Feeding Behaviors in Cellular Slime Molds: a Microbial System to Study Competiion

Feeding Behaviors in Cellular Slime Molds: A Microbial System To Study Competiion

Donna M. Bozzone

O NE challengefaced in the teachingof introduc- goes a series of shape changes, specific genes are tory biology laboratory is in finding ecology turned on while others are turned off, and cells differ- exercises that are experimental, open-ended, entiate. The result is a fruiting body, a structure relatively inexpensive, and which can be completed consisting of a ball of spores borne atop a delicate and even repeated in a reasonable time-frame. A cellular stalk (Bonner 1944). The fruiting body is a survey of Introductory Biology laboratory manuals resting structure for the organism. Unless food be- shows exercises that undertake studies of pond eco- comes available, the amoebae will remain dormant Downloaded from http://online.ucpress.edu/abt/article-pdf/59/9/565/48121/4450384.pdf by guest on 26 September 2021 systems (Eberhard1990; Morgan & Carter1993), water within the spores. analysis (Abramhoff& Thomson 1994), forest ecosys- The problem investigated in this student project is tems (Morgan& Carter1993), and survivorship(Perry an ecological one. Examination of soil samples col- & Martin 1992). However, none takes full advantage lected from a variety of locations reveals that different of the ease with which microbial eukaryotes can be species of cellular slime molds exist very close to- used for laboratoryinstruction. Described in this pa- gether in nature and in all probability, inhabit the per is a laboratoryproject undertaken by our first year same physical space (Eisenberg 1976; Kuserk 1980; biology students. In this project,students study com- Buss 1982;Eisenberg et al. 1989;Ketcham & Eisenberg petition among various cellular slime molds. After a 1989). The question arises then, how do different brief introductionto the topic of competitionand basic species share or compete for resources to permit life history information about cellular slime molds, survival; how do these organisms partition their students either chose a suggested question, or posed niches?At least for some cases, it has been determined their own, and designed original experiments to an- that different species of cellular slime molds exhibit swer these questions. Without exception,the results of dissimilargrowth rates and competitiveabilities when student projectslead easily to additionaltestable ques- fed different species of bacteria (Horn 1971; Kuserk tions. 1980). Therefore,the similarity of the physical space occupied may not be the most critical factor; the different species avoid direct competition by having Background differentfood preferences.While this differentialuse If you were to isolate the organisms found living in of resourcesmight help to produce the microdistribu- the soil, some of the very interesting microbial eu- tion of cellular slime molds in nature, direct interfer- karyotesyou would encounterare the social amoebae, ence between species is another possible mechanism. or cellular slime molds (Raper 1984). As part of their Indeed, McQueen (1971a,b) studied competition be- unusual life cycle, cellular slime molds exhibit a tween two cellularslime mold species and argued that variety of cellular behaviors that has made them a direct interspecificcompetition is an important strat- very popular experimentalsystem for studying basic egy used by some cellular slime molds. The experi- questions about cell biology, gene expression, devel- ment outlined below describes a method to study opment, ecology, and evolution (Bonner1967; Loomis these behaviors. 1982;Spudich 1987).During the asexual component of the life cycle (Figure 1), spores germinate releasing amoebae. These feed on bacteria and produce more Methods amoebaeby binary fission. If the food supply becomes Student Instructions depleted, the amoebae enter their social phase. They aggregate, or come together, thus producing a multi- Setting Up Cultures. USE STERILETECHNIQUE cellular organism. This multicellularstructure under- FOR THE PROCEDURES DESCRIBED BELOW. 1. Each group of students will be provided with suspensions of various types of bacteria. For Donna M. Bozzone is Associate Professor and Chair of bacterial species #1, vortex or agitate the sus- Biology at St. Michael's College, Colchester, VT 05439; to the bacteria.Using a e-mail: [email protected]. pension gently suspend sterile loop, streak a thin line of the bacterial

