tected from bright light and so are naturally "self- Blepharisma in stained" and much easier for a beginning student to see than colorless . They are also slower swimmers than ; they are, therefore, much easier to follow and pick up if isolation is Introductory Biology desired. The mouthparts (peristome) are more com- plex than in Paramecium, with membranelles (con- sisting of rows of interdigitated "fused" cilia forming paddles, which make a food-gathering vortex) on the left side of the oral groove, and an undulating ARTHUR C. GIESE membrane (also made of "fused" cilia to form a tongue, which helps in food engulfment) on the right ANNE MULLER SMITH side. Usually at any time five to seven metachronal Downloaded from http://online.ucpress.edu/abt/article-pdf/35/7/407/30722/4444465.pdf by guest on 26 September 2021

ALTHOUGH Blepharisma (fig. 1) is available in biology supply houses and sometimes appears in introductory courses, it is more often used as a curiosity, not as an example of a , in part - . tUndulatingi f because Paramecium is traditional and illustrated in membrane all elementary textbooks, in part for lack of a source of information. Now that a monograph on the MembranellIe

Blepharisma is available (Giese et al. 1973), perhaps - cytostome a short discussion of how it might be used for teach- ing purposes, as Stolte (1926) long ago suggested, now has more point than before. Blepharisma, a pink, bottom-dwelling protozoan, is commonly found in freshwater ponds. It is a mem- ber of the class Ciliata, subclass Spirotricha, order Heterotricha. The ciliary rows on the spindle-shaped body are spirally arranged, and in the region of the oral groove the cilia are fused and differentiated into membranelles and an undulating membrane to form a peristome, which gathers particulate food, mostly . When ponds dry up or freeze, it forms cysts, from which active cells emerge on re- turn of favorable conditions. The classification of the ~~Food- ~~~I:t. vacuole genus is still incomplete, because many species were sketchily described from a few field specimens; but a total of 21 species have been differentiated into four main groups on the basis of macronuclear shape -that is, whether it is dumbbell-shaped, elongate, beaded or compact (see below).

Cytopyge General Characteristics Fig. 1. . Note the elongate filiform Blepharisma is easy to grow in an infusion of macronucleus. The micronuclei are the minute bodies clus- powdered cereal greens. Rich cultures can be ob- tered along the length of the macronucleus. An undulating tained in test tubes three membrane protrudes from the right side of the mouth region days after inoculation. At (peristome), and a band of membranelles lines the left side. this time the blepharismas (to use the generic name The oral groove terminates in a cone-shaped gullet, at the as popular name) are often aggregated at the top of bottom of which is the mouth, where the food vacuoles are the tube and can be readily decanted into a dispenser. formed. A contractile vacuole is present at the very posterior The species of Blepharisma-B. americanum, com- end of the cell. Note that the entire body is covered with cilia and that the cilia occur in rows forming lines on the mon in this country, and B. japonicum, common in body. Between the rows of cilia are rows of pigment granules India, Japan and Africa-are pink or red when pro- (not illustrated).

