JOURNAL OF BACTERIOLOGY, Mar. 1969, p. 1411-1418 Vol. 97, No. 3 Copyright @ 1969 American Society for Microbiology Printed in U.S.A. Growth of Large Plasmodia of the Myxomycete Physarum polycephalum' JOYCE MOHBERG AND HAROLD P. RUSCH McArdle Laboratory, Medical School, University of Wisconsin, Madison, Wisconsin 53706 Received for publication 29 November 1968 A method has been developed for growing Physarum polycephalum plasmodia that are 8 to 10 times larger than those obtained in the petri dish cultures used by Nygaard, Guttes, and Rusch. In the large-scale procedure, plasmodia were grown in metal trays on a membrane supported by filter paper on stainless-steel screen. Plasmodia were started from a ring of inoculum to allow inward and outward mi- gration and were incubated on a rocker so that nutrient medium would flow back and forth, wetting the undersurface of the plasmodium. Rocker and petri dish cul- tures had similar growth characteristics: (i) the interphase time between mitoses I and II and between II and III was about 8 hr; (ii) ribonucleic acid and protein increased essentially logarithmically throughout the cell cycle; and (iii) deoxy- ribonucleic acid increased only during early interphase and it doubled in approxi- mately 3 hr after each mitosis. Rocker cultures were not as nearly synchronous as petri dish cultures and had a range in metaphase time (at mitosis III) within indi- vidual plasmodia of 15 to 45 min, as compared with 5 to 10 min in petri dish cultures. Synchronous plasmodial cultures of Physarum polycephalum can be grown quite simply in petri dishes on filter paper, supported on the surface of nutrient medium by glass beads (4-6, 11). However, such cultures, even when they have reached their maximal size, contain only about 300 ,ug of deoxyribonucleic acid (DNA). For studies of nuclear histones, 10 plasmodia were needed for a single histone preparation, and 40 to 60 were needed for an experiment. Since in- dividual plasmodia, although synchronous within themselves, went through mitosis at different times, it was necessary not only to examine smears (5, 6) of all cultures to establish metaphase times but also to chill plasmodia with earlier mitoses to stop their development until enough cultures for a histone preparation had been accumulated. The time thus required for handling cultures virtually precluded certain experiments with metabolic inhibitors (3) and radioisotopes. It also was questionable whether these pools of plasmodia, made up of cultures which had gone through mitosis at different times and which were harvested at a fixed time after individual mitoses, could be considered synchronous. In an effort to avoid these problems, we be- gan work on a system for growing plasmodia 1 A preliminary report of this work was presented at the 7th Annual Meeting of the American Society for Cell Biology in Denver, Colo. (J. Cell Biol. 35:96A, 1967). that were 10 times larger than those obtained with petri dishes. Preliminary experiments showed that plasmodia of the desired size could not be obtained simply by expansion of the petri dish system, i.e., by using a larger dish and more inoculum, since plasmodia so prepared were very thick, slow growing, and poorly synchro- nized. However, a plasmodium with good ap- pearance and synchrony could be grown when (i) the inoculum was applied in an open ring so that the plasmodium could migrate toward both the center and the periphery, and (ii) the culture vessel was incubated on a rocker so that the me- dium was continually agitated. MATERIALS AND METHODS Maintenance of cultures. Stock submerged cultures of Physarum strain M3c (4) were grown at 22 C in 20-ml shaken cultures in semidefined medium with hemin and citrate. Transfers (0.8 ml) were made at 3-day intervals. The methods of plasmodial culture were similar to those used by Mittermayer et al. (10). Inoculum for plasmodial cultures was prepared by inoculating shake flasks with 3 ml of 3-day stock cul- tures and incubating the flasks for 24 to 27 hr, to a protein level of about 1.5 mg per ml of medium. Eight flasks were inoculated to make six large plasmodia (see below). Petri dish cultures were grown on mem- branes (Millipore Corp., Bedford, Mass.) supported on 4-mesh stainless-steel screen. Apparatus. The rocker platform (Beilco # 6600 1411 on September 9, 2020 by guest http://jb.asm.org/ Downloaded from
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JOURNAL OF BACTERIOLOGY, Mar. 1969, p. 1411-1418 Vol. 97, No. 3Copyright @ 1969 American Society for Microbiology Printed in U.S.A.