CELLULARSLIME MOLDS 565 into a sterile test tube. This suspension will be used by the entire class. 5. Once the bacterial streak is dry, deposit, at one end of the MORPHOGENESIS SLUG streak,10 ,lI of a cellularslime MIGRATION mold spore suspension. (You can use an automatic pipet, a sterile capillary pipet, or a sterile loop to do so.) Repeat this step for each bacterial streak. 6. If a sterile bench is available, return the petri dishes to it, and once again blow sterile air over the open dishes to facilitateevaporation of liquid Downloaded from http://online.ucpress.edu/abt/article-pdf/59/9/565/48121/4450384.pdf by guest on 26 September 2021 from the spore suspension. (If FRUITINGBODY AGGREGATION no hood is available, be care- ful when moving the dishes so that the liquid from the spore suspension won't spread. It will eventually dry on its own.) 7. Steps 1-6 can be repeated to study another cellular slime mold strain or species. 8. Determine the rate of feeding by measuring the movement SPORES of amoebae down the bacterial streak.The position of the amoebae on the bacterial streak is called the feeding front. Measurements should AMOEBAE be taken daily for 7-10 days. Figure 1. Life cycle of a cellular slime mold. 9. Examine the morphology of the different cellular slime mold species used in your ex- suspension on a petri dish of lactose-peptone periment to determine the identity of the cellu- (2LP)agar. lar slime mold fruiting bodies that develop at 2. Repeat Step 1 for bacterial species 2, 3, and so the far end of the bacterial streak (after the one. Markeach petri dish to indicate the type of streakhas been eaten). Comparetheir morphol- bacteriapresent. ogy to the fruiting bodies on the stock plates. 3. If a sterile bench equipped with a fan is avail- 10. Whateverquestion you decide to pursue, exam- able, place the petri dishes streaked with bacte- ine Figure 2 for possible ways to set up cultures ria within it. Turn the fan on, uncover the and choose or design one most appropriatefor dishes, and allow the liquid from the bacterial the question you are pursuing. Also, be sure to suspension to evaporate. design and set up your experiment so that you 4. Each group of students will also be provided have at least two replicates. with a petri dish containing cellular slime mold fruiting bodies that you will use to collect Ideas for Experiments spores. Each lab group will prepare a different spore suspension for general use. In order to 1. Does a given species of cellular slime mold show prepare the spore suspension, add 8 ml of any food preference? Bonner's Salt Solution (Bonner 1947; Table 1). 2. Does a given species of bacteria vary in its Using a sterile glass elbow, gently dislodge the suitability as a food source for differentcellular fruiting bodies. Pour the resulting suspension slime molds?

566 THEAMERICAN BIOLOGY TEACHER, VOLUME 59, NO. 9, NOVEMBER/DECEMBER1997 3. Will two or more species of cellular slime mold solutions. Table 1. Recipes for culture media and directly influence each other's feeding behavior Lactose-PeptoneAgar (double strength; 2LP) on a given bacterium? If so, is the "winner" determined by the type of bacterium? Lactose 2 g 4. Will two or more species of cellular slime mold Peptone 2 g KH2PO4 0.28 g indirectly influence each other's feeding behavior Na2HPO4 0.28 g on a given bacterium? If so, is the "winner" Agar 15 g determined by the type of bacterium? cellu- Add distilled or deionized H20 up to 1 liter. Autoclave 5. Will two opposite mating type strains of a 20-25 minutes, slow exhaust (liquid cycle). (Raper 1984). lar slime mold species show any difference in food preference? Sussman's Medium (half-strength;SM/2) 6. Will two opposite mating type strains of a cellu- Glucose 0.5 g lar slime mold species indirectly influence each Peptone 0.5 g other's feeding, and if so, is the "winner" deter- Yeast extract 0.05 g mined by the type of food? MgSO4 7H20 0.05 g 7. Can two species co-exist if provided with mixed KH2PO4 0.1 g Downloaded from http://online.ucpress.edu/abt/article-pdf/59/9/565/48121/4450384.pdf by guest on 26 September 2021 K2HPO4 0.05 g bacterial suspensions? 8. After approval by your laboratory instructor, Add distilled or deionized H20 up to 100 ml. Autoclave pursue a question of your own design. 20-25 minutes, slow exhaust (liquid cycle). (Sussman 1987). Bonner's Salt Solution Materials Available for Each Group CaCl2 0.3 g (Studentsdo not need to use everything.) KCI 0.75 g NaCl 0.6 g * 10 lactose peptone plates * Cellular slime molds: Add distilled or deionized H20 up to 1 liter. Autoclave 20-25 minutes, slow exhaust (liquid cycle). (Bonner 1947). Polysphondyliumpallidum Dictyosteliumdiscoideum NC4

