"Light," Baum writes, "is an immediate environmental factor in the life of most organisms. In the case of plants one thinks chiefly of photosynthesis, but the less overt processes of phototropism (plants turning toward light) and photoperiodism (the response of plants and animals to variations in the relative duration of day and night) are also controlled by light. The common denominator of these varied effects of light is the absorption of light energy in one or more photochemical, temperature-independent reactions and the conversion thereby of light energy into the energy of chemical bonds.

"In the case of phototaxis, the entire organism moves in response to light. Positive phototaxis is exhibited by the flight of insects toward a light source at night and by such behavior patterns as the vertical layering of zooplankton in surface waters during the day. Negative phototaxis is exhibited by darkling beetles and their larvae burrowing under the oatmeal on which they are being cultured and by the myriad of arthropods living under a log or rock.

"My interest in phototaxis was stimulated by the microscopic, single-celled organism called Euglena, which propels itself through water with a whiplike flagellum. Euglena has chloroplasts and can carry on photosynthesis, so that it qualifies in this respect as a plant. It also is capable of growing on appropriate media in the dark, so that it qualifies in this respect as an animal. Its high degree of motility, anterior gullet, contractile vacuoles, and its external, probably noncellulosic pellicle, or exoskeleton, are 'rule of thumb' animal characteristics [see Figure 1].

"It is likely that generations of biology teachers have plagued or stimulated their students by asking them if Euglena is a plant or an animal in the hope that they will perceive from this loaded question the arbitrary nature of biological classification at certain levels and the continuity of morphological and physiological features in various organisms The characteristics of Euglena combine to make it not only an interesting and instructive organism but also an excellent experimental subject.

"One of the more conspicuous features of most species of Euglena as observed under the microscope is an orange-red body called the stigma, or eyespot. In the electron microscope it appears as a loose aggregation of about 50 granules. The color of the eyespot is due to one or more carotene derivatives, the exact identities of which are still in doubt. As a group these pigments are involved in a number of photochemical processes in both plants and animals. Evidence suggests that the eyespot is closely related to phototactic responses in Euglena as well as in other organisms, although phototactic responses occur in organisms without eyespots.

"In addition to the eyespot a dense nodule near the base of the flagellum on the side facing the eyespot is variously designated as the photoreceptor and the paraflagellar body. Some investigators consider it of major importance in the mechanism of the phototactic response. According to one concept, positive phototaxis is related to a periodic darkening of the photoreceptor by the eyespot as the Euglena moves forward in its spiral path. This active orientation and swimming of Euglena in relation to the light source is designated as topophototaxis. It is distinguished from the phobophototaxis of certain bacteria: a spontaneous return to a region of greater light intensity whenever the light intensity else where diminishes. Thus the organism is 'trapped' in the light spot. The phototaxis of some organisms may involve both mechanisms.

"Although experiments on phototaxis using Euglena predate the work of the German biologist T. W. Englemann, it was he who in the l88O's carried out classical experiments on both photosynthesis and phototaxis. His experiments have served subsequent investigators as models of ingenuity and experimental design. Englemann worked with a purple sulfur bacterium, with Paramecium ursaria and with Euglena as well as with other organisms. He correlated the phototaxis of Euglena with the eyespot. In the purple sulfur bacteria he demonstrated both phototaxis and a correlation of phototaxis with photosynthesis in the near-infrared portion of the spectrum. He made an inference, subsequently confirmed, that in photosynthetic bacteria light of a color that most strongly attracts the organism also promotes photosynthesis most effectively. The energy involved in both processes is absorbed by the same pigment system.

"Investigators since Englemann have concentrated on pinpointing the absorption and action spectra and on elucidating the mechanism of the phototactic response, particularly the functions of the eyespot and the photoreceptor in organisms that have those parts. The work also includes studies of phototaxis in organisms that lack eyespots, such as the purple sulfur bacteria, desmids and blue-green algae, and of other organisms that possess eyespots, such as Chlamydomonas and Volvox. Comparative studies of phototaxis have been made on strains of Euglena with chloroplasts, eyespots and photoreceptors; on strains without chloroplasts but with eyespots and receptors, and on strains with chloroplasts and photoreceptors but without eyespots. Chlorophyll-free Euglena can be prepared by the bleaching action of certain chemicals such as streptomycin. Strains without eyespots have been obtained by subjecting specimens to ultraviolet radiation.