407 beats (like the waves caused by wind on a grain Arthur C. Giese is professor emeritus of field) may be seen on the membranelles. The con- biology, Department of Biological Sciences, tractile vacuole is terminal and large but contracts Stanford University, Stanford, Calif. 94305. more slowly than in Paramecium-about ev-.ry 90 A 1928 graduate of the University of Chi-. seconds at room temperature. Blepharismas will eat cago, he received his Ph.D. in 1933 from not only Stanford, where he taught from 1929 until staple food bacteria but also small proto- 1970, when he retired (although he is con- zoans, becoming enlarged as they do. When diverse tinuing his research there). Giese has pub- in size after starvation, blepharismas may eat each lished some 200 articles, chiefly on photo- other. (Cannibals appear routinely in our cultures physiology and on the reproductive cycles of marine in- after three weeks.) Cannibalism and encystment (a vertebrates; and his book Cell Physiology (Saunders) is now in its fourth edition. Since 1964 he has edited the annual reaction of some stocks to worsening environment) review Photophysiology; and he is coeditor, with John might make interesting student research topics. Pearse, of the seven-volume treatise Reproduction in Marine Blepharisma varies in size, some species being less Invertebrates, forthcoming from Academic Press. than 100 ,um in length and other species reaching a A biographic footnote (delayed) about coauthor Anne Muller Smith length of almost 0.33 mm. Cannibal blepharismas are will appear with a forthcoming article. sometimes more than twice as long and wide as bacteria-fed individuals of the same species. plasm. Cell divison (fig. 2) may be observed in The shape of Blepharisma is variable, depending actively growing cultures of Blepharisma, although Downloaded from http://online.ucpress.edu/abt/article-pdf/35/7/407/30722/4444465.pdf by guest on 26 September 2021 on nutritive conditions. When well-nourished, they the fraction in division at any one time is small. become pyriform or spindle-shaped, expanded pos- teriorly; when undernourished, they become elon- Culture gate and cylindric. Generally, the anterior half of the body is flattened laterally, and the posterior half Materials for the culture of Blepharisma are test is expanded and almost circular in cross-section. tubes (2 cm by 15 cm are good) with steel or plastic Like other ciliates, Blepharisma has two kinds of caps or cotton plugs; O.1-M NaH2PO4 and 0.1-M nuclei: macronuclei and micronuclei. The macro- Na2HPO4; powdered cereal greens or lettuce (Chlo- nuclei serve housekeeping functions; the micronuclei rovim, a mixture of powdered organic grasses with transfer genetic information. As in other ciliates, the additives, or alfalfa tablets [about 0.5 g each] are macronucleus arises anew during conjugation (sex- both suitable, and can be bought at health food ual reproduction) from the zygotic nucleus. Macro- stores); 1-L graduate; serologic pipettes (gradu- nuclei are large and can readily be observed in live ated), 1 ml and 5 ml; 2-L beaker; balance; and non- animals even under low power. toxic water. Water is always a problem in protozoan The macronucleus of Blepharisma varies in shape, culture. Distilled water may be toxic, as may tap depending upon the species. Four types are recog- water, even after boiling to remove chlorine. Spring nizable in the vegetative cell: (i) dumbbell-shaped water or unpolluted water from a pond or stream (halteriform), with two terminal nodes connected may be good but should be tested, and may vary by a thick strand; (ii) elongate-cylindric (filiform), with the season. We have tested a variety of com- sometimes with more or less enlarged ends; (iii) mercially available water types and found Feather beaded (moniliform), consisting of a string of macro- River Canyon Purified Water (deionized), Feather nuclear nodes connected by slender strands; and River Spring Water Co., Sacramento, Calif. 95814, (iv) ovoid-compact, a single mass. A filiform macro- and Sierra Spring Water Company, Inc., 1825 "R" nucleus is characteristic of B. japonicum; a beaded Street, Sacramento, Calif., both very good. Distilled nucleus is characteristic of B. americanum, the water from a local high school (testing should be species most likely to be available in supply houses. done on any local distilled or deionized water sup- Micronuclei are numerous and close to the macro- ply), and deionized water from a local high school, nucleus in vegetative cells. Because of their small were both good, though cultures did not grow as size, they can be seen only in stained specimens, best well as in the commercial samples listed. However, under oil immersion. In many species they are Black Mountain Spring Water, San Carlos, Calif. vesicular: the dense internal portion is surrounded 94070, proved variable but still satisfactory, inas- by a clearly defined halo. During cell division and much as the culture medium made up with it sup- conjugation the micronuclei move from the proxi- ported growth. mity of the macronucleus to the surrounding cyto- To the beaker add 0.5 g of cereal green (or lettuce) Fig. 2 (opposite). Division of a Blepharisma americanum with a beaded macronucleus. This species is smaller than the one illus- trated in fig. 1. A, vegetative stage; B, early division stage, showing at a an early anlage (forerunner) of the new peristome; C, later stage, showing clearly the developed peristome anlagen (mza, uma, pfa); D, still later division stage in peristome anlage, which is well formed, and the anterior peristome is also reorganizing and the macronucleus (ma) condensed; E, stage showing the beginning of the division line (tl) (note elongated but nodeless macronucleus); F, final (dumbbell) stage of division (note that the macronucleus is again noded). The following abbreviations have been used: a, anlage, or forerunner, of each structure; um, undulating membrane (uma is the anlage of the um); mz, membranellar zone or band (mza is the arlage of the mz); pf, peristomal field (pfa is the anlage of the pf); cp, cytopyge (cpa is the anlage of the cp); ma, macronucleus; ps, pigment stripe; cs, ciliary stripe; cp, contractile vacuole; ta, division anlage; ra, reorganization anlage of the peristome of the anterior in- dividual; tl, division line; tr, end of the membranellar band; VTp, peristome of the anterior individual.

