& Data Analysis
Protein gel analysis
Keeping a lab notebook
Writing research papers
Dimensions & units
Using figures (graphs)
Examples of graphs
Principles of microscopy
Solutions & dilutions
Fractionation & centrifugation
Radioisotopes and detection
Guide to the study
Lab part 1
Lab part 2
"Pond" Cultures of Microscopic Invertebrates
The diversity of microscopic life in samples from a well-illuminated, unpolluted source of fresh water can be astounding. So too can be the contrast between the quantity and diversity of life in such a sample and one from a source that is polluted, protected from sunlight, or lacking in key nutrients. For both quantity and diversity, an unpolluted heterotrophic pond or lake in full sunlight is ideal. sunlight, of course, provides a source of energy for autotrophs - both protists and plants - and allows the establishment of a food chain. Photosynthetic organisms, being pigmented, are easily seen with a dedicated bright field microscope or even with a hand lens. A freshly collected sample should show a lot of action in any microscope field.
Sampling should be done the day the observations are to be made, since a balanced system depends on many factors in order to maintain diversity. We have found that a day or two in a lab in artificial light and constant temperature is sufficient to reduce the diversity in a sample considerably. The photosyhthetics are the first to go, followed by many of the non-photosynthetic protist and animal species, leaving just a few heterotrophs to feed on the organic material they leave behind.
We use a plastic beaker attached to a long pole for collecting. Plan to take multiple samples. A scoopful from open water may contain very little life, while a scoopful from an algae mat may be teeming. Collect debris from the bottom, including rotting leaves and branches. Samples collected from a shady part of the source will contain different species than those collected from a sunny location. Try a number of sources, including permanent ponds, lakes, slow streams, and even wet areas that dry up in summer. Take notes on what you find at what times of the year. A fine source in spring might be hopelessly devoid of interest in fall.
Samples are best kept separately in large jug or jars, with high surface-to-volume ratio. That is, fill them part way so that gas exhchange can take place. Avoid temperature extremes, and place samples in wide bowls with strong illumination (but watch the heat!) once they are in the laboratory.
Place the aquarium in a window where it will receive sunlight for most of the day. Alternatively or additionally you may place a grow light over the tank. Add gravel to cover the bottom to a depth of about one inch. Add water from a natural source such as a lake or pond, if available. If not, you can use bottled drinking water (not distilled or carbonated water), or use tap water treated to remove chlorine. If you do not treat the tap water let it stand for a couple of days before adding samples. If your water contains chloramines, the use of tap water is not recommended. A tropical fish store can test your water for chloramine.
If you have an air pump, anchor the air stone at the bottom of the tank. Don't use an undergravel filter. The idea is to have everything growing naturally. Undergravel filters trap bacteria below the gravel where they process ammonia and other deadly chemicals.
To diversify the environment, you can cover the top and sides of one end of the tank with aluminum foil so that there is a gradient of light from one end of the tank to the other. Light cycles are important - put the light on a timer so that the light cycle is close to the daily outdoor light cycle for the time of year.
Add pond samples and other materials to your tank.
You can get samples from almost any safe freshwater source, since even rainwater collected in your back yard will grow a variety of plants and animals if you toss a bit of dirt in the bottom of the container. The best source, however, is a shallow pond in a sunny location. Collect samples from the surface - pond scum teems with algae and animal life. Collect some of the debris on the bottom of the pond such as rotting leaves or twigs - living things congregate around anchoring sites, not usually in the open water. Collect some vegetation as well, including vascular plants and mats of algae. Dirt adhering to the plants should be kept, not washed off.
Add samples to your tank. You only need a small quantity of each type of sample. If the samples make the tank cloudy don't worry, as it will clear in a few hours or days. To add some interest to your tank you might toss in a few large invertebrates such as snails or a crayfish or two.
To observe the organisms, periodically take samples from microenvironments within the tank. For example, a dish containing some of the floating material on the surface should be rich with green (photosynthetic) organisms, since the light is strongest there. At the bottom, scavengers live among the gravel and in the clumps of rotting vegetation. Floating mats of algae may have a different collection of plants and animals. Look at the leaves and roots of plants as well. You should be able to see a succession of different species as each population peaks then dies back.
A large outside or greenhouse tank with renewable water source offers more promise in terms of maintaining a balanced system with both quantity and diversity of life.
Direct observation of culturesChanges in the color and texture of the surface or liquid provide hints as to the presence of living organisms. In freshwater samples, look for plant growth or movement in "microenvironments" such as on top of gravel, around rocks, sticks, or plants, or floating on the surface of an aquarium. You might find a difference in color or texture of the water near a light source or air stone. Any differences in an otherwise uniform environment are a hint that there might be a microscopic "zoo" right there under your nose.
A good way to shake things loose is to shake a plant in a bucket of clean water, and see what comes out. Soil or gravel can be washed in a bucket, releasing "beasties" into the water. You can scoop some scum from the surface of the water into a shallow dish and watch carefully for movement. Decaying material at the bottom of a tank or pond is usually a rich and diverse source of freshwater invertebrates.
Using a magnifying lens - 2x to 4xYou would be surprised what you can see just by magnifying something a few times. In water samples you may see creatures swimming around that you couldn't detect without the magnifier. It is best to use a shallow dish to examine water samples, and keep in mind there are different levels. If you focus on the surface, you may miss something that is on the bottom. A drawback to looking at living things is that they often move! If something moves too fast, you might try catching it and confining it to a small space, such as one well of a depression slide.
Using a stereomicroscope - 10x to 40xA low power view of a shallow (petri) dish or depression slide containing a pond water sample can reveal an incredible microscopic "zoo." Transmitted light (through the bottom of the dish) is best for viewing such a sample, since direct light produces reflections. Move the dish around until some material is in the field of view, focus, then watch carefully without touching the dish. When the dish is still, you will see that things in the water are still moving. As you become more skilled in observing the sample, you will be surprised at how many things are moving.
Using a compound microscope - 40x to 1000xIf you have access to a compound microscope, you might prepare a wet mount (also called a vaseline mount) for long term (e.g., several hours) observation of cultures. Specialized lighting (phase contrast or dark field) is best for viewing organisms that require 100x magnification or better, since they generally have very little pigmentation. You will find more and more diverse forms of life as your observing skills improve.