"Phototaxis in Euglena is frequently illustrated by exposing a culture in a transparent container to a beam of light. The active organisms promptly move to the side of the container nearest the light source, where, even though individually microscopic in size, they congregate in such numbers as to be readily apparent to the naked eye. It occurred to me that the effect might be emphasized by completely darkening the culture except for a small spot on the side wall of the container. I covered a culture contained in a 100-milliliter glass jar with a mask made of black paper. A single 1/4-inch opening was punched in the paper mask. When the culture was exposed to light, a dense aggregation of Euglena collected near the opening in less than an hour.

"To demonstrate the influence of color I next made a black paper sleeve with windows covered by transparent plastic of various colors. A series of six 1/4-inch holes 3/8 inch apart was punched in a piece of black construction paper with an ordinary paper punch. A small strip of clear or colored cellophane was placed over each hole and secured with cellophane tape [see illustration above]. Dennison-packaged Du Pont cellophanes, available in different colors, were used as crude filters.

"The assembled device was carefully fitted to a glass vial so that the interior was completely darkened with the exception of the colored windows. The vials were about 3 1/2 inches high and about an inch in outside diameter; both round and rectangular types were used. The fitted paper sleeve was assembled with masking tape so that it could be slid from the vial without agitating the culture.

"A Euglena culture was placed in a vial, the vial was stoppered and the window side of the fitted sleeve was exposed to a beam of white light. Almost any light source was found to be satisfactory. A fluorescent lamp is weak in red wavelengths. When one uses a tungsten lamp, one should take care to assure uniform distribution over the exposed side of the culture vessel, and the lamp should not be so close that it heats the vial. Light intensity can be controlled in part, and the effect of intensity differences can be studied, by placing cultures at various distances from the light source.

"The approximate intensity of the light passing through the color filters at the distances involved (and presumably reaching the organisms in the vial) was measured by a light meter. Meters of the type used for determining photographic exposures can be used. The clear filter transmitted the most intense beam, followed by blue, then yellow, green and red in order of decreasing intensity. The approximate corresponding range of wavelengths was determined by measuring sample strips of the colored plastic in a colorimeter that indicated the percentage of light transmitted. The light turned out to be far from monochromatic. For example, the blue plastic transmitted blue light as follows: at a colorimeter setting of 400 to 450 millimicrons (blue), 90 percent; at 550 millimicrons (green), 60 percent; at 650 millimicrons (red), 23 percent. For a given range of wavelengths the intensity was varied by using two or more thicknesses of the filtering material. Complete data on the light-transmission characteristics of the plastic material are usually available from the manufacturer.

"Active Euglena cultures moved to the colored windows in less than 15 minutes. White light produced the quickest response, followed by blue, green, yellow and red. The number of organisms that assembled at each window was also greatest in the case of white light and declined in the same sequence: blue after white, then green, yellow and red. The sequence was the same whether the culture was lighted for only a few hours or overnight. The maximum phototactic response in blue light coincides with the maximum absorption of blue light by the eyespot. If aggregation of the organisms is allowed to continue for periods of more than 30 minutes, the Euglena adhere to the walls of the glass vials even when the culture is mildly agitated. The cultures can then be carefully poured off and the vials inverted on a paper towel for drying. After drying, the Euglena aggregates can be fixed to the glass by placing the vial next to a low heat source for a few minutes.