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CELLULARSLIME MOLDS 567 TESTFEEDING OF DIFEERENT CSM ON THESAME TYPE OF BACTERIA

1 1 ~~2 2 1 1&2 1&2

ALONE ALONE INDIRECT DIRECT COMPETITION COMPETITION Downloaded from http://online.ucpress.edu/abt/article-pdf/59/9/565/48121/4450384.pdf by guest on 26 September 2021

TESTABILITY OF DIFFERENT TYPES OF BACTERIA TO SUPPORTCSM GROWTH

SINGLEBACTERIA MIXED BACTERIA CULTURE CULTURE

TESTWHETHER CSM CAN PARTITION RESOURCES AND CO-EXIST

568 THEAMERICAN BIOLOGY TEACHER, VOLUME 59, NO. 9, NOVEMBER/DECEMBER1997 Dictyostelium discoideum V12 (NC4 and V12 are opposite mating types) Dictyostelium rosarium Bacterial suspensions:

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... SS Bacillus megaterium

. ..:':.i:.:. . '.... _,::.. .. .:......

Bacillus subtilis _M .:;.....,...... :.:ii...... ;... :._

: : ~~~~~~~~~~~~~~~~~~~~...... ''. :::..s': ;.:.:. : .:::'... :..':. ::i.: _ ...... , k .. r .... .~~~~~~~~~~~~~~~~~~~~~~....., ...... _... Escherichiacoli (mucoid) FigurE 3. Dictyostelium roarum (left) and Polysphondylium.. pallidum ( ) f b C lu e .... . w.e.r.e. g...... d..' ...... s...... : .... Escherichiacoli (non-mucoid) :. ^w?8..... ' .....,,:<,

Pseudomonasfluorescens ~~~~~L'M...'.:lSe~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~... ..S,.S ;,. ...

..:...... :, :-.:,....: : :,, ~~~~....: ...... Serratia marcescens

Data Analysis Figure 3. Dictyostelium rosarium (left) and Polysphondylium pallidum (right) feeding on Klebsiellaaerogenes. Cultures were six days old.

1. Prepare a table presenting the position of the Downloaded from http://online.ucpress.edu/abt/article-pdf/59/9/565/48121/4450384.pdf by guest on 26 September 2021 amoebae feeding front each day for each bacterial streak. Calculate the averages for your replicates. uled laboratory. Cultures can be incubated at room If pertinent, also indicate the species of fruiting temperature. bodies appearing at the far end of the streak. Cellular slime molds can either be obtained from 2. For each cellular slime mold bacterial combina- Carolina Biological or from ATTC (12301 Parklawn tion, plot the total distance moved by the amoe- Drive). To culture, inoculate petri dishes containing bae feeding front vs. time. Interpret your results double strength lactose-peptone medium (Table 1) fully in the context of both the specific question and 1 ml of Klebsiella aerogenes bacterial suspension, addressed in your experiment and the general and a loop of cellular slime mold spores (Figure 3). problem of cellular slime mold competition and Using a sterile glass elbow, spread the spore-bacterial co-existence in nature. suspension evenly over the medium. These cultures 3. If your experiment was the first one in a long- should be prepared 1-2 weeks before the scheduled term project, what would you do next (besides laboratory. They can be incubated at room tempera- repeat the experiment you already did)? ture. A more detailed description of how to culture cellular slime molds can be found in Bozzone (1993). Instructor's Instructions Other Suggestions Preparation of Cultures The success of this experiment depends somewhat The bacterial cultures used in this experiment were on sterile technique. Review the basics of aseptic obtained from Carolina Biological Supply Company culture with the class before they begin and provide a (2700 York Rd., Burlington, NC 27215). These specific couple of practice plates to each group so they can cultures were chosen because they had been previ- practice making the bacterial streaks, etc., rather than ously shown to be potential food sources for cellular gouge the plates they are going to need. Many labo- slime molds (Raper 1984). For use in this experiment, ratory manuals contain general instructions for sterile bacterial suspensions were prepared by inoculating culture techniques. half-strength Sussman's Medium (Table 1) with a The method described here uses a sterile transfer transfer loop of bacteria 48-72 hours before the sched- hood. If you don't have one, simply pour the plates

