Tetraselmis, both green algae that can live in either freshwater or salt water. And I've raised a little saltwater rotifer known as Brachionus plicatilis. You may also want to grow diatoms--a type of algae that strengthens its cell walls with fantastically beautiful silica structures. If so, a good choice might be Chaetoceros. Many other organisms are also available for experiment. You can purchase phytoplankton as cultures on Petri dishes called algae plates, or suspended in liquid. Believe me, you want to work with the algae plates. They are much less expensive, and since they can survive for six months they are much more forgiving than the liquid cultures that must be processed almost as soon as they arrive.

Clear plastic soda bottles in the two-liter size make ideal culture flasks. To prevent yours from being taken over by bacteria, you'll need to sterilize everything before you begin. So get some sanitizing solution from either pool supply store, an aqua culture supplier, or from your local trader in beer and wine making supplies. The aqua culture folks as well as the beer and wine makers sell chemicals like potassium metabisulphite or branded sterilizing agents like C-Brite, both of which come with detailed instructions for their safe use. The chemicals will evaporate from the solution over a period of hours to a day or two, so make sure not to inoculate your cultures with live organisms until the instructions say it is safe.

If you shop at a pool supply store, then you'll need to follow the instructions here. First, purchase a small quantity of granular chlorine. Dissolve as much of the solid as possible into 30 milliliters (about an ounce) of warm water. Then prepare a 10-to-1 dilution by mixing five milliliters (one teaspoon) of the concentrated chlorine solution into 45 milliliters of distilled water. Be careful you don't transfer any undissolved crystals into the sterilizing solution you are preparing. Next, fill your two-liter containers nearly to the top with either distilled water or seawater and add five drops of the sterilizing solution to each. Wait two hours for the chlorine to do its work. Chlorine evaporates quickly from the solution, so you'll have to make up a fresh batch of sterilizing fluid every time you need some. In this sense, evaporation is a nuisance, but you can take advantage of it to remove the chlorine in the flasks by bubbling air through the water for about 24 hours. A few drops of bottled dechlorinating agent from a tropical-fish store will do the job in seconds. Either way, don't introduce your plankton until you've verified, using a kit for testing home pools, that no chlorine is detectable.

A single pump for a 10-gallon aquarium can easily aerate 10 culture flasks. Use a multi-port manifold (a common piece of aquarium plumbing with one input and many outputs) to distribute the air to the different cultures. Some stiff plastic tubing (also available at the aquarium store) will allow you to inject the air at the bottom of each flask. But you should pump it through a filter with 0.5-micron openings to keep bacteria from invading your sterilized containers. The Aquaculture Supply Web site sells the laboratory-style units (part no. RD-SF10045 or RD-SF10056, pg. 98 of their catalogue) that pass air through a porous wafer with pores of known diameter. These work extremely well, but frankly they are more than you need. Personally, I prefer to use a very simple filter that I make myself by passing the air through a stiff tube about half an inch wide and about four inches long stuffed with sterile cotton. Florida Aqua Farms sells the perfect tubes with hose connections and adapters included (part no. CK-DT4) for $2.25 each.

Now enrich each flask with the appropriate nutrients. There are several commercial products available from the aquaculture supply world and every supplier sells them. I've used Micro Algae Grow. It works well with most kinds of green algae. For diatoms consider using Liquid Silicate Solution. Directions come with the packages. Make sure you follow them to the letter!

If you purchase your starter cultures on algae plates, then your plankton samples will arrive in the mail growing in small plastic dishes filled with gelatin. To remove the living cells, submerge the gel beneath a thin layer of your growing solution and allow it to soak for 12 hours. The microorganisms will then easily rub off the gel under the gentle pressure of a sterile cotton swab. Inoculate each flask with about 10 milliliters (two teaspoons) of the resulting cell-laden solution. Make sure at every step that all your instruments are germ-free by carefully washing them with detergent and sterilizing solution and then rinsing them with distilled water.

Ideally, your culture should be incubated at 19 degrees Celsius (about 66 degrees Fahrenheit), but I had no problems just letting mine sit at room temperature. Avoid exposure to direct sunlight because the sun's rays can quickly heat your small starter cultures to lethal levels. Instead place the flasks in front of a bright fluorescent lamp for 18 hours a day. A standard bulb of at least 2,500 lumens always worked fine for me, but some aquarists recommend "grow-lights," which produce more of the energetic blue photons used in photosynthesis. A light timer, like the kind sold in hardware stores, will insure your cultures get the right amount of illumination.