"A more versatile color filter was next constructed in which different-colored plastic strips 5/8 inch wide were placed across rectangular slots, measuring about two inches by 1/4 inch, cut in a black paper cylinder. The strips were arranged horizontally and overlapped about 1/16 inch. They were held in place by strips of gummed paper placed along the overlapped edges. Still a third variation of the filter assembly was prepared by securing narrow strips of colored plastic across a wide slot (about 1/4 inches) cut in a three-by-five-inch card. This proved to be a very flexible device, easy to place over slots in opaque sleeves that fit a variety of containers in assorted shapes and sizes.

"The results of experiments made with the color filters were so encouraging that I decided to subject cultures to an actual spectrum in the hope of observing the sharpest possible differentiation of phototactic response with respect to the wavelength of light. A simple apparatus for dispersing the light was improvised from materials that were at hand. The rays of a 60-watt incandescent lamp were refracted into a beam of parallel rays by means of the lens from a reading glass and were dispersed into the spectral colors by a glass prism that measured 1 1/2 inches by two inches. The colors were then projected onto a culture of Euglena. A pronounced differential response was immediately apparent. Maximum aggregation occurred in the red area of the culture! This was contrary both to the results previously observed and to the literature. The puzzle was resolved by measuring intensity across the spectrum: the light meter indicated that the red portion was almost 20 times more intense than the blue. The difference in energy was therefore masking the effects of the difference in wavelength."

(If the amateur does not own a prism, an alternate source of intense spectral light can be improvised by equipping an ordinary 35-millimeter projector with a vertical slit in the position normally occupied by the slide and placing a replica diffraction grating in front of the projection lens. This scheme is suggested by Roger Hayward, who illustrates this department. Transmission gratings of adequate quality for this application can be obtained for $1.50, in a sheet that measures eight by 11 inches, from the Edmund Scientific Co. in Barrington, N.J. Details of the arrangement are depicted in Figure 4.)

"On one occasion, when a very dense, older culture of Euglena was exposed to light overnight, I was surprised to find no phototactic response. Although apparently all experimental conditions were in order, the organisms failed to aggregate and to adhere to the side of the vessel. This and related evidence suggested that not all cultures are phototactically equivalent. Therefore care should be taken in the design of experiments to equate only the results of cultures that are comparable in terms of age, nutrition and similar factors.

"Since there is some indication that the inorganic environment may influence phototaxis (and other processes as well) I decided to investigate whether or not this might be a factor in the lack of response of the inactive cultures. The inactive organisms were older, denser cultures in which the pea extract I used as a nutrient in the medium had not been replenished for several weeks. In lieu of waiting for a culture to become nutritionally depleted one could separate the organisms from the culture solution by aggregating active Euglena on the sides of a vessel and either pouring off the balance of the culture, centrifuging or filtering. The aggregated organisms could then be resuspended in tap water or distilled water.

"To investigate the effect of mineral nutrition I dissolved a commercial N-P-K (nitrogen-phosphorus-potassium) plant-fertilizer tablet in water an added the equivalent of half a tablet t a 400-milliliter suspension of photo tactically inactive Euglena. I kept an other suspension without fertilizer as control. Essentially no phototaxis an aggregation were observed in the control, but the experimental culture to which the plant tablet had been added displayed marked activity [see Figure 5]. This suggests that mineral nutrients may be a decided factor in phototaxis.

"In another experiment made to disclose the effect of mineral salts on inactive organisms, nitrogen, phosphorus and potassium were added to separate phototactically inactive cultures in amounts equivalent to their concentration in the plant-fertilizer tablet. To each 100 milliliters of cultures of inactive Euglena I added 15 milligrams of phosphoric acid, 16 milligrams of potassium chloride and 190 milligrams of ammonium sulfate. Light intensity was maintained at 400 foot-candles. The light source was placed 20 centimeters from the cultures. The light exposure was five hours. The salts most effective in promoting phototaxis and aggregation were, in descending order, phosphorus, nitrogen and potassium. All produced more aggregation and phototaxis than appeared in the control. These results indicate not only the general effect of mineral nutrients on phototactic response but also the differential effects of the several ions, particularly the pronounced influence of phosphorus and nitrogen.

"Sets of inorganic 'sufficient-and-deficient' plant-growth salts are available from biological supply houses. The results of experiments made with them not only are readily apparent to the eye but also are automatically plotted by the differential adhesion of the organisms to the glass. Permanent records of the responses can be made by simply photographing the aggregated organisms. The same general procedure can be used for investigating the effect on phototaxis of light intensity, pH, drugs, vitamins, hormones and age of cultures.

"Active Euglena organisms as well as many other cultures are stocked by the larger biological supply houses. Pure cultures of both green and colorless species of Euglena for research uses are available from the Culture Collection of Algae at Indiana University. Algae and other organisms can also be collected in the field. The specimens are placed in an aquarium, concentrated phototactically by a light source placed at one end of the aquarium in an otherwise darkened room and pipetted into a simple culture medium. Alternatively, the organisms can be placed in a volumetric flask that is darkened except at the neck; after they have aggregated they can be pipetted out of the neck.

"Two nutrient media are widely used for culturing Euglena. The split-pea medium is made up of the fluid obtained by boiling 40 split-pea halves in one liter of tap or pond water for a few minutes and then discarding the solid residue. The second medium, known as the soil-water type, is also highly recommended for Euglena as well as for a wide variety of other algae. It is prepared by adding successively to a test tube a pinch of calcium sulfate, a half-inch of good garden soil and a quarter of a dried split pea. About 75 milliliters of water (tap, pond or distilled) is then added along the side wall of the test tube. After the test tube has been loosely plugged with cotton it is steamed (do not autoclave) for one hour on each of two successive days. After the resulting fluid has cooled and cleared by settling it can be inoculated with the desired organisms.

"I have used split-pea medium with consistently satisfactory results. One may observe a temporary rapid increase in bacteria after adding split-pea medium to a culture. The bacteria will diminish over a period of several days as the concentration of the Euglena in creases until they are no longer apparent.

"The experiments need not be confined to Euglena. Although most species of Paramecium will be found to be indifferent to moderate light intensities, P. bursaria, which plays host to enough green algae to give it a green and plantcell-like appearance, is positively phototactic. In these organisms phototaxis has been found to be a response related to the oxygen produced by the symbiotic algae in photosynthesis. It is sometimes referred to as secondary phototaxis arising from chemotaxis or aerotaxis. I once observed the response by placing a culture of P. bursaria in a miniature beaker that was completely darkened except for a single 1/4-inch hole punched in the black paper on the bottom. The culture was placed on the stage of a stereoscopic binocular microscope equipped with a transilluminating substage. The hole was illuminated overnight from below, the spot of light being centered in the field of view. By morning the spot was covered with P. bursaria.

"In a similar experiment made with five green Hydra only three organisms were found in the light spot after an overnight exposure. Incidentally, the prior condition of organisms tends to influence their response to a given set of conditions. In my experience more uniform patterns are observed when the organisms are kept in darkness for a 24-hour period before their use in an experiment.

"Many refinements of the above techniques can be developed for investigating the phototactic responses of an entire range of smaller organisms. Given a choice of various wavelengths, where would P. bursaria and Hydra viridissima preferentially aggregate? Flatworms are considered negatively phototactic. If given no opportunity to remain in the dark, in what wavelength, if any, would they preferentially remain? Many smaller crustaceans exhibit phototaxis; brine shrimp, for example, are readily cultured in the laboratory. What effect, if any, would a competing population of other organisms have on their phototactic responses?

"The fundamental question of just why and how the energy of light triggers and then guides the swimming motion of these organisms still awaits explanation. Doubtless the full answer will come when data from experiments such as these are correlated with comparable information derived from the disciplines of cellular physiology, biochemistry, biophysics and electron microscopy."

Bibliography

EUGLENA: AN EXPERIMENTAL ORGANISM FOR BIOCHEMICAL AND BIOPHYSICAL STUDIES. Jerome J. Wolken. Rutgers University Press, 1961.

PHOTOTAXIS. Selina W. Bendix in The Botanical Review, Vol. 26, No. 2,: pages 145-208; April-June, 1960.

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