408 THEAMERICAN BIOLOGY TEACHER. OCTOBER 1973 um~~~~~~~~~~~~~~m

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to~~~~~~~~~~~~~~~~~~~~~~~ powder. Add 50 ml of boiling water and stir with a pipettes, 1 m long, one per student; medicine drop- glass rod. After a few minutes add 4.7 ml of 0.1-M per, one per student; test tube 2.5 cm in diameter Na2HPO4 and 0.3 ml of 0.1-M NaH2PO4. Add 945 ml and 20 cm long, to store pipettes, one per student; of water and dispense about 15 ml per test tube, cap nonabsorbent cotton to pad bottom of above tube and sterilize in a pressure cooker, or autoclave under and to cap it; 100-ml beaker with about 40 ml of 15 lb pressure for 30 minutes. Sterilized medium petroleum jelly to make moist chambers, one per keeps well and may be stored in a refrigerator for class; standard glass slides; and cover slips. months. If no pressure cooker or autoclave is avail- For cutting Blepharisma a glass needle is quite able, boil the medium to kill extraneous organisms effective. To make a needle, heat locally at the tip, and make up in batches of less than 1 L, because the in a Bunsen flame, a 7-cm length of 5-mm soft glass medium will not keep without sterilization. rod and then bring the tip against another, similar Inoculate with available bacteria 12-24 hours be- rod heated slightly less than the first one (fig. 3). A fore use. Although single strains of bacteria such as quick pull will give you a needle about 1-2 cm long Pseudomonas ovalis or Serratia marcescens have coming to almost an invisible tip, at the same time been used to inoculate the infusion 12-24 hours be- leaving a bit of glass on the other rod. To be func- fore the blepharismas are added, blepharismas also tional, such a needle must be quite flexible; this can grow very well on a mixed flora from the air. be tested by bouncing the needle gently on a glass Blepharismas are added by pouring about 4 ml of a slide. You can make a receptacle for the needles by Downloaded from http://online.ucpress.edu/abt/article-pdf/35/7/407/30722/4444465.pdf by guest on 26 September 2021 good culture into a tube of medium previously inocu- gluing to a block of wood a piece of open corrugated lated with bacteria. Cultures can be maintained paper. The handles of the needles are then supported, easily by transferring as described above once every but the needles protrude and are protected from two weeks or so. Addition of one grain of sterilized being broken by rolling on the desk. It is best to wheat will provide additional nutrient for long- make a number of needles because they break, es- standing cultures. pecially when one is not accustomed to their fragility. The pipettes for transferring blepharismas or Regeneration Studies

The very presence of readily visible complex organelles (described above) has led to the exten- sive use of Blepharisma in experiments on regenera- fl tion. Blepharisma can regenerate all its mouth parts A in a single afternoon (5-6 hours at room tempera- ture), making it a convenient test object, because the regenerated cells can be observed the following day. It is presumed that all cells can regenerate to some extent, but organelles of most cells are too B small to see with low-power light microscopy, except ) ______2 for fibers of nerve cells, which, however, regenerate ~ ~~~ 2 - much more slowly and require more equipment than is usually available. For classroom studies on re- generation Blepharisma is almost ideal and requires D only the simplest of equipment, as follows: < ~ ~2 ~ ~~~; Dissecting microscope 10-15 X, preferably with substage mirror; light source (Stocker Yale Lite- mite with two 4-watt cool white fluorescent lamps is excellent for the purpose because it is cool; an in- / /B candescent lamp must be shielded with a water filter); dishes with a concavity (our preference is for Columbia watch glasses 4 cm square, with a 3-cm- diameter concave depression, because there is ample room for maneuvering the needle in cutting; how- ever, depression slides are much cheaper and satis- factory); Petri dish to hold watch glass or depres- sion slide; soft glass rods 7 cm long and 4-5 mm in Fig. 3. Making a glass needle. Two pieces of soft glass tubing diameter, four per student; Bunsen burner; wood are brought into the Bunsen flame (A), no. 2 being heated rectangles 1 inch thick, 1.5 by 3 inches, one per stu- somewhat more than no. 1; (B) the two are touched together dent; corrugated paper, open on top, to be glued to when softened and no. 2 is pulled rapidly from no. 1. If it the top of the above wood block for holding the glass is done right, a fine needle, flexible yet strong enough for needles; glue; soft glass tubing 14 cm long, 6 mm in cutting, can be made (C, D). As shown in A, B, C, and D, the needle protrudes linearly from the piece of glass. Some outer diameter, for making pipettes, four per stu- individuals find it easier to make the needle at an angle to dent; rubber tubing to fit over the large end of the the rod, as shown at B' and C'.