Growth of Large Plasmodia of the MyxomycetePhysarum polycephalum'JOYCE MOHBERG AND HAROLD P. RUSCH
McArdle Laboratory, Medical School, University of Wisconsin, Madison, Wisconsin 53706
Received for publication 29 November 1968
A method has been developed for growing Physarum polycephalum plasmodiathat are 8 to 10 times larger than those obtained in the petri dish cultures used byNygaard, Guttes, and Rusch. In the large-scale procedure, plasmodia were grownin metal trays on a membrane supported by filter paper on stainless-steel screen.Plasmodia were started from a ring of inoculum to allow inward and outward mi-gration and were incubated on a rocker so that nutrient medium would flow backand forth, wetting the undersurface of the plasmodium. Rocker and petri dish cul-tures had similar growth characteristics: (i) the interphase time between mitosesI and II and between II and III was about 8 hr; (ii) ribonucleic acid and proteinincreased essentially logarithmically throughout the cell cycle; and (iii) deoxy-ribonucleic acid increased only during early interphase and it doubled in approxi-mately 3 hr after each mitosis. Rocker cultures were not as nearly synchronous aspetri dish cultures and had a range in metaphase time (at mitosis III) within indi-vidual plasmodia of 15 to 45 min, as compared with 5 to 10 min in petri dish cultures.
Synchronous plasmodial cultures of Physarumpolycephalum can be grown quite simply in petridishes on filter paper, supported on the surfaceof nutrient medium by glass beads (4-6, 11).However, such cultures, even when they havereached their maximal size, contain only about300 ,ug of deoxyribonucleic acid (DNA). Forstudies of nuclear histones, 10 plasmodia wereneeded for a single histone preparation, and 40to 60 were needed for an experiment. Since in-dividual plasmodia, although synchronous withinthemselves, went through mitosis at differenttimes, it was necessary not only to examine smears(5, 6) of all cultures to establish metaphase timesbut also to chill plasmodia with earlier mitosesto stop their development until enough culturesfor a histone preparation had been accumulated.The time thus required for handling culturesvirtually precluded certain experiments withmetabolic inhibitors (3) and radioisotopes. Italso was questionable whether these pools ofplasmodia, made up of cultures which had gonethrough mitosis at different times and whichwere harvested at a fixed time after individualmitoses, could be considered synchronous.
In an effort to avoid these problems, we be-gan work on a system for growing plasmodia
1 A preliminary report of this work was presented at the 7thAnnual Meeting of the American Society for Cell Biology inDenver, Colo. (J. Cell Biol. 35:96A, 1967).
that were 10 times larger than those obtainedwith petri dishes. Preliminary experiments showedthat plasmodia of the desired size could not beobtained simply by expansion of the petri dishsystem, i.e., by using a larger dish and moreinoculum, since plasmodia so prepared werevery thick, slow growing, and poorly synchro-nized. However, a plasmodium with good ap-pearance and synchrony could be grown when(i) the inoculum was applied in an open ring sothat the plasmodium could migrate toward boththe center and the periphery, and (ii) the culturevessel was incubated on a rocker so that the me-dium was continually agitated.
MATERIALS AND METHODS
Maintenance of cultures. Stock submerged culturesof Physarum strain M3c (4) were grown at 22 C in20-ml shaken cultures in semidefined medium withhemin and citrate. Transfers (0.8 ml) were made at3-day intervals. The methods of plasmodial culturewere similar to those used by Mittermayer et al. (10).Inoculum for plasmodial cultures was prepared byinoculating shake flasks with 3 ml of 3-day stock cul-tures and incubating the flasks for 24 to 27 hr, to aprotein level of about 1.5 mg per ml of medium. Eightflasks were inoculated to make six large plasmodia(see below). Petri dish cultures were grown on mem-branes (Millipore Corp., Bedford, Mass.) supportedon 4-mesh stainless-steel screen.