Once you start things going, you should keep aerating the water constantly. In about a week, your container should attain a deep green hue, which indicates that the culture is mature and ready to feed to other aquatic creatures. In as few as 10 days, the cellular population explosion can generate enough waste to poison itself; so don't wait too long before making use of it. If you extract 10 milliliters of mature culture to start a new batch, you'll never need to purchase another starter gel.

The professionals grow larger quantities of algae in 20-liter (five-gallon) containers called carboys. Some scientific supply companies charge $100 for these transparent plastic bottles, but you could just as well use a discarded five-gallon jug from a water cooler. I have used these, but I frankly prefer to use glass carboys that I purchased from my local brewer supply store. Glass is easier to clean than plastic, and even though it is a little heaver, I must confess that I also prefer the esthetics of glass containers filled with the greening cultures. These suppliers stock glass containers in 1, 2, 5, and 6 gallon sizes. Aquarists usually install a special arrangement of tubing into their carboys to pass the air through without risking contamination. I used a hot-air gun to bend a stiff plastic aquarium tube and achieved the same result [see illustration].

So, want to grow a whole lot of plankton? Fill an empty one-gallon water jug with distilled water or salt water and add five milliliters of fresh sterilizing solution. As before, let things stand at least two hours, then dechlorinate the water and test it. Add the necessary nutrients and inoculate the jug with the contents of one complete flask of mature culture. Connect the air pump and make sure the container gets plenty of fluorescent light.

You can track the rate of growth with a special dipstick that is based on the concept of the Secchi disk. Florida Aqua Farms manufactures the gold standard in this field (part no. AC-DM9), which sells for just $8.00. The simple instrument is calibrated to measure cell density. As the flat disk is lowered into the water, the turbidity obscures the white dot in the center. The depth at which the dot disappears corresponds to a concentration of algae floating in the water. Depth is recorded off the scale on the side of the dipstick and the approximate cell density is obtained from the chart provided for the following genera: Nannochloropsis, Chlorella, Isochrysis, Tetraselmis, Pyramimonas, and Nannochloris. After about a week, my water carboy had more than 10 million cells living in each milliliter--some 200 billion cells in all.

With a stable supply of algae, even if it's only two liters' worth, you'll be able to raise rotifers. Although procedures for rearing these sophisticated aquatic predators are straightforward, they are more complex than the simple steps described here for raising their algal food. The interested amateur should consult Hoff and Snell's excellent book for pointers.

OK, to answer my earlier question, how do you super do-it-yourselfers who live near an ocean isolate and grow a single species of algae from the biologically diverse samples you almost certain to get in your plankton net? It's simple. Different organisms tend to prefer somewhat different physical conditions. So if one type of algae prefers the particular conditions of light, temperature, salinity, etc. in your culture bottle it will tend to out compete the others and be better represented in the next generation of culture than the first. So, if you take a small sample, say, just a drop or two, from your first culture bottle, it will likely be less biologically diverse than your original sample, and contain a higher fraction of the happiest organisms. If you repeat this processes and grow identical cultures in sequence, each generation will have a higher fraction of the favored organism until at last, you will find yourself growing up a mono-culture. You may have to go through five or more generations, but the method always works. You'll need a microscope to know when you've produced a pure strain.

If you'd like more information about rearing plankton, or if you are looking for suggestions about what to do with your microscopic menagerie, check out the following articles from "The Amateur Scientist" in Scientific American magazine. All of these can be found on The Amateur Scientist 2.0 CD-ROM:

The Pleasures of Pond Scum, S. Carlson, Mar., 1998.

About Cultivating Algae in the Soil, A. G. Ingalls, Dec. 1954 [This is the article that got me turned on to algae. SC].

How to Build and Maintain Aquariums for Organisms That Live in the Ocean, C. L. Stong, Nov. 1962.

Experiments in Phototaxis: the Response of Organisms to Changes in Illumination, C. L. Stong, Oct. 1964.

An Observatory Built in a Pond Provides a Good View of Aquatic Animals and Plants, C. L. Stong. Oct. 1972.

The Pleasures of Exploring Ponds, S. Carlson, Sept. 1996.

How to Study the Life of a Pond and to Cultivate Aquatic Insects, C. L, Stong, Mar. 1970.

On Experiments with Gibberellic Acid Which Stimulates the Growth of Plants, C. L. Stong, Dec. 1958.

Looking into the Ways of Water Striders, the Insects That Walk (and Run) on Water, J. Walker, Nov. 1983.

How to Build a Polarograph, a Sensitive Instrument for Making Chemical Analyses, C. L. Stong, Sept. 1962.

On the Fascination of Microscopy, by A. G. Ingalls, Jan., 1953 [Explains how to build a Leeuwenhoek microscope!].