JOURNAL OF BACTERIOLoGY, July 1968, p. 86-92Copyright 1968 American Society for Microbiology

Factors Affecting the Rate of Heat-induced SporeGermination in Dictyostelium discoideuml

DAVID A. COTTER2 AND KENNETH B. RAPERDepartments of Bacteriology and Botany, University of Wisconsin, Madison, Wisconsin 53706

Received for publication 18 April 1968

Washed spores of Dictyostelium discoideum, strains NC-4H, NC-4D, and V-12,germinated rapidly after being heat shocked at or near 45.0 C for 30 min. Culturesof the slime molds were grown in association with Escherichia coli B/r as the hostbacterium; spores taken from plates of synthetic medium had a higher final germina-tion value than spores from complex medium containing peptone and yeast extract.Young spores germinated more rapidly than older spores. Optimal germinationoccurred between pH 6.0 and 7.0, and, of the buffers tested, potassium phosphateallowed the most rapid germination. After heat shocking, spores were diluted intofresh oxygenated buffer to provide enough oxygen for completion of germination.Germination occurred most rapidly between incubation temperatures of 22 and 25 C.

Washed dormant spores of Dictyosteliumdiscoideum Raper can be induced to germinateby plating them on agar media containing pep-tones or amino acids (4; Cotter, M.S. Thesis,Univ. of Wisconsin, Madison, 1965; Cotter,Bacteriol. Proc., p. 17, 1966). Mild heat shockalso stimulates germination of D. discoideumspores (4; Cotter, M.S. Thesis).

This communication reports the results offurther investigations that define more preciselyoptimal conditions for obtaining relatively highgermination rates with mild heating.

MATERIALS AND METHODS

Production ofspores. Many students of the cellularslime molds have observed that media low in nutrients,although not optimal for the bacterial host, are re-quired by many slime molds for optimal growth andfruiting.The first medium used in this study was diluted

from its original concentration so that it containedthe following components: 0.2% peptone (Difco),0.2% glucose, 0.2% yeast extract (Difco), 2% agar(Difco), and 10 mm potassium phosphate buffer atpH 6.5. This agar medium was called 0.2% PGY byCotter and Raper (4).The exact reason(s) why rich media often repress

growth and normal fruiting of the slime molds is notknown. It cannot be assumed that the cellular slimemolds use only the bacterial cells and not some of thesoluble components of the medium as nutrients for

I Part of a thesis submitted by the senior author inpartial fulfillment of the requirements for the Ph.D.degree, University of Wisconsin, Madison.

2Present address: Pacific Biomedical ResearchCenter, University of Hawaii, Honolulu, Hawaii.

growth and development. However, it is known thatcertain products of bacterial growth, if in excess,render media unsuitable for slime mold growth. Thus,a second medium, originally devised by Adams (1) forbacteriophage study, was tried for simultaneousgrowth of Escherichia coli B/r and D. discoideum.This balanced medium, which will be termed syntheticmedium throughout the text, was used to achieve richbacterial growth without the production of a highbackground of inhibitory products or unusednutrients. The composition of the synthetic mediumfollows: NH4Cl, 1.0 g; MgSO4, 0.13 g; KH2PO4, 3.0 g;Na2HPO4, 6.0 g; glucose, 4.0 g; and distilled water,1,000 ml. Difco agar at 1.5% was added when a solidsubstrate was desired, in which case the glucose wasadded just before the plates were poured. The pH ofthe medium was 6.5 before and after growth. Themedium was used in liquid form for the growth of E.coli B/r alone; for the production of vegetative myxa-moebae the bacteria and slime mold were grown to-gether in shaken flasks.

Spores of D. discoideum used in the germinationstudies were obtained from stock cultures preparedas follows: 1.0-ml samples of a mixed suspension ofhost bacteria from liquid synthetic medium and 104nonshocked spores were spread onto plates of syn-thetic medium which were then incubated in the lightat 25 C. The large number of spores used as inoculumproduced an even growth of vegetative myxamoebae,and, subsequently, an even distribution of fruitingstructures occurred over the entire surface of the plate.Migration of the pseudoplasmodia was minimizedbecause of the relatively high ionic strength of themedium. Synchronous fruiting was necessary so thatall the newly produced spores would be of the sameage. The usual incubation time required for fruitingwas 3.5 days. The stock cultures were stored at 25 Cuntil the spores were to be harvested.