from Bellco Glass Inc., Vineland, N.J.) with a rackfor five culture trays or 10 plasmodia is shown inFig. 1. The culture vessel was a tray, 6.5 X 32 X53 cm, of either enameled or stainless steel (# 2002-1,Vollrath Co., Sheboygan, Wis.), with a stainless-steellid. The tray was fitted with a 4-mesh stainless-steel( # 304) screen, 25 X 45 cm. On the screen was laida 19 X 38 cm sheet of # 576 chromatography paper(Schleicher and Schuell Co., Keene, N.H.), and onthis was laid a 15 X 33 cm piece of Millipore mem-brane (HA, 0.45 uim pore size) on which two circles5 cm in diameter had been drawn with a no. 2 leadpencil 6.5 cm from each end. To prevent wrinkling ofthe Millipore membrane during autoclaving, one ortwo 23 X 46 cm sheets of the blue glassine paper usedas backing in rolls of Millipore membrane were laidon top of the membrane and weighted with a secondpiece of stainless-steel screen. The lid was put on thepan, and the entire assembly was slipped into anenvelope made of two thicknesses of brown kraftpaper. The packet was autoclaved at 120 C for 15 minand was dried by exhausting for 15 min.
Neither the wrapping paper nor the culture vesselcomponents was given any special treatment to re-move toxic materials, except that the stainless-steelitems were soaked overnight in detergent solutionbefore they were used for the first time. Subsequently,screens, lids, and trays were washed in a mechanicaldishwasher (Better Built Machinery Co., New York,N.Y.).
Preparation of rocker cultures. Just before inocula-tion, culture pans were slipped out of their envelopes.
FIG. 1. Rocker platform with one culture tray. Thealuminum rack consisted of horizontal bars (I X 2.5 X30 cm), fastened with set screws to vertical rods (0.6 X40 cm). The rack had shelves forfive culture trays.
Weighting screens and glassine papers were removedwith sterile forceps and put aside for reuse. The eightinoculum cultures were then removed from the shakerand immediately poured into four sterile, conical50-ml polypropylene tubes. The tubes were cappedwith heavy aluminum foil and centrifuged at 300 X gfor 1 min. The medium was discarded, and thepellets were suspended in twice their volume of steriledistilled water by gentle swirling in a 100-ml Erlen-meyer flask. A 3-ml amount of microplasmodial sus-pension was added with a wide-tipped pipette aroundthe periphery of each penciled circle (Fig. 2), and panswere set aside at room temperature to allow coales-cence of the microplasmodia. After 1.5 hr, 300 ml ofmedium of the same composition as that used forshaken cultures was poured into each pan, care beingtaken that medium flowed under the filter paper andnot on top of the Millipore membrane or the plas-modium. Trays were mounted on the rocker and incu-bated at 9 to 11 oscillations per min in the dark at26.5 to 27 C. (Cultures were kept in theincubatorroomthroughout the growth period. Medium changes andpreparation of smears were all done in the incubatorroom.) After the trays had been rocked for 1 to 2 hr,air bubbles trapped under the filter paper or betweenfilter paper and Millipore membrane were released byraising the papers with a sterile forceps. After about15 hr of incubation, the medium was aspirated fromthe trays, and 250 ml of fresh medium was added toeach pan. Once or twice during the incubation period,trays were moved to different shelves on the rockerto equalize any effects of the difference in the arcs ofrocking.
Chemical analyses. Plasmodia were prepared forchemical analysis as described by Sachsenmaier andRusch (12): after plasmodia had been washed withcold trichloroacetic acid in acetone and with cold0.25 M perchloric acid (PCA), nucleic acids were ex-tracted with 0.5 M PCA at 70 C. The 0.5 M PCAextract was analyzed for DNA with diphenylamine (1)and for ribonucleic acid (RNA) with orcinol (2).Diphenylamine color was developed for 8 hr insteadof the recommended 22 hr (1) to reduce interferenceby a cytoplasmic component that slowly developed ablue color with diphenylamine. Salmon sperm DNA,from Calbiochem, Los Angeles, Calif., was used as astandard. In the RNA analysis, the isoamyl alcoholextraction step was omitted to increase the range oflinearity. D-Ribose, A grade from Calbiochem, wasused as the RNA standard. Ribose equivalents wereconverted to RNA by multiplying by 3.5, on the as-sumption that 60% of the RNA ribose reacted withorcinol (2). The residue from the 0.5 M PCA extrac-tion was dissolved in 0.4 N NaOH and was analyzedfor protein by use of phenol reagent (8) with bovineserum albumin (Cohn Fraction V, from NutritionalBiochemicals Corp., Cleveland, Ohio) as standard.
