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The Physiological Tolerance of Two Species of Protozoans

Sergio A. Orminati

Laboratory Exercise for:

  • Biology
  • AP Biology
  • Ecological / evolutionary/ taxonomic studies


Protozoans are single-celled heterotrophic organisms that belong to several Phyla. They vary widely in their morphology and method of reproduction as well as in their ecological niches. The two species that you will study belong to different phyla and obviously have very different habitats.


The purpose of this inquiry is to examine the differences in pH tolerance between the two species and to relate these differences to their respective niches.


An organism which is free-living is subjected to, and must therefore adapt to, a wider environmental variation than one which is an internal symbiont.


  1. 6 microscope slides and cover slips
  2. 3 dropper bottles with isoosmotic saline, marked pH 6, 7, and 8
  3. 3 termites
  4. Paramecium culture
  5. Forceps and teasing needles


  1. Lay out the six slides and place one drop of pH 6 saline on each of them, repeat for pH 7 and pH 8 saline.

  2. Using forceps, take one termite from the container and place it in the pH 6 saline drop. Decapitate and rip open the abdominal region of the termite with the teasing needles, discard the head and cover the remains with a cover slip. Repeat the process with the two other termites in the other two pH concentrations.

  3. Take a drop of Paramecium culture and place it on each of the remaining three slides. Cover each drop with a cover slip.

  4. Observe each of the six slides under the low power objective of your microscope and make note of what you observe. Repeat the observation after 5 and then 10 minutes.


  • Q1. Which of the two organisms - free-living or symbiont - would you expect to have a wider range of pH tolerance? Why?

  • Q2. Why was the termite unable to escape from the drop of water in which it was placed?

  • Q3. The name of the organism in the termites gut is Trichonympha, it is a true Mastigophora (notice the numerous long flagella arranged in grooves on its surface) and a symbiont of the termite. What type of symbiotic relationship do you think it has with the termite? Why?

  • Q4. Do your observations of the two organisms confirm your hypothesis in Q1?

  • Q5. Would you expect the same results if another environmental factor, say temperature or salinity, was tested in the same way?


What, in your opinion, is the evolutionary significance of the reduced environmental tolerance of Trichonympha? Can you see any relationship between the presence of this symbiont and the colonial lifestyle of the insect in which it lives (think back to your answer to Q3)?

Teacher's Guide for Lab


Before or during the experiment you might want to stress the economic significance of termites. Ridding our houses of these pests is a multi million dollar industry. The problem with termites is that they cannot tell the difference between a fallen log and the lumber we use for building.


  • Termites and Paramecium cultures are now both available from scientific supply houses, but termites are easily collected in the field by turning over logs, old cardboard, etc. and by using an aspirator.

  • The isoosmotic solution is made by dissolving 9 grams of NaCl in one liter of water.

  • The three pH levels are adjusted by adding quantities of H Cl and Na OH to the saline and testing with pHydrion paper.

  • The pH values can be approximated, as they are not critical to the experimental results.


The results should show that Paramecium has a wider tolerance to pH than Trichonympha, the latter dies within 5-10 minutes at pH 7 and almost immediately at pH 8.


  • Q1) The first question may be used to review the properties of water with your students. Surface tension prevents the termite from escaping from the drop on the slide.

  • Q2) The second question may be obvious if your students have studied termites in your curriculum, but the reason that it must be mutualistic may not be immediately apparent. Point out the huge number of flagellates and the size of the host to discourage thoughts of parasitism and commensalism.

  • Q3&4) An internal symbiont has its host supplying a stable environment and would thus be expected to have a narrower tolerance to environmental changes.

  • Q5) Evolutionary theory would predict the results that were obtained. The mutualistic relationship is one in which the symbiont gives up its self-sufficient status as it becomes more finely tuned to the physiology of the host. This is an example of coevolution. (See below for a brief discussion of the evolution of the termite society)

For further discussion:

Termites evolved from cockroach-like ancestors and developed the mutualistic relationship with Trichonympha and their social behavior very early in their evolution. There are some cockroaches that also evolved the capacity to digest cellulose by the use of gut symbionts in the same manner, but relatively recently. These wood roaches live in groups and have a social system that requires the young to associate with the adults that is very similar to that of termites.

You might present this information to your class after they have completed the lab and then ask for possible reasons for this parallel evolution of social behavior. The reason is that Trichonympha cannot be inherited, or passed trans ovarially and must, therefore, be ingested by the young from the cultures present in the gut of the adults (without Trichonympha to digest the cellulose, termites and wood roaches would have no niche). This inoculation of the symbiont is accomplished by the ingestion of adult fecal material in both young wood roaches and termites.


Trichonympha may be used as an example of a typical Mastigophora. It can be used as an example of this Phylum rather than Euglena, which is really an alga.


Wilson, E.O. The Insect Societies, Harvard UP, Cambridge, 1971.

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