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Preparation of the spores for heat shocking. Thesorocarps were allowed to age for at least 1 day at25 C before the spores were harvested. All operationswere performed at room temperature because earlywork had shown that preparation of the spores at lowtemperature caused suboptimal germination rates.A similar slowdown in germination rate for peptone-activated spores has also been noted by other investi-gators (3).The spores were removed from the sori by holding

a glass slide endwise at the level of the sori and rotatingthe petri dish. During this operation, we proceededwith care to avoid picking up stalks. This procedurewas more difficult for the diploid variety of D. dis-coideum, NC-4D, than for the two haploid varieties(A. T. Weber, M.S. Thesis, Univ. of Wisconsin,Madison, 1967). The spores were washed from theslides into a beaker of distilled water; the resultingspore suspension was then passed through cheesecloth,which allowed passage of single spores but retainedmost of the spore clumps and any stalk materialpresent. It was then placed in a 15-ml centrifuge tubewhich was touched to a Vortex Junior mixer to breakup any remaining spore clumps and to wash the spores.This suspension was then centrifuged at 1,000 X g for5 min to sediment the spores and to remove any water-soluble inhibitor(s) (2, 3, 7). The supernatant fractionwas saved for studies of possible inhibitors, and thespores were suspended in 15 ml of 10 mm potassiumphosphate buffer, pH 6.5. The spores were thenwashed twice by centrifugation in the above buffer andfinally were suspended in 5 ml of the same buffer forheat shocking. The top and sides of the centrifuge tubeabove the liquid level were wiped with tissue to removeany spores that might not undergo heat shocking.The spore suspension was then shocked, generally at45.0 C for 30 min in a Precision Freas model 161 waterbath in which the temperature was controlled to±0.02 C.

Methods of incubation after heat shocking. Theshocked spores were then usually pipetted into 10 mmpotassium phosphate buffer, pH 6.5, contained inbeakers, in flasks, or in the 10 X 1 cm tubes used inthe Bausch & Lomb colorimeter (Spectronic-20).This dilution procedure cooled the spores and alsoresulted in a final concentration of from 106 to 107spores per ml, as measured by a Bright-line hemacy-tometer. Higher spore concentrations did not germi-nate rapidly because of oxygen depletion (Cotter,Ph.D. Thesis, Univ. of Wisconsin, Madison, 1967).The beakers, flasks, or tubes contained small magneticstirring bars which were spun just fast enough to pre-vent the spores from settling. The object of the stirringwas to provide some gaseous exchange into and outof the medium without causing destruction of thenewly emerged myxamoebae. This gentle stirring didnot prevent clumping of the myxamoebae whichemerged from the swollen spores.

Flasks on rotary shakers could also be used, butthese were not generally employed since the shakerhad to be stopped for frequent sampling.The spore suspensions were usually incubated at

temperatures between 22 and 25 C for periods up to5 hr.

In certain experiments, 0.5 ml of spore suspensionwas evenly plated onto buffered agar (10 mm potas-sium phosphate, pH 6.5; 2% Difco purified agar).Incubation of the plates was at 24 C.

Scoring germination. The various stages of germina-tion (4) were determined by removing 1 ml of the sporesuspension from the incubation vessel. The sample waspipetted into a small tube which was stirred with aVortex Junior mixer for 30 sec to break up clumps ofspores and myxamoebae; these clumps usually formedafter 3 hr of incubation when the temperature wasbetween 22 and 25 C. Then, 0.05 ml of this dispersedspore suspension was removed from the tube, placedon a glass slide, and examined under a glass coverslip. An AO Spencer microscope equipped with a43 X objective and 15 X eyepieces was used to countat least 200 objects. Counting took less than 3 min.The objects were divided into three groups: morpho-logically dormant spores, swollen spores, and emergedmyxamoebae (Fig. 1).

In the plate experiments, the germinating sporeswere examined on the buffered agar at a magnificationof 645 X and then were divided into only two groups,myxamoebae and spores.A spore was considered to have germinated com-

pletely (emergence) only when a viable myxamoebawas counted. Empty spore cases were not countedbecause they do not necessarily represent livingmyxamoebae. Myxamoeba emergence in these experi-ments was thus equivalent to outgrowth in a bacterialspore or to germ tube formation in a fungal spore.

