The protozoan body consists of a single cell, but it is a mistake to think of these organisms as simple. Without benefit of multicellular tissues or organs, many protozoa achieve structural complexity that rivals that of some multicellular animals.
Some biologists prefer to think of protozoan organization as “acellular” rather than “unicellular.” Whichever way you look at it, the protozoa are well worth studying. Most protozoans are microscopic, but certain amoebae reach 4 to 5 mm in diameter, and the shells of their cousins the foraminifera may be 10 cm across. Such shells accumulate on the ocean bottom, forming limestone. Incredibly enough, the Egyptian pyramids were largely built of such foraminiferan limestone; yet, this is only a relatively minor example of the important role that protozoans play in the life of mankind. It would be difficult to overestimate their importance in the food chains of soils, oceans, and fresh waters, upon which people depend.
Many protozoans are agents of disease; malaria, for example is caused by the protozoan Plasmodium vivax. The protozoans have traditionally been classified according to their means of locomotion. The shape of Amoeba proteus, a sarcodinian, constantly changes as it extends false feet (pseudopodia) from any part of its body. The pseudopodia engulf particles of food, a process known as phagocytosis. The flagellates, including Euglena and Volvox, move by means of one or more whiplike structures, flagella, which propel them through the water. Euglena moves in a spiral path. Ciliates, such as Stentor, Vorticella, and Paramecium, possess short, hairlike cilia which beat in unison, moving them rapidly through the water. Paramecia can swim either forward or backward while simultaneously rotating on their long axes.
Open your shipment at once and remove your culture jar(s). We ship a plastic dropping pipet for each culture. Label each pipet with the name of a matching culture; that is, if you receive an Amoeba culture and a Euglena culture, label 1 pipet “Amoeba” and the other “Euglena.” Use the pipet only with its matching culture. This is to avoid cross-contamination of your cultures, which could lead to confusion.
Remove the lids from the culture jars and aerate each culture using its designated dropping pipet. Replace the lids onto the jars but do not tighten them. Keep them at room temperature (20–22°C) and use them in lab as soon as possible. Never place the cultures in a refrigerator or in direct sunlight.
Allow 15–20 minutes after aeration for the protozoa to settle, and then inspect the contents using a stereomicroscope (dissecting microscope) at the lowest magnification of the scope, generally 10x. This examination will allow you to confirm that the organisms are in good condition and will also show where they are concentrated.
Begin by focusing on the water surface and then slowly focus down through the water column to the bottom of the jar. For many of our cultures, you will find 2 or more small ovate bodies on the bottom. These are wheat seeds. Various harmless bacteria feed on the breakdown products of these seeds, and the protozoans graze on the bacteria; thus, there is a simple food chain in your culture jar. Other cultures, such as Euglena, are cultured on different media and there are no wheat seeds.
Once you have located your protozoan, increase the magnification of your scope for a better view. Lacking a stereomicroscope, you may be able to use a good hand lens or digital magnifier, although this will be more tedious. Using a hand lens, you may have to examine the jar from the side to find the protozoa.
Begin at the surface and focus slowly down the water column. You may find a few amoebas floating about midway down. They send out pseudopodia in all directions, assuming an asterisk shape: *. Most of the amoebas will be on the bottom of the jar. Focus on the bottom debris. After several seconds, you will notice that some of that debris is slowly moving. Increase magnification for a better look. Their slow creeping motion is fascinating to watch. You may notice some smaller and faster organisms swimming in the culture. This is Chilomonas, a food source for Amoeba.
Euglenas are usually so numerous in the culture that there is no need to concentrate them. Should you wish to do so, place a cardboard cylinder, with a 2– to 3-mm wide slit on one side, over the jar so the slit faces a lamp. After 10 minutes, carefully lift the cylinder and examine the jar. Euglenas exhibit positive phototaxis and should be concentrated in a thin, green line along the side of the container where the light entered. You can then pick them up with a dropping pipet.
Euglena illustration showing internal structure. Note the long flagellum and the many chloroplasts that fill much of the cell.
These concentrate in the culture jar in the area around the wheat seeds, where the most bacteria are found.
Paramecium illustration showing a large macronucleus and a small micronucleus, a condition characteristic of many ciliates.
Stentor is often sessile, extending from its attachment point into a trumpet shape. This shape, reminiscent of a megaphone, is responsible for the name Stentor, which means loud-voiced.
Stentor can also be found freely swimming, at which time it assumes a pear-shape.
To dislodge sessile individuals, agitate the contents of the jar with a dropping pipet. Leave the jar near a lamp for 5 minutes. Stentors will move away from the light, exhibiting negative phototaxis. Now you can pipet them from their concentrated gathering on the side opposite the lamp.
Volvox can be seen without magnification as small green dots by looking down through the shipping jar. Upon arrival, they may be on or near the bottom of the jar. If so, they will soon resume swimming when returned to normal conditions of light and temperature. Because of their relatively large size, they may be crushed under a coverslip on a standard microscope slide and may require use of a depression slide.
This interesting ciliate has a bell-shaped body with a long, tail-like appendage that attaches to a substrate. The tail can quickly contract like a spring, pulling the cell body away from possible danger. The beating of its oral cilia creates a vortex, accounting for the name. You may have to search the water surface as well as the bottom and sides of the jars to find Vorticella.
Vorticella photo showing the bell-shaped body and the slender stalk.
If a lab assistant is to conduct the lab, have them examine the cultures with a stereomicroscope as described above so they know the characteristics of the culture and where the organisms are concentrated.
It is best to set up separate, widely spaced workstations for each protozoan culture. Assign each student group a different culture to begin with. This will prevent students from crowding around one culture and make it less likely that they will mix dropping pipets among the cultures. Each student group should make one slide, return to their work area, and examine the slide before returning to make a slide from a different culture.
Either distribute slides and coverslips at the beginning of the lab or set up one or more stations for pickup during the lab.
Have all students rinse their hands under running water (no soap or detergent) and dry them before beginning the lab. Soap, salts, or lotion residue can kill protozoans. If one group consistently reports not finding anything living on their slides, suspect that this may be the problem. To further prevent this problem, instruct students to handle slides and coverslips by their edges only.
Advise students to avoid stirring the culture with the dropping pipet as doing so will break up concentrations of protozoans, making it more difficult for others to locate them.
Reserve the last 15–20 minutes of the lab period for cleanup. Have all students rinse and dry their hands before leaving.
If you are willing to branch out a bit, we have numerous cultures that go well beyond the big three of Amoeba, Paramecium, and Euglena.
A sarcodine that secretes a shell (test) and has lobose pseudopodia.
A very large barrel-shaped ciliate that feeds on Paramecium.
Also known as Pelomyxa, Chaos looks like Amoeba but us much larger and has many nuclei.
A bizarre ciliate with a long, tapering proboscis equipped with toxicysts that enable it to capture prey.
One of the most complex of the ciliates. Many of its cilia are bundled into leg-like appendages. An interesting lab activity is to pair Euplotes with a rotifer and ask, “Which is the animal, and how do you know?”
A marine, bioluminescent dinoflagellate. Under proper conditions, Pyrocystis produces a striking blue light. Highly recommended for studies of bioluminescence.
Many protozoans form protective cysts that allow them to survive during periods of harsh environmental conditions (e.g., lack of water or food). Didinium is an ideal organism for demonstrating cyst formation. The cultures of Didinium you receive contain numerous paramecia as a food source, but if you pour the liquid into a culture dish and do not add paramecia, the Didinium will eventually starve. They will reduce in size to 20 to 25% of their “well-fed” diameter and will form tiny black spheres on the bottom of the dish. The cysts remain viable for months. To revive them, prepare a hay infusion medium by heating dry grass or hay in boiling springwater for 10 minutes. Pour some of the yellowish water and a few hay straws into a clean culture dish and let cool to room temperature. Add a thick concentration of Paramecium caudatum or P. multimicronucleatum and pipet in a number of Didinium cysts. Within 24 hours, most of the cysts will open and tiny Didinium will swim about, preying on the paramecia.
Maintaining most protozoan cultures for a few weeks is relatively easy. Maintaining one culture for a year or more is quite different, and maintaining several cultures long–term requires a significant investment of labor and other resources. Before initiating a series of long-term protozoan cultures, we recommend beginning with 1 or 2 of the more easily cultured organisms, such as Paramecium or Blepharisma, to determine feasibility. Cultures require daily attention. Media that give excellent results at the start may not be satisfactory for continuous cultures. It may be best to multiply a single protozoan culture to obtain sufficient quantities for several student labs and then allow the culture to lapse, and begin anew when a culture is needed again.
The following information applies to both brief and long-term culturing.
Note: Neither Vitachrome® cultures nor mixtures are suitable for culturing. Vitachrome-stained protozoans will not reproduce or will reproduce poorly, and in mixtures, one protozoan will, over time, replace the others.
Except for the photosynthetic species, protozoans are best cultured under conditions of dim to moderate light, a neutral or slightly alkaline pH, and temperatures of 20 to 21°C. Amoebas are especially prone to lose their vitality or die at higher temperatures. We maintain our cultures in 41/2– to 2-inch dishes stacked on top of each other. The top dish of each stack is left empty and serves as a cover.
See our culture media for protozoans.
| Protozoan-Sarcodinids | Recommended Protozoan Medium |
|---|---|
|
Actinosphaerium |
Wheat Medium |
|
Amoeba |
Wheat Medium |
|
Arcella Hay |
Wheat Medium |
|
Centropyxis |
Hay–Wheat Medium |
|
Difflugia |
Sand–Spirogyra Medium |
|
Pelomyxa |
Concentrated Paramecium |
| Protozoan- Flagellates | Recommended Protozoan Medium |
|---|---|
|
Algae |
Alga-Gro® Freshwater Medium (153752) for freshwater algae and dinoflagellates |
|
Chilomonas |
Wheat Medium |
|
Dinoflagellates |
Alga-Gro® Seawater Medium (153754) for marine algae and dinoflagellates |
|
Euglena |
Euglena Medium |
|
Peranema |
Wheat Medium |
|
Pelomyxa |
Concentrated Paramecium |
| Protozoan- Ciliates | Recommended Protozoan Medium |
|---|---|
|
Blepharisma |
Wheat Medium |
|
Bursaria |
Concentrated Paramecium |
|
Colpidium |
Wheat Medium; Protozoan Pellets |
|
Didinium |
Concentrated Paramecium |
|
Dileptus |
Wheat Medium; Protozoan Pellets |
|
Euplotes |
Wheat Medium; Protozoan Pellets |
|
Paramecium |
Double Wheat Medium; Protozoan Pellets |
|
Spirostomum |
Hay–Wheat Medium; Protozoan Pellets |
|
Stentor |
Wheat Medium; Protozoan Pellets |
|
Tetrahymena |
Wheat Medium |
|
Vorticella |
Wheat Medium |
For most protozoans, place 3 or 4 grains of previously boiled wheat in each culture dish. For paramecia, use 6 to 8 grains (double wheat medium). Pasteurize springwater, and while it is hot pour 200 mL into each dish. Cool to room temperature and inoculate.
Culturing protozoa in wheat medium.
Pasteurize springwater and while it is hot pour approximately 200 mL into each culture dish. Add two grains of wheat and two 3-cm stems of timothy hay that have been previously boiled. When cool, inoculate.
Add one jar of Euglena Medium Concentrate to 3.8 L (1 gallon) of springwater. Boil for 5 minutes. Let stand overnight and inoculate. Deep culture vessels or wide-mouth gallon jugs work well. Keep Euglena cultures in a well-lighted area, but out of direct sunlight because high temperatures are harmful. Artificial illumination is adequate.
Thick cultures of Paramecium caudatum are required. With a dropping pipet, remove a large number of paramecia without getting too much fluid. Place in fresh springwater. Strain through a cloth handkerchief to remove debris and large masses of bacteria. Let the filtrate stand until paramecia congregate in white masses on the bottom of the dish. With a dropping pipet, transfer the concentrated paramecia to fresh springwater in another dish and inoculate with Didinium or Bursaria. More paramecia must be added every few days, and subcultures should be prepared every 2 weeks.
A microscope magnifies not only the size but also the apparent speed of living, motile subjects. Ciliates may zoom through your field of view. Protoslo slows them for better viewing at higher magnification.
A vital stain is one that does not kill the organism yet makes cytoplasmic structures more visible. Available for Amoeba, Chaos (Pelomyxa), and Paramecium cultures.
Pairs a live culture with a prepared, stained microscope slide of the protozoan to highlight internal structure. Available for Amoeba, Euglena, and Mixed Protozoa.
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