Introduction

Protozoans belong to the domain Eukarya. They possess characteristic eukaryotic organelles, histones with their chromosomal DNA, nuclear envelopes, eukaryotic ribosomal RNA and endoplasmic membranes. Protozoans cannot be simply defined as unicellular organisms. There are many examples of multicellular similarities that have arisen independently. Some are multinucleate (for example large ciliates) and others form cell colonies. These colonial forms however do not form tissues of specialized cells. Cells specialization has been found in some groups. The single-celled organism concept of protozoans gives people the impression of primitive organisms. However, protozoans have evolved to adapt to all types of environments. Aerobic metabolism is a primary function of most free living protozoans.

All protozoans show some motility. Flagella and cilia are the most important swimming organelles in protozoa. Sliding motion of microtubules is responsible for movement. Flagella and cilia differ in function. There are usually only one or two flagella per cell compared to a large number of cilia per cell. Flagellates can be distinguished from ciliates by movement. Flagella motion is characterized by undulating waves. On the other hand, cilia bend at the base only and are then drawn back to its initial position. Both cilia and flagella function to propel the organism through water or to generate water currents from which food particles may be strained. Swimming velocities of ciliates and flagellates vary with cell size. There are some protozoans that use pseudopodia (extensions of the cytoplasm) for locomotion as well as for capturing food.

Motility is used to catch food particles and move to a new location. An example that can be done in the classroom is to ingest a tiny drop of acetic acid into a larger suspension of paramecia and note that the cell will form a ring around the drop of acid compatible with their pH. Photosynthetic protists orient themselves toward light.

Feeding occurs mainly by phagocytosis. In ciliates and some flagellates, this occurs on the "cytostome"-a special site on the cell surface. Protozoans play a key role in food chains. They are found at several trophic levels of food chains based on their size. For example, a protozoan that is 3 microns feed on bacteria, decomposing material, and microalgae whereas larger protozoans (up to 1 cm) feed on other protozoans.

In this investigation, we enriched for ciliates and flagellates by making a baiting trap of decomposing material. Ciliates and flagellates feed on the bacteria that is attracted to the detritus and also to the detritus itself. Two food sources were used – water based tuna fish and cheese. We hypothesized that the baiting traps with tuna would attract more ciliates and flagellates. Extensions were done with broccoli and ham.

Methods and Materials

Using a sharp razor, a square window was cut on the side of a film canister about two centimeters from its bottom. This window was cut toward the top of the canister no larger than about 1cm by 1cm. Nylon stocking was used as a screen. It was cut to fit the opening and adhered to the canister with fingernail polish. (Nytex and water insoluble glue may also be used.) Care was taken to coat the area around the window to avoid leakage. Several coats were applied and canisters were left to soak overnight to allow release of solvents from fingernail polish. Four aquatic habitats were chosen: tap water (control), still water pond, artificial pond water (made from soil and water), and bucket pond water (sample taken from a pond). All chosen habitats were placed in containers that were about the same size.

The next day baiting traps were prepared. Two canisters were placed in each aquatic habitat. A small amount of cheese or tuna was placed in the bottom side of each canister. A small stone was placed inside to weigh the canister down. Broccoli and ham were also placed in the still water pond and tuna was placed in a sample of algae from Central Park pond. The open canisters were submerged in the aquatic environments and filled with water to avoid bubbles before capping. Twine was used to affix canisters placed in the pond so they would not float away. After one week, canisters were retrieved and observed for ciliates and flagellates by pipetting water from the bottom of canisters.

Results
 

AQUATIC ENVIRONMENT	FOOD SOURCE
	Tuna	Cheese
Tap Water	none	none
Artificial Pond Water	Few, small ciliates (one per field)	same
Bucket Pond Water	Many, small ciliates (three per field)	same
Natural Pond Water	none*	none*

*canisters became exposed

Extension of Investigation
 

Aquatic Environment	Food Source
Natural Pond Water	Broccoli – ciliates and flagellates Ham - ciliates
Algae	Tuna - flagellates

As expected, there was no presence of protozoans in tap water. There were ciliates in the bucket pond water and artificial pond water. However, the artificial pond water had very few and they were very small. The pond had receded and although water remained in the baiting traps, no ciliates or flagellates could be found. In the extension investigation, ciliates and flagellates were found in the broccoli and ham that was placed in the natural pond water. Flagellates were found in the tuna placed in the algae.

Discussion

Fresh water protozoans can live under extremely varied environmental conditions ranging from clear springs to sewage. Temperature, salinity, light, and pH are important factors that determine habitats. However, the level of purity of the water has even greater influence on the protozoan fauna. This can be used as an index of the degree of contamination with organic matter that leads to bacterial decay and which influences the oxygen content of the water. Some protozoans live in waters with a high level of protein composition. Others grow in water in which oxidation processes occur as well as the break down of organic matter. Tap water, therefore, was used as a control because of its treatment for contamination. The artificial pond water ciliates were small. We hypothesized that any protozoans found in the soil may not have been aquatic and could not survive. Numerous ciliates and flagellates are found in the soil. Perhaps, if water had been aerated, more would have survived. Both tuna and cheese were good sources for ciliates in the bucket pond water. Because this water was taken from the pond, it should contain sufficient amount of protozoans. This method of trapping can work well in the classroom.

Natural pond water had no protozoans in either food source. We hypothesized that bacteria proliferated and consumed the oxygen so that protozoans could not survive. A dissolved oxygen test was not performed due to time constraints. Pond water does appear to be a good source for enrichment because broccoli and ham placed in the pond contained numerous ciliates and flagellates. These traps were still submerged in water when we obtained our samples. Also, the tuna placed in algae from Central Park pond contained large amounts of flagellates.

In conclusion, we found that natural pond water may provide a good source for enrichment of ciliates and flagellates. A sample can be taken from the pond and used in the classroom. The sample should be retrieved in one or two days instead of one week. However, if traps need to remain longer, it is suggested that the aquatic environments be aerated.
 
 

References

Campbell, Neil A. (1996) Biology (4^th ed.) Benjamin/Cummings Publishing

Company, Inc., Menlo Park, Ca

Fenchel, Tom (1987) Ecology of Protozoa, Science Tech Publishers, Madison, Wi.

Rainis, Kenneth G. and Bruce J. Russell (1996) Guide to Microlife, Franklin Watts,

Danbury, Conn.

Westphal, Albert (1976) Protozoa, Blackie, Glasgow
 

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