410 THE AMERICANBIOLOGY TEACHER, OCTOBER 1973 a medium with organisms while looking under the dissecting microscope. B A moist chamber base can be made by blowing a ring of petroleum jelly (such as Vaseline) onto a slide (fig. 5). This is best done by heating the jelly D ( N2g//////// until it melts. Then heat a small pipette and, using the mouth E ( I ~ I (0 4 6cm control method, suck in some petroleum jelly, warm gently in the flame, and blow it out in a ring on a slide. If you do this carefully you can make F

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Fig. 4. Equipment for regeneration and cell-division experi- ments. Culture tubes (A-E): soft 5-mm glass tubing 10-12 cm long is sealed at both ends and cut; the cut ends are A ~ - heated and flared by inserting a tapered carbon rod (hatched rod). Pipette for culture medium (F-H): a piece of 6-mm tubing 15-16 cm long is heated and gently pulled to a di- ameter of less than 1 mm, and the middle of the narrow section is scratched with a file and broken. Such pipettes carry more medium than those used for cells. Pipette for transferring blepharismas (I-L): a 10-cm piece of 6-mm tubing is heated and pulled gently to half its diameter; when it has become rigid again, the center of the narrow segment is carefully heated and rapidly drawn to a very fine diameter. The tip is scratched at an appropriate place and broken. Both culture tubes and pipettes should be plugged with rolled cotton and sterilized. Tips of fine pipettes may be fire-polished if desired, but passage through the flame must be very rapid, lest the tips become sealed. pieces of them are made from 10-12-cm lengths of 5-6-mm-O.D. soft glass tubing by two draws: the lAi over th2ld B n h erlu eligmd first to narrow the diameter of the tube to about it ecl() oesuet one-third its original diameter (fig. 4), the second aitgtb,etepesr draw after heating a small central section of the nar- rowed portion made rapidly to produce a very fine tip (fig. 4). The tip can be broken cleanly by first scratching it lightly with a small file and pressing gently. The tip size should be several times the diameter of Blepharisma (about 200 ,um in diame- ter). The wide end of such pipettes can be plugged with cotton and the pipettes sterilized in a large test Fig. 5. Making a moist chamber. A pipette is heated gently in a flame and filled with melted petroleum jelly (Vase- tube (20-25 cm long, 2.5 cm in diameter), the narrow line) from a heated beaker. The tip is gently heated end of the pipettes down on a cotton wad at the again, and a thin layer is blown from the pipette onto a bottom of the test tube. Sterilization prevents con- glass slide (A) to form a ring or a square smaller than the tamination but is not required. cover slip (size of which is shown in dotted lines). The The preparation containing several postperistomal pieces of "mouth pipette" consists of a 1-m length of amounBlepharisma of nutrient nispipetteamist presbent.Appteietdgnlonto the slide.- The. ovepr sdip is the-n rubber tubing of a diameter to fit snugly over the wide end of the transfer pipette. The mouth end of in ~ 411m~ n ~ ild ~ ihmle~ ~~~LPAIM eroemjly(ae the rubber tube is tipped with a medicine dropper, wide end outwards, for holding in the mouth. By sucking or blowing gently on the attached pipette, it is possible to take up or expel fluid or culture an even ring. An alternate method is to make the cell (see fig. 2). Meanwhile, the anterior end of the ring at the edge of a well slide. fragment gradually assumes a new shape, although For regeneration studies, use a large species of there are stages of what looks like puckering. Finally, Blepharisma, such as B. japonicum. (Small species after about 4-5 hours the new anterior end takes work as well but are slightly more difficult to definite shape. Even after regeneration is complete handle.) Pour a bit of a culture of Blepharisma into (about 6 hours) the new peristome is too small and a concave depression of a slide or a Columbia watch may undergo further alteration. This is of interest glass. Cutting is best done at 15-20 magnifications theoretically, because it demonstrates that the "blue- under a dissecting microscope. print" in the cell requires not only the general struc- To cut a Blepharisma (fig. 6) let it swim under ture but one that is in proportion to the cell size and your needle (dipping in the medium) in the con- is functional. The fact that this can all occur in the cavity of the dish; when it is in the right position, course of an afternoon emphasizes the remarkable gently rock the needle back and forth, cutting the capacity of cells to maintain the normal pattern of Blepharisma into two parts. It is best to cut just organization. below the peristome to sever the Blepharisma into anterior and In posterior (postperistomal) portions. Observation of Cell Division the postperistomal piece of Blepharisma it is much Downloaded from http://online.ucpress.edu/abt/article-pdf/35/7/407/30722/4444465.pdf by guest on 26 September 2021 easier to see the course of events in the regeneration Population growth by cell division, which occurs of mouth parts starting from a "blank" (cut) surface about once a day, can be directly observed over a than to watch the modification of the existing ones period of 4-5 days by using small sterile culture in the anterior fragment of Blepharisma. Transfer tubes (fig. 4, E), which are commercially available to a moist chamber by gently blowing the drop of from Kimball or can be made by students, using culture medium containing the cut piece(s) onto a soft glass tubing: 10 of these are filled with bac- clean cover slip, over which the slide with the terized culture medium, using a mouth pipette as in petroleum jelly ring should be placed and gently fig. 4, H. Each small tube is inoculated with one pressed. The slide can then be inverted and the Blepharisma, using a pipette as in fig. 4, L, incu- Blepharisma fragment observed even under high bated at room temperature, and the number of power. If the seal is tight, a moist chamber is formed; organisms present counted once a day for several it will last for days. At first the cut portion heals and days. Because the tubes are very narrow, they can develops a protective pellicle like that over the be held horizontally for counting under the dis- normal surface. The granules (kinetosomes) under- secting microscope without danger of losing fluid lying the cilia in the region just below the former from them. The organisms will show exponential peristome multiply to form the forerunner of the growth, with the number present equal to 2n, where new peristome, much as in the formation of a new n represents the number of divisions. If N equals peristome in the posterior daughter of a dividing the number of blepharismas, then log N divided by log 2 equals the number of divisions that have oc- NEEDLE curred. Number of divisions plotted against time will give a fairly straight line during the exponen- COLUMBIA tial growth period. Details of this type of experiment WATCHGLASS and its implications have already been described in this journal (Mertens 1966). The small culture tubes can be cleared of medium