RESULTSGrowth. Figure 2 shows two large plasmodia at
the end of the coalescence period, at the time ofaddition of medium. Appearance was essentiallythe same as at the time of inoculation, since
FIG. 2. Two plasmodia, immediately after additionof medium. Inoculum was pipetted around 5-cm circles,drawn on the Millipore membrane. The membrane wassupported on Schleicher & Schuell filter paper, whichrested on 4-mesh stainless-steel screen. Details of di-mensions and culture procedures are given underMaterials and Methods.
there was no measurable growth or migrationduring the coalescence period. Figure 3 showsplasmodia after approximately 30 hr of incuba-tion (about 4 hr before mitosis IV), by which timegrowth toward the center and periphery hadproduced a disc with a diameter of about 14cm. Inoculum rings appear dark in the photo-graphs because they were somewhat thickened.Actually, the color was quite similar to that ofpetri dish cultures. The only obvious differencein appearance was that cultures grown on therocker were thicker; at 6 hr after mitosis III,they had a ratio of protein to area of 1.3 mg percm2, as compared with 0.7 mg per cm2 in sta-tionary cultures.
Figure 4 presents growth curves of rocker andpetri dish cultures, prepared from the sameinoculum pool, grown at the same temperature(26 C), and given a change of medium after 15
FIG. 3. Two plasmodia after 30 hr of growth. Plas-modia, prepared as shown in Fig. 2 and incubated on therocker at 26 C for about 30 hr, had migrated towardthe center and periphery to produce a disc with a 14-cmdiameter.
hr of growth. The two types of culture were al-most identical in growth rate: both went throughmitosis I between 4.75 and 5 hr after addition ofmedium to the coalesced plasmodia, throughmitosis II between 12.5 and 13 hr, and throughmitosis III between 20 and 21.5 hr, giving aninterphase time of about 8 hr. When mediumwas not changed 3 hr after mitosis II, the inter-phase time between mitosis II and mitosis IIIwas 10 hr or more. (Other experiments showedthat the large plasmodia went through mitosisIV 13 hr or more after mitosis III, even whenmedium was changed twice between mitoses IIIand IV.)Both rocker and petri dish cultures showed
essentially logarithmic increases in RNA andprotein. Mittermayer et al. (9) showed that in-corporation of 3H-uridine and 3H-leucine into
FiG. 4. Growth curves of rocker and petri dish cul-tures. Six rocker and 12 petri dish plasmodia were pre-
pared and grown as described under Materials andMethods. All cultures were started from the same poolor inoculum, with 3.0 ml being used for the large plas-modia and 0.3 ml for petri dish cultures. Plasmodiawere harvested andprocessedfor analysis (see Materialsand Methods) at the times indicated on the graph. Fourpetri dish cultures were pooled for analysis at the firstsampling point, and two cultures were pooled for eachsubsequent point. (The graph also gives data fromanother experiment with petri dish cultures, "Expt.II$," where shorter sampling intervals were used and onlymitosis I was studied.) One whole large plasmodiumwas taken for both the 4- and 8-hr points; at each sub-sequent point, halves of two different plasmodia wereharvested and analyzed separately. Plasmodia were cutin half with sterile scissors, following guide lines drawnon the Millipore membrane before it was sterilized.
Physarum plasmodia is biphasic with a minimalrate at mid-interphase, and mid-interphase pla-teaus in the RNA and protein growth curveswould therefore be expected. Such plateaus arenot obvious in Fig. 4, perhaps owing to the longintervals between the times samples were taken.DNA almost doubled in both large and smallplasmodia during the first 3 hr after mitoses I,
II, and III (and IV, as well, in other experiments),and had completely doubled by 6 hr after eachmitosis. Experiments in which samples were takenat closer intervals showed that DNA replicationwas 50% complete in 1 hr and 75 to 80% com-plete by 2 hr after mitosis.