RESULTS AND DIscussIoN

Morphogenetic capability of emergent myxa-moebae. The myxamoebae produced during thesegermination studies were viable and capable ofaggregating when they were placed on bufferedagar, despite their smaller dimensions and anabsence of prefeeding. The times required foraggregation were identical to those required forvegetative myxamoebae grown in liquid syntheticmedium with E. coli B/r. Aggregation centerswere formed about 2.5 hr after the myxamoebaewere deposited onto buffered agar, and sorocarpsof normal pattern were formed within 18 hr. Amajority of the spores from such sorocarps wereable to germinate after heat shocking.

Type of buffering compound and its effect upongermination. Spore germination in D. discoideumdepends, to some extent, upon the solution inwhich the spores are suspended during and afterheat shocking (Table 1). Of course, germinationdoes not occur at all under these conditions unlessthe spores are shocked (4). Oxygenated distilledwater permitted high germination but allowedclumping of the spores. This nonspecific clump-ing, i.e., in the absence of myxamoebae, made itdifficult to determine the amount of germination,since agitation of the culture with a Vortex Jr.mixer failed to separate the clumps into singlespores.

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COTrER AND RAPER

___in ' m m n__ _~~~~~~~~~~~~~~~~~~~~~~~M.._ .............FIG. 1. Sequence of changes observed during spore germination in Dictyostelium discoideum, strain NC-4H.

(A) Dormant spore, 8 ,u long; (B-E) early, intermediate, and late swelling; (F) emergence of the myxamoeba;(G-H) postemergence. In (H) the myxamoeba has movedawayfrom the empty spore case. X 1,000. Phase micros-copy.

When less than 10 mm potassium phosphatebuffer was used, clumping again occurred, some-times as soon as incubation began. A buffersolution containing 10 mm potassium phosphate,pH 6.5, produced high germination without non-specific spore clumping. Higher concentrationsof phosphate (50 mM) seemed to inhibit thegermination process (Cotter, M.S. Thesis,Univ. of Wisconsin, Madison, 1965).

The use of tris(hydroxymethyl)aminomethane(Tris) adjusted to pH 7.0 with HCI did not allowas high germination as phosphate buffer. Inaddition, nonspecific clumping occurred in theTris-chloride buffer.

Potassium citrate (pH 6.5) and sodium pyro-phosphate (pH 7.0) did not allow emergence ofmyxamoebae from swollen spores until after 5 hrof incubation. The slow release of myxamoebae

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TABLE 1. Spore germination in liquid culture as afunction of the suspending mediuma

Percentage Percentage ofSuspending medium of swollen emerged

spores myxamoebae

Distilled water. 8.0 90.0Potassium phosphate, 5 mM. 10.0 88.0Potassium phosphate, 10 mm 8.0 90.0Tris-chloride, 10 mM....... 40.0 58.0Potassium citrate,10 mM.... 96.0 0.0Sodium pyrophosphate, 10mM.................... 80.0 0.0

a The spores were placed in the above mediaand shocked at 45.0 C for 30 min. Subsequentincubation was at 25 C, and percentage of germi-nation was determined 5 hr after dilution.

100

80..a60..1

spores produced on 0.2% PGY agar and com-pares them to those spores produced on syntheticmedium. The final amount of spore swelling, aswell as the final amount of myxamoebae emer-gence, is greater for spores produced on syntheticmedium. Cycloheximide was used to allow thenormal swelling rate while preventing myxa-moeba emergence (4).Beyond this point, most experiments were

conducted with spores produced on syntheticmedium that had not aged for more than 3 days.Accumulated evidence had shown that theseconditions of age and substrate provided sporesthat were optimal for rapid germination.For the production of myxamoebae, liquid

synthetic medium was superior to 0.2% PGYmedium. With the latter medium, considerablenumbers of the host bacteria still remained, as arule, along with the vegetative myxamoebae atthe end of their growth period. However, withsynthetic medium, very few bacteria were ob-served in the culture at the stationary phase ofmyxamoeba growth. No other liquid nutrientmedium investigated in this laboratory producedsimilar results.For studies of cell aggregation, also, the

presence of few bacteria after growth of the

0 1 2 3 4 5 6HOURS

FIG. 2. Effect of spore age and the composition ofthe growth medium on the rate (%) of emergence ofmyxamoebae from swollen spores. Symbols: 0, 1-day-old spores produced on synthetic medium agar; *,4-day-old spores produced on synthetic medium agar;El, 1-day-old spores produced on 0.2% PGY agar; and*, 4-day-old spores produced on 0.2% PGY agar.Incubation temperature, 25 C.

was not a result of pH change, although thesecompounds had little buffering capacity in thepH range used.