DEPRESSION by shaking, brushed with a pipe cleaner (available LIDES from a tobacconist), and rinsed with water from a plastic "squeeze" bottle with a narrow spout. The cotton stoppers may also be reused.

Photoreactions POSTPERISTOMAL CD PIECE Colorless cells, such as the cells of our skin and paramecia, are killed by direct exposure to sunlight Fig. 6. Cutting a Blepharisma. Blepharismas in a concave but are not killed by sunlight passed through win- depression, as in a Columbia watch glass, are allowed to dow glass, which removes the "sunburn" radiations swim under the needle dipping in the medium. By a rocking if motion they can be cut into an anterior and a posterior at the short end of the sun's spectrum. However, section (postperistomal). The postperistomal pieces (or the a pigment that absorbs sunlight passed through converse, if desired) are placed two each in culture medium window glass is present in cells, it may sensitize in depressions on a slide and examined periodically for them to such light. This is true of Blepharisma, regeneration. The slide should be moist-chambered in a which has a red pigment that absorbs the blue- Petri dish (upside down), which should contain some filter paper or a cellulose wipe moistened with water to saturate violet and near-ultraviolet (longwave) radiation in the atmosphere. window-glass-transmitted sunlight. Blepharismas are

412 THE AMERICANBIOLOGY TEACHER, OCTOBER 1973 therefore killed by very bright sunlight passed morning and observe midday. On a bright day mnany through window glass. The experiment is best per- of those exposed midmorning are likely to be dead, formed with the blepharismas in a shallow glass dish but those exposed the night before have hidden covered by a pane of glass and outdoors where the behind stones. Dense aggregation can be seen par- sunlight may reach the organisms from all sides. ticularly well among light-colored stones. In the They are not readily affected by exposure at a test tubes with stones exposed midmorning, many window. of the cells are likely to be dead but others are quite For these experiments it is important to keep the healthy: only those that got to the hiding places by cultures of Blepharisma immersed in a vat of water, chance and returned to their hiding places each the temperature of which is not appreciably in- time they ventured forth in the light survive. In creased by exposure to sunlight. In very bright sun- cultures lacking hiding places, the blepharismas are light blepharismas die in less than an hour's ex- killed even though they often aggregate at some posure, but in dim light of cloudy skies they may last region in the tube where the intensity of the light is for days: the exposure time for killing varies with lowest. the time of day and the season of year, as well as with the weather. In all seasons the intensity of sun- Effects of Blepharisma Pigment light is greatest at noon, falling off on either side. In