Table 2. Growth of Dictyostelium discoideum (V12) on streaks of various types of bacteria. Distance of Feeding Front from Origin of Bacterial Streak (cm) Days of Culture E. coli P. fluorescens B. megaterium K. aerogenes B. subtilis 1 .08 0.5 0.3 0.4 0 2 4.2 0.8 1.0 0.9 0 3 5.1 1.7 1.2 1.4 0 4 7la 4.2 3.5 3.0 0.2 5 7 5.4 4.1 3.7 0.4 6 7 7 5.0 4.0 0.6 a/maximum length of bacterial streak.

CELLULARSLIME MOLDS 569 here is pathogenic if used as discussed, we generally sterilize everything before regular cleaning or dis- posal. Add full strength germicidal soap to bacterial suspensions. Flame transfer loops or if disposable ones are used soak them in germicidal soap or auto- E clave them. Similarly, soak pipet tips or capillary pipets and autoclave petri dish cultures before dis- posal. 0 E 04 Results This experimentwas done by students in our Gen- eral Biology course. While this course is designed for majors, fewer than half of the students are declared 1 2 3 4 5 6 biology majors while enrolled in the course. Labora- tory meets once a week for three hours. There are

Days of Culture usually 20-24 students in a lab with one instructor Downloaded from http://online.ucpress.edu/abt/article-pdf/59/9/565/48121/4450384.pdf by guest on 26 September 2021 and one undergraduate assistant. Students work in E.coli IF P.fluorescens * B.megaterium teams of three or four. The results reported here are K.aerogenes - B.subtlfis the actual data collected by four lab groups. Group #1 decided to examine whether a particular Figure 4. Effectof bacteriatype on the growth of Dictyoste- type of cellular slime mold would exhibit differential (V12). lium discoideum growth rates on different types of bacteria. They preparedstreaks of five differenttypes of bacteria,and inoculated each streakwith D. discoideum(V12). Their results clearly show that this type of cellular slime mold was able to utilize these food resources with very differentefficiencies (Table 2, Figure 4). Group #2 asked whether a particulartype of bacterium was an equally good food source for different 2 4 types of cellularslime molds. They preparedstreaks of E Pseudomonasfluorescens and inoculated them with four 3 differentcellular slime molds. Again, it was very clear that a specific type of bacteriumcould serve as a food source with varying efficiencydepending on the type of cellular slime mold (Table3; Figure 5). In Group #2's experiment, one of the unexpected 0 outcomes was that the two opposite mating type 1 2 3 4 5 6 7 strains of D. discoideum,NC4 and V12, grew at very different rates on P. fluorescens(Table 3; Figure 5). of Culture Days Group#3 pursued this furtherby testing the growth of NC4 and V12 on another species of bacterium,Esche- D.rosarium P.pallidum richiacoli. They also observed better final growth of V12 on the bacteria as compared with NC4 (Table 4; D.discoideum(NC4) * D.discoideum(V12) Figure 6). It remains to be seen whether this growth difference is true for other bacteria and what would Figure 5. Ability of several cellular slime mold species to happen to the growth rate if NC4 and V12 were mixed feed on Pseudomonasfluorescens. together. Finally, Group #4 asked whether different cellular the students will use a week early and don't refriger- slime molds could influence each other's growth. To ate them. The dishes will dry out a little bit and the test this, D. rosariumand P. pallidumwere inoculated liquid from the bacterial streaks will soak in more on streaks of E. coli. When each was the only cellular quickly. Alternatively, students can prepare their bac- slime mold on the plate, D. rosariumadvanced 4.8 cm terial streaks 24 hours before they inoculate them with down the streakand P. pallidum,2.0 (Table5; Figure7). cellular slime mold spores. When cultures were set up so that there were two Finally, a word about cleanup. While none of the bacterialstreaks, one with D. rosarium,and one with P. bacterial or cellular slime mold cultures described pallidum,on the same plate, results were very different.

570 THEAMERICAN BIOLOGY TEACHER, VOLUME 59, NO. 9, NOVEMBER/DECEMBER1997 Table 3. Growth of various cellular slime mold species on Pseudomonasfluorescens.

Distance of Feeding Front from Origin of Bacterial Streak (cm) Days of Culture D. rosarium D. discoideum (NC4) D. discoideum (V12) P. pallidum (PN200) 1 0.3 0.4 0.1 0.4 2 0.4 1.2 0.9 0.5 3 1.6 1.5 1.4 1.2 4 2.1 1.9 1.9 1.8 5 5.5 2.5 6.4 1.9 6 5.8 2.5 6.8 2.1 7 6.2 2.6 7.0 2.1

to pursue. Some of these questions have included the Table 4. Growth of two strains of D. discoideumon E. coli. following:

Distance of Feeding Front from Would we get the same results if we inoculated the Days of Culture Origin of BacterialStreak (cm) bacterial streaks with amoebae instead of spores (in Downloaded from http://online.ucpress.edu/abt/article-pdf/59/9/565/48121/4450384.pdf by guest on 26 September 2021 NC4 V12 other words, eliminate any growth differencescaused 1 0.9 0.8 by differentialspore germination)? 2 1.9 0.8 Do the fastest eaters win a "head-to-head"competi- 3 1.9 0.8 tion (mix the spores together and then inoculate the 4 2.3 1.1 bacteria)? 5 2.7 2.3 6 2.8 4.0 Will varying light, temperature or humidity have any influence on competition? While the of D. rosariumon the plate had no Would we observe these same phenomena if we presence were to isolate local soil bacteria and cellular slime effect on the growth of P. pallidum,the converse was molds to use in these experiments? not true; the presence of P. pallidumreduced the As can be seen, the students have no shortage of growth of P. rosarium(Table 5; Figure 7). ideas and we look forward to seeing what they come up with next year. Discussion This project has been a great success for our stu- References dents. Using relatively easy methods, they were able Abramhoff,P. & Thomson,R.G. (1994). Laboratory Outlines in to design their own experiments to answer their own BiologyVI. New York,NY: W.H. Freemanand Company. questions about competition and interspecificinterac- Bonner,J.T. (1944). A descriptive study of the development tions. Since this project uses a microbial system, it of the slime mold Dictyosteliumdiscoideum. American Jour- doesn't take up too much room; it is not very expen- nal of Botany,31, 175-182. Bonner,J.T. (1947). Evidence for the formationof cell aggre- sive; and should an experimentfail, it can be repeated gates by chemotaxisin the developmentof the slime mold immediately. It is an added benefit that if students (or Dictyosteliumdiscoideum, Journal of ExperimentalZoology, instructors) so desire, additional experiments can be 106, 1-26. run to answer questions raised by initial experiments. Bonner,J.T. (1967). The CellularSlime Molds. Princeton, NJ: Withoutexception, the individual experimentsdone PrincetonUniversity Press. Bozzone,D.M. (1993).A practicalguide to the use of cellular by student groups produced data that could be ana- slime molds for laboratoryexercises and experiments.In lyzed and interpreted.More importantly,these exper- C.A. Goldman (Ed.),Tested Studies for LaboratoryTeaching, iments lead to a rich array of ideas for other avenues Volume14 (pp. 1-19). Proceedingsof the 14th Workshop!

Table 5. Growth of different cellular slime mold species cultured together or alone on E. coli.

Days of Culture Distance of Feeding Front from Origin of Bacterial Streak (cm) D. rosarium D. rosarium P. pallidum P. pallidum (alone) (with P. pallidum) (alone) (with D. rosarium) 1 0.9 0.7 0.5 1.1 2 1.0 0.7 0.5 1.1 3 1.2 1.3 1.1 1.9 4 1.3 1.5 1.2 1.9 5 2.8 1.9 1.5 1.9 6 3.3 2.4 2.0 1.9 7 4.8 2.9 2.0 2.2

CELLULARSLIME MOLDS 571 Conferenceof Associationfor BiologyLaboratory Education 4 (ABLE). Buss, L. (1982).Somatic cell parasitismand the evolution of 3.5 somatic tissue compability. Proceedingsof the National C Academyof Science,USA, 79, 5337-5341. Eberhard,C. (1990). SaundersGeneral Biology Manual. Fort 0 Worth, TX:Saunders College Publishing. 0 2.5 Eisenberg,R.M. (1976).Two-dimensional microdistribution E of cellular slime molds in forest soil. Ecology,57, 380-384. Eisenberg, R.M., Hurd, L.E. & Ketcham, R.B. (1989). The cellular slime mold guild and its bacterialprey: Growth rate variationat the inter- and intraspecificlevels. Oecolo- 2 21.5 gia, 79, 458-462. I Horn, E.G. (1971). Food competition among the cellular slime molds (Acrasiae).Ecology, 52, 475-484. Ketcham,R.B. & Eisenberg,R.M. (1989).Clonal diversity in 0.5 populations of Polysphondyliumpallidum, a cellular slime 1 2 3 4 5 6 mold. Ecology,70,1425-1433. Days of Culture Kuserk, F.T. (1980). The relationship between cellular

slime molds and bacteria in forest soil. Ecology, 61, Downloaded from http://online.ucpress.edu/abt/article-pdf/59/9/565/48121/4450384.pdf by guest on 26 September 2021 NC4 - V12 1474-1485. Loomis, W.F. (Ed.). (1982). TheDevelopment of Dictyostelium Figure 6. Growth of opposite mating type strains of Dictyo- discoideum.New York:Academic Press. steliumdiscoideum on Escherichiacoli. McQueen,D.J. (1971a).A components study of competition in two cellularslime mold species:Dictyostelium discoidium and Polysphondyliumpallidum. Canadian Journal of Zoology, 49, 1163-1177. 5 McQueen,D.J. (1971b).Effects of continuous competitionin two species of cellular slime mold: Dictyosteliumdiscoi- deum and Polysphondyliumpallidum. Canadian Journal of Zoology,49, 1305-1315. Morgan, J.G. & Carter, E. (1993). InvestigatingBiology: A 0 LaboratoryManual for Biology.Redwood City, CA: Ben- E 3 0 jamin/Cummings Publishing Company, Inc. Perry,J.W. & Morton,D. (1992).Laboratory Manual for Starr E and Taggart'sBiology: The Unity and Diversityof Lifeand Starr's Biology:Concepts and Applications.Belmont, CA: WadsworthPublishing Company. Raper,K.B. (1984). The Dictyostelids. Princeton, NJ: Princeton University Press. Spudich, J.A. (Ed.). (1987). Methodsin Cell BiologyVolume 28-Dictyostelium discoideum:Molecular Approaches to Cell 0 Biology.New York:Academic Press. 1 2 3 4 5 6 7 Sussman, M. (1987). Coltivation and synchronous morphogenesis of Dictyosteliumin controlled experimental Days of Culture conditions. In J.A. Spudich (Ed.), Methodsin Cell Biology, D.rosarium (alone) v D.rosanium(with P.pal.) Volume 28 -Dictyostelium discoideum; Molecular Ap- proachesto Cell Biology(pp. 9-29). New York:Academic P.pallidum(alone) * P.pallidum(with D.ros.) Press.

Figure 7. Growth of Dictyosteliumrosarium and Polyspondy- liumpallidum on Escherichiacoli. Two streaksof bacteriawere inoculated on each plate. In the plates designated "along," both streaks of bacteria were inoculated with either D. rosariumor P. pallidum.In other plates, one streakof bacteria was inoculated with D. rosarium,and the other with P. pallidum.

572 THEAMERICAN BIOLOGY TEACHER, VOLUME 59, NO. 9, NOVEMBER/DECEMBER1997