Both the large and small plasmodia showed anincrease in DNA between the time of inoculationand mitosis I. This would be expected if S-phasenuclei in the inoculum finished duplicating theirDNA before they participated in mitosis I.However, in other experiments, such as experi-ment II in Fig. 4, there was either no increase oran increase of only about 10%. Since the bulkof DNA synthesis occurs in the first 2 hr of the8-hr generation time, one would expect at least25% of the nuclei to be in S phase. This suggeststhat the inoculum transfer schedule sometimesgives partial synchrony of the shaken, micro-plasmodial cultures so that they are in G2 phaseat the time they are used to start plasmodia.This question has not been explored further.
Figure 4 shows a somewhat larger DNA toprotein ratio in petri dish plasmodia than inrocker cultures. This difference was seen con-sisteutly, but the cause has not been determined.
Mitotic synchrony. It was evident, both fromthe smears made for the growth curve experi-ment (Fig. 4) and from the stepwise increase inDNA during growth, that the large plasmodiawere at least partially synchronous. Synchronywas checked more thoroughly in a second seriesof experiments in which time of mitosis wasdetermined at several points within individualrocker and petri dish plasmodia. Before a com-parison of synchrony in small and large plas-modia could be made, it seemed advisable todetermine the duration of the various mitoticstages in petri dish cultures, because the originalobservations of Guttes, Guttes, and Rusch (5, 6)were done with cultures having a 12-hr instead ofan 8-hr interphase time.
Accordingly, petri dish cultures were startedand incubated until shortly before mitosis III.Sampling for microscopic examination was thenbegun, with smears being taken from one quad-rant of each plasmodium at 1- or 2-min intervalsuntil 30 min after metaphase, at which time thesampling interval was increased to 10 or 15 min.Smears were constantly monitored by phase-contrast microscopy, and when mitosis was ob-served smears were made simultaneously fromthree points of the plasmodium-two oppositeedges and the center or inoculum spot. Datawere thus obtained both on the duration of themitotic stages (Table 1) and on the synchronywithin plasmodia (Table 2). (A more accuratedetermination of duration of the mitotic stagescould be made if single nuclei were followed bytime-lapse cinematography, but we do not as yethave techniques for eliminating the effects of thelight and heat that would be associated withsuch a procedure.)
metaphase in all sampling areas, metaphase timeoften had to be estimated from other mitoticstages. To make such estimates more accurate,the diagram in Fig. 5 was constructed, by useof data from Table 1 and drawings, traced fromphase-contrast photomicrographs, of nuclei atdifferent stages in mitosis. Since detailed studiesof karyokinesis in Physarum plasmodia havealready been published (6, 7), Fig. 5 is intendedonly to illustrate the following events. At ap-proximately 30 min before metaphase, the nu-cleus contracts, leaving a space between itselfand the nuclear membrane. (Stippled outlinesof nuclei in the drawings are intended to repre-sent the edge of the surrounding cytoplasm, andnot the nuclear membranes.) At the same time,
TABLE 1. Duration of mitotic stages in Physarumplasmodia"
Duration of each mitotic stageCulture _ _ _ _ Nuclearculur reconstruc-
a SiX petri dish cultures were started in twosets of three each, with 2 hr between inoculationof the two sets. Microscopic observations werebegun shortly before mitosis III. Smears weremade from one quadrant of each plasmodiumat 1- or 2-min intervals until 30 min after meta-phase, and then the sampling interval was in-creased to 10 or 15 min.
b Not determined.
the nucleolus moves toward the periphery of thenucleus, and during the period from 20 to 5 minbefore metaphase, it decreases in size and dis-appears, leaving the finely granular pattern ofprophase. During the next 5 min, the granularmaterial condenses to give rise to the mitoticspindle. Metaphase persists for about 7 minbefore the two sets of chromosomes separate inanaphase. After 3 min, the chromosomes havearrived at opposite mitotic poles. During thenext 5 min, the dense chromatin pattern of telo-phase becomes looser, as nuclear reconstructionbegins. After this point, visible changes are moregradual and less dramatic. Clumps of nucleolarmaterial appear at about 20 min after metaphase.These fuse to form a nucleolus that is at firstU-shaped, then bean-shaped (30 to 45 min aftermetaphase), and finally spherical (by 60 to 90min after metaphase).Table 2 shows synchrony of petri dish cultures
examined at three points. Metaphase times of theopposite edges of the six plasmodia differed bynot more than 10 min. This finding agreed withthe results of Guttes, Guttes, and Rusch (6)and represents very close synchrony when oneconsiders that observations were made at mitosisIII, after 21 hr of growth, and that the interphasetime is 8 hr. However, the inoculum centers werenot synchronous in most cultures, and "nests"of nuclei in all mitotic stages from late to earlyinterphase could be found in smears of five of thesix plasmodia examined. It would seem advisable,therefore, when precise experiments are to bedone, to discard the inoculum center when theculture is harvested.
Table 3 shows the degree of synchrony of thelarge plasmodia at both mitosis II and mitosisIII. For this experiment, six plasmodia were
started, and each was checked for mitosis at sixpoints-four on the periphery, one in the center,and one in the inoculum ring. When metaphase
TABLE 2. Synchrony of plasmodia grown in petri dishesa
Culture no.Location
1 2 3 4 5 6
Edge................... P(-5) T(+5) T(+5) R(+ 10) M MCenter.I T(+5) I-T I - R I - T LI - ElEdge (opposite).T(+5) M R(+ 10) T(+5) T(+5) MA
a Data were obtained from the same experiment described for Table 1. When mitosis was observed atone edge of the plasmodium, smears were also made of the opposite edge and of the center (inoculumspot). When metaphase was missed, the time of metaphase was estimated from other mitotic stages (seeFig. 5). The numbers in parentheses refer to minutes before and after metaphase. Abbreviations: P,prophase; M, metaphase; A, anaphase; T, telophase; R, reconstruction; I, interphase; E, early; L, late.Arrows indicate the presence of intermediate mitotic stages as described in the text.
FIG. 5. Mitotic stages in the nucleus of the Physarum plasmodium. Alcohol-fixed plasmodial smears were pho-tographed through a phase-contrast microscope. Representative nuclei on the photographs were traced to give thedrawings of the figure. All drawings are at the same magnification. Stippled outlines of nuclei represent surround-ing cytoplasm and not nuclear membranes. On the first line below each row of nuclei is the duration in minutes ofeach stage of mitosis, and on the second line is the number of minutes before the beginning or after the ending ofmetaphase. Data are from Table 1.
was missed, it was estimated from other mitoticstages with the aid of Fig. 5. Estimated minutesbefore or after metaphase are given in paren-theses after the observed mitotic stage. Samplingfor mitosis II was begun too late to allow deter-mination of the range in metaphase times in anyof the plasmodia, but the ranges seemed to beabout 10 min in culture 1 and 20 min in culture 3.Similar results have been obtained in other ex-periments. In mitosis III, which was more accu-rately timed, the range in metaphase times was15 min in culture 4, which had the best syn-chrony, and 40 min in culture 3, which had thepoorest synchrony. (In contrast to the resultsfor the petri dish plasmodia, asynchrony wasnot seen in the inoculum ring.) The six plasmo-dia appeared to go through mitoses II and IIIat times almost as close together as the times fordifferent regions within individual plasmodia.The 50 to 60 petri dish cultures that would beneeded to produce an equivalent amount ofplasmodial material could be expected to differby as much as 1.5 hr in the time of occurrence ofmitosis III.
DISCUSSIONThe main problem in growing a large Physarum
plasmodium seemed to be to prevent its becomingso thick that the growth rate would be decreasedbecause of the slow diffusion of oxygen and nu-trients into the organism. Applying the inoculumas an open ring gave a thinner plasmodium,and growing the plasmodium on the rocker im-
proved aeration by exposing both the upper andlower surfaces to air.Rocking probably also helped to preserve
synchrony by eliminating localized temperaturedifferences in the medium. Temperature controlis almost certainly essential for maintenance ofsynchrony, since even small temperature differ-ences caused marked differences in the time ofmitosis in petri dish cultures.The rocker culture system was developed
to reduce the time required for handling culturesduring experimentation. After the appropriatevolume of culture medium and types and dimen-sions of supporting screens and culture trays hadbeen determined, it was found that the rockermethod involved less time and labor, not only inthe growing of cultures, but also in the prepar-ing (washing, assembling, sterilizing) of appara-tus for use. The method was therefore preferableto the petri dish procedure whenever quantityof material and not precision of synchrony wasof prime importance, and, in some cases, alsopreferable to the microplasmodial system (4)when quantity of material was the only interest.The rocker system was designed to produce a
plasmodium yielding about 0.5 mg of histonejust prior to mitosis III. Since the range in meta-phase times within individual plasmodia at mito-sis III is 15 to 45 min, it may be advisable whenbetter synchrony is needed to start with a largeinoculum and to study mitosis II, where syn-chrony is similar to that of petri dish cultures atmitosis III.
a Six rocker cultures were grown as described under Materials and Methods. At the times indicated,smears were made at four points on the periphery (a-d, one in each quadrant), at one point in the inocu-lum ring (e), and in the center (f) of each plasmodium. Sampling was continued until each point hadgone through metaphase. When metaphase was missed, the number of minutes before or after metaphasewas estimated from other mitotic stages (see Fig. 5).bTime after addition of medium to coalesced cultures.¢ Mitotic stages, abbreviated as in the footnote to Table 2. Numbers in parentheses are estimated
minutes after metaphase.
ACKNOWLEDGMENTS LITERATURE CITEDThis investigation was supported by Public Health Service 1. Burton, K. 1956. A study of the conditions and mechanisms
grant CA-07175 from the National Cancer Institute and by of the diphenylamine reaction for the colorimetric estima-funds from the Alexander and Margaret Stewart Trust. tion of deoxyribonucleic acid. Biochem. J. 62:315-322.We thank Eugene Goodman for help with photomicrography 2. Ceriotti, G. 1955. Determination of nucleic acids in animnal
and Judith Blomqwst for technical assistance. tissues. J. Biol. Chem. 214:59-70.
3. Cummins, J. E., J. C. Blomquist, and H. P. Rusch. 1966.Anaphase delay after inhibition of protein synthesis be-tween late prophase and prometaphase. Science 154:1343-1344.
4. Daniel, J. W., and H. H. Baldwin. 1964. Methods of culturefor plasmodial myxomycetes, p. 9-41. In D. M. Prescott(ed.), Methods in cell physiology, vol. 1. Academic PressInc., New York.
5. Guttes, E., and S. Guttes. 1964. Mitotic synchrony in theplasmodia of Physarum polycephalum and mitotic syn-
chronization by coalescence of microplasmodia, p. 43-54.In D. M. Prescott (ed.), Methods in cell physiology, vol. 1.Academic Press Inc., New York.
6. Guttes, E., S. Guttes, and H. P. Rusch. 1961. Morphologicalobservations on growth and differentiation of Physarumpolycephalum grown in pure culture. Develop. Biol. 3:588-614.
7. Guttes, S., E. Guttes, and R. A. Elis. 1968. Electron micro-
scope study of mitosis in Physarum polycephalum. J.Ultrastruct. Res. 22:508-529.
8. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folin phenolreagent. J. Biol. Chem. 193:265-275.
9. Mittermayer, C., R. Braun, T. G. Chayka, and H. P. Rusch.1966. Polysome patterns and protein synthesis duringthe mitotic cycle of Physarum polycephalum. Nature210:1133-1137.
10. Mittermayer, C., R. Braun, and H. P. Rusch. 1965. The effectof actinomycin D on the timing of mitosis in Physarumpolycephalum. Exptl. Cell Res. 38:33-41.
11. Nygaard, 0. F., S. Guttes, and H. P. Rusch. 1960. Nucleicacid metabolism in a slime mold with synchronous mitosis.Biochim. Biophys. Acta 38:298-306.
12. Sachsenmaier, W., and H. P. Rusch. 1964. The effect of 5-fluoro-2'-deoxyuridine on synchronous mitosis in Phy-sarum polycephalum. Exptl. Cell Res. 36:124-133.