winter, when the sun is at a low angle, causing its When Blepharisma is exposed briefly to freezing Downloaded from http://online.ucpress.edu/abt/article-pdf/35/7/407/30722/4444465.pdf by guest on 26 September 2021 radiation to pass through a maximal thickness of temperatures the pigment is extruded into the atmosphere, the ultraviolet component of the spec- medium. For this purpose it is best to concentrate trum is attenuated and the injurious effect on the blepharismas first by gentle centrifugation (10 X Blepharisma and other cells is less marked. In sum- gravity: turn the standard lab centrifuge barely on, mer, when the sun passes through a minimal layer count to five, turn off). Combine cultures until you of atmosphere, its action is maximal. Clouds, es- have a dense suspension of blepharismas; then pecially thick ones, reduce sunlight intensity, water plunge the tip of the centrifuge tube into ice for 2 vapor absorbing the heat rays strongly, but con- minutes. If left longer in the cold, the cells will die. siderable ultraviolet still gets through. (Smog ab- The pigment extract has an intense red color and sorbs considerable sunburn ultraviolet.) can be separated from the blepharismas by gentle In disorders of porphyrin metabolism in the liver, centrifuging; it remains in the supernatant, from porphyrins appear in the skin of man and domestic which it may be removed by a mouth pipette and animals and sensitize it to sunlight passed through placed in a small vessel on ice for future use. window glass. The effects on the skin are much like The pigment concentrate obtained in this manner those of sunburn but are produced by longer wave- is toxic to other cells (for example, paramecia) even lengths. in the dark. When diluted, the pigment is not toxic Sunlight from overcast, cloudy winter skies does to other cells in the dark but it will photosensitize not kill blepharismas even after prolonged exposure; other cells to bright light, causing their death, even however, the photochemical action of the light ab- though controls lacking the pigment but exposed in sorbed is evident in the change in color from red to the same manner are not affected by the light. blue-grey as a result of a photooxidation of the The skin of man and domestic animals (or other Blepharisma pigment. At Stanford during the rainy animals in experiments) may be sensitized by eating winter of 1973, it took 1-4 days of exposure to change plants, such as St. Johnswort, that liberate a pig- blepharismas from red to blue-grey even when fully ment similar to Blepharisma pigment that gets onto exposed (with no objects behind which they could the skin, or by coming in contact with certain mem- hide). On partially sunny winter days the blephar- bers of the family Umbelliferae (to which parsley ismas were photooxidized much more rapidly. On and carrots belong) and several other families. completely sunny days they had to be kept in indi- These plants have glands that secrete furocoumarins. rect light to avoid killing; killing can occur without The furocoumarins spread on the skin and absorb evident change in color. sunlight, particularly in the near-ultraviolet, causing Although the mechanism by which Blepharisma skin damage resembling sunburn (Giese 1970). avoids light and hides behind physical objects in its environment has not been analyzed in detail, it ap- Conclusion pears to be by trial and error. To demonstrate the reactions to light, fill one third of a test tube with It is evident from what has been discussed above small aquarium stones and add enough of a rich that Blepharisma offers unique opportunities for suspension of Blepharisma to fill the tube two-thirds studies with cells. The equipment needed for these full. For controls use an equivalent suspension with- studies is commonplace and is either available in all out material behind which the blepharismas can laboratories or can be made by the students. The hide. Place in a large vat of water and expose one organisms are available from biology supply houses, set of two tubes late in the afternoon or in the even- and culturing them is easy, provided care is taken ing and allow the morning sun to strike them, ob- serving at midday. Place another set of tubes mid- (Concluded on p. 419)