Effect of spore age and growth medium on therate of spore germination. A dramatic effect wasproduced when the spores for germination testswere obtained from two contrasting growthmedia. Young spores (1 day old) from the twodifferent media had similar germination ratesafter they were heat shocked at 45 C for 30 min.However, 4-day-old spores showed a differencein the rate of germination, depending upon themedium on which they were produced (Fig. 2).Figure 3 presents a summary ofexperiments with

HOURSFIG. 3. Germination of 1- to 3-day-old spores from

synthetic medium agar compared to spores from 0.2%PGY agar. The incubation temperature after heatshocking was 25 C. The mean and standard deviationabove and below this mean for 10 experiments areplotted. Symbols: spores from synthetic medium arerepresented by circles, while spores from 0.2% PGYmedium are represented by squares. Without cyclohexi-mide; open symbols, percentage of the spore populationswhich is swollen; closed symbols, percentage of thespore populations present as myxamoebae. With 100jig of cycloheximide per ml: half-closed symbols, per-centage ofthe sporepopulations which is swollen.

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COTITER AND RAPER

myxamoebae is a distinct advantage, since onlyone low-speed washing is needed to separate mostof the residual bacteria from the myxamoebae.

Influence ofpH on spore germination. Severalexperiments demonstrated that the rate, as wellas the final amount of germination, was the samewhen dormant spores were heat shocked andincubated atpH 6.0, 6.5, and 7.0. WhenpH below6.0 or above 7.0 was used for heat shocking andincUibation, the rate and final amount of germina-.tionia Were reduced. Thus, the pH range, which

optitnal for spore germination, was similart_o that for growth (6).

Activation occurred at a low pH, although,spore swelling and myxamoeba emergence were"slowed down. The results of an experiment inwhich spores were heat shocked at pH 4.0 arepresented in Fig. 4. Incubation was at 25 C. Theinitial reaction was pH 4.0, and at various timeintervals the pH was adjusted to 6.5 with KOH.Although a small amount of swelling occurred atpH 4.0, rapid swelling occurred only after pHadjustment to 6.5.

Optimal temperature for heat shocking. It hasbeen shown that temperatures of 40.0 and 50.0 Cfor 15 min did not result in germination whenplated spores were examined after 7 hr of incuba-tion at 25 C (4). A recheck of heat shocking at50.0 C for 30 min showed that spores not onlywere activated but also were damaged at thistemperature. Swelling of the spores and someemergence of myxamoebae from the swollen

100

80

60

0 I

HOURSFiJ. 4. Heat shock activation of spores in 10 mm

potassium phosphate buffer, pH 4.0. The abscissa refersto the time after shocking. Symbols: 0, percentage ofswollen spores plus free myxamoebae when thepH wasadjusted to 6.5 directly after heat shocking; 0, per-centage of swollen spores when the pH was adjustedto 6.5 after 3 hr ofincubation atpH 4.0; A, percentageofswollen spores when thepH was maintained at 4.0.

TABLE 2. Comparison of the effects of differentheat shock temperatures upon spore germination

in Dictyostelium discoideuma

Time after Heat-shock No. of Mean percentageplating temp determinations of emergenceofmyxamoebae

hr C

10 25b 1 0.010 45.0 2 99.010 50.0 3 0.010 55.0 3 0.024 25b 1 0.524 45.0 2 99.024 50.0 3 17.024 55.0 3 0.0

a The spores were produced on synthetic me-dium and allowed to age for 2 days before har-vesting. After heat shocking at the indicated tem-peratures for 30 min, the spores were plated onbuffer agar and incubated at 24.0 C.

b Control.

spores occurred, but this action took place muchlater than usual (Table 2). A temperature of 55.0C for 30 min caused death of the spores, sincethey never germinated after plating and incuba-tion at 25 C.

Experiments with temperatures closer to 45.0 Cshowed that the optimal temperature range forheat shocking was very narrow. Some quantitativedata that illustrate this point, i.e., for shockingtemperatures very near 45.0 C, are given in Table3. On the average, temperatures slightly below45.0 C resulted in rapid spore swelling and rapidemergence of myxamoebae from the swollenspores, but not all the spores in the populationwere activated; thus, the final percentage ofgermination was reduced. The remaining sporesdid not swell. Temperatures slightly above 45.0 Ccaused a lower rate of swelling of the spores, andemergence of the myxamoebae was slower also.However, high levels of germination were ob-tained with extended incubation (24 hr), and thesmall number of ungerminated spores had swol-len. The data also show that emergence is usually2 to 3% higher on plates when compared to ratestudies such as those conducted in liquid (Fig. 6).

