John D. Spikes (deceased)

From his review: Photosensitization, in The Science of Photobiology (1st ed, 1977), (KC Smith, ed), Plenum Press, New York, pp. 87-112.

One of the simplest demonstrations of photosensitization in biology is the photodynamic killing of paramecium (the same used by Raab in his discovery of photodynamic action) (see Photosensitization - What Stopped the Wiggling?). This experiment can be done in many ways; the following directions should be regarded only as a general outline. Prepare a 1 x 10-4 M sock solution of the dye Rose Bengal (MW = 974; ~10 mg/100 ml in distilled water). Add 10 drops of a culture of paramecium (can be obtained at little cost from any biological supply house) to each of 3 small dishes or shallow vials numbered 1-3. Then in dim light, add 10 drops of distilled water to dish #1, and 10 drops of Rose Bengal solution to dishes #2 and #3, and mix well. Immediately cover dish #3, and place it in the dark to serve as the dark control. Place dishes #1 and #2 under a light source, and examine at intervals with a low-power microscope. If the dishes are placed ~5 cm from a 15-W cool white fluorescent bulb, a swelling of the cells, and a decrease in the swimming rate will be observed in ~1 min with Paramecium caudatum; by 2 min most of the organisms will be immobile or swimming erratically, and by 5 min the majority will be immobile, and some will have broken open.

The reciprocal of the time of illumination (in minutes or seconds) necessary for the immobilization of 90-100% of the paramecia can be used as the rate of photodynamic immobilization. You should observe not only motility, but also the morphology of the cell, the behavior of the contractile vacuole, etc. Dish #1 serves as a light control (without dye); when most of the animals are dead in dish #2, examine those in the dark control (dish #3). The dye solution may be too concentrated for some cultures of paramecia. If the reaction goes too fast, or if appreciable effects are observed in the dark control, try 3 x 10-5 M or 1 x 10-5 M dye.

This experiment is quite open-ended in that the rate of photodynamic immobilization, and the development of morphological changes can be measured as a function of dye concentration, dye type (e.g., methylene blue, neutral red, eosin Y, etc., can be used), light intensity, light color, temperature, etc. Also, the sensitivity of other organisms can be compared (e.g., other protozoans, Euglena, Daphnia, brine shrimp, nematodes, small tadpoles, small fish, etc.); bacteria could also be used, but samples would have to be plated out after successively increasing periods of illumination to determine inactivation.


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