BLEPHARISMA 413 boys, and replaced all plastic tubing. We have learned, however, that all of this work can be avoided Blepharisma from page 413 by the following simple procedure: (i) pumping old to avoid water polluted with chlorine, heavy metals, nutrient solution out of the system; (ii) soaking industrial pollutants, and the like. Both students and and then flushing with a dilute sodium hypochlorite teachers should find experiments with Blepharisma solution (approximately 0.5%); and (iii) rinsing intriguing and successful. with water. Our second use for household bleach can be recommended only as a technique of last resort: to Acknowledgments.-Fig. 1 and 4 are from, and fig. 6 is remove contaminatingmicroorganisms and the build- adapted from, Blepharisma: the Biology of a Light-Sensitive on columns. It Protozoan, by Arthur C. Giese et al.; used with the permis- up of discoloring residues Sephadex sion of the publisher, Stanford University Press; ? 1973 by is possible to autoclave Sephadex gels, but heat the Board of Trustees of the Leland Stanford Junior Uni- sterilization does not remove the discoloration that versity. Permission to reprint fig. 1 has also been given by accompanies long-term use of Sephadex for purifi- Shoichiro Suzuki. cation of plant extracts. Furthermore, dismantling Fig. 2 is from a 1962 article by Raimund Eberhardt in Archiv fiur Protistenkunde (106:241-341); used with the per- the column, autoclaving and washing the gel, and mission of the author and of Gustav Fischer Verlag, Jena, repouring the column are time-consuming opera- publisher, tions. We have found that application of a volume of Fig. 5 is adapted from a 1955 article by T. M. Sonneborn in Downloaded from http://online.ucpress.edu/abt/article-pdf/35/7/407/30722/4444465.pdf by guest on 26 September 2021 dilute sodium hypochlorite (maximum concentra- American Biology Teacher (17:187-190). tion, 0.5%) equal to the bed volume of the column, followed by rinsing with several bed volumes of dis- REFERENCES tilled water, restores flow rates, removes all traces GIESE, A. C. 1971. Photosensitization by natural pigments. In of contaminating microorganisms, washes away all Photophysiology, ed. by A. C. Giese. Academic Press, New discoloring materials that adhere to dextrans after York. Vol. 6, p. 77-129. -- et al. 1973. Blepharisma: the biology of a light- prolonged use, clears scintered glass plates, and sensitive protozoan. Stanford University Press, Stanford, rinses away all films that accumulate on glass sur- Calif. faces when contaminantsare present. MERTENS,T. R. 1966. Population explosion in a test tube. We have corresponded with Pharmacia Fine American Biology Teacher 28:103. the deleterious STOLTE,H. A. 1926. Die Kultur von Blepharisma undulans Chemicals, Inc., concerning possible and sein Verwendung in zoologischen Kursen. Zoologische effects of sodium hypochlorite on Sephadex. This Anzeiger 65:213. oxidizing agent must be used with caution because of the potential oxidation of terminal glucose resi- dues in the gel. The formation of carboxyl groups may induce ion-exclusion and ion-exchange proper- ties. These properties are most noticeable at pHs that produce ionization of the carboxyl group and Footnotes to Genesis 6-8 at low ionic strength of the eluant. However, in our qualitative use of Sephadex columns we routinely * The following was contributed by John H. Rosen- use distilled water as an eluant-a condition that gren, Biology Dept., William Paterson College, Wayne, N.J. 07470. should enhance such effects. We have noticed no significant change in the effectiveness of G-10, G-25, After many days of rain and the two worst floods or G-50 Sephadex columns to remove small mole- in 20 years, the following notice was placed on the cules from crude plant enzyme preparations. How- bulletin board in the Biology Department at William ever, there are observable changes in the physical Paterson College, in New Jersey-as it continued to properties of these gels: there is a noticeable swell- rain: ing, a slightly more gelatinous consistency, a signifi- cantly enhanced flow rate, and a restoration to the To: ALL pure white of a new gel. Meet at 9 a.m. at the water tower with nails, Because the changes in Sephadex gels are unpre- boards, animals, and "plants" (insisted on by the dictable, this method of decontamination should be botanists). NO WORK, NO GO. - NOAH used with caution. The method does provide an Within 15 minutes the following was added: alternative to discarding gels that are no longer usable, however, and the deleterious effects of dilute Dear NOAH: sodium hypochlorite appear to be less serious than Let the botanists build their own ark. UNLESS the action of contaminatingbacteria and fungi. they march the plants on, two by two. - THE ANIMALS. Helen M. Habermann and Doreen Berg Sekulow This was followed in 10 minutes by: Department of Biological Sciences Goucher College Dear NOAH: Towson, Baltimore, Md. 21204 You need us. But we DON'T need you. - THE PLANTS.

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