Optimal temperature for incubation after shock-ing. In these experiments, the heat-shockingtemperature was again 45.0 C for 30 min; but theincubation temperature was varied, and thepercentage of germination was compared to thatobtained at 25.0 C. The controls for these experi-ments are shown in Fig. 3 as the curve forgermination of spores from synthetic medium at25.0 C. Thus, an incubation temperature of 25.0C resulted in a population containing 90%

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VOL. 96, 1968 HEAT-INDUCED SPORE GERMINATION IN D. DISCOIDEUM

myxamoebae 5 hr after heat shocking. The effectof incubation at 22.0 C was compared with thatat 28.0 C (Fig. 5). The data suggest that highincubation temperatures favor rapid swelling ofthe heat-activated spores, but these temperaturescause a decrease in the rate of myxamoebae

TABLE 3. Comparative effects upon germination ofheat shocking spores ofDictyostelium discoideum

at temperatures near 45.0 Ca

Time afterplating

hr

4444

55

5

777

2424242424

Spores heatshocked at

C

44.545.045.546.0

44.044.545.044.044.545.0

44.044.545.045.546.0

No. ofdetermi-nations

333

1

1

1

1

222

33322

Age ofspores

days

2222

1

1

1

1

1

1

22

2

2

2

Mean percentageof emergence

of myxamoebae

88.875.258.821.0

91.094.099.090.794.098.8

94.399.199.599.898.0

a The spores were shocked for 30 min at theindicated temperatures, plated on buffer agar,and incubated at 24 C.

emergence from these swollen spores. At 22.0 C,after 5 hr of incubation, 95.5% of the spores hadgerminated to release myxamoebae.A compromise incubation temperature of 23.5

C produced rapid and synchronous germinationafter heat shocking at 45.0 C for 30 min (Fig. 6).The kinetics of spore swelling resembled those atan incubation temperature of 25.0 C. The kineticsof myxamoeba emergence, however, more closely

100

s0

60.

100

40

20 l

0 _1

0 1 2 3 4 5 6HOURS

FIG. 6. Spore germination as a function of the incu-bation temperature, 23.5 C. The mean and one standarddeviation above and below the mean for six experimentsare presented. Symbols: 0, percentage of the sporepopulation which is swollen; 0, percentage of the popu-lation present as myxamoebae.

0

0 I 2 3 4 5 6HOURS

FIG. 5. Spore germination as a function of the incu-bation temperature after heat shocking. Symbols: 0,percentage of swollen spores at 22.0 C; 0, percentageof myxamoebae at 22.0 C; l, percentage of swollenspores at 28.0 C; *, percentage of myxamoebae at28.0 C. For comparison of spore germination at 25 Csee Fig. 3.

TABLE 4. Presence of bacteria during the germina-tion of heat-shocked spores of Dictyostelium

discoideuma

Percentage Bacteria/mlTime after of Bacteria/ml consumed by theshocking myxamoe- myxamoebae

bae present (approximate)

hr

0 0.0 2.4X 102 01 0.0 2.6 X102 02 0.0 2.3 X 102 03 37.5 2.3 X 102 04 75.0 1.6 X 102 1.0 X 1025 89.0 0.8 X 102 1.8 X 102

a Two-day-old spores were heat shocked at45.0 C for 30 min and then diluted with bufferto a final spore concentration of 3.9 X 106/mit;20 ml of this suspension was incubated at 25 n C .Viable bacteria were determined by plating I mlof suspension in nutrient agar and incubatingat 35 C; this procedure would rapidly kill themyxamoebae. The triplicate plates were read after2-day incubations, and the results were averaged.

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resembled those obtained at 220 C.- After -5 -hrof incubation at 23.5 C, 96.5% of the heat-acti-vated spores had germinated to myxamoebae.

Germination in various strains ofD. discoideum.Every strain of D. discoideum tested responded tothe heat shocking process. Strains NC-4H,NC-4D, V-12, and AC-4 from Raper's collectiongerminated rapidly after heat shocking. Strain B(from Maurice Sussman) and ACR-12 (also fromRaper's collection) responded to the heat shock-ing, but these strains germinated more slowly thanthe preceding four strains. This topic will becovered more fully in a later paper in this series.

Bacterial contamination during spore germina-tion. Bacterial contamination should be minimalfor physiological studies during spore germina-tion. This was not a problem in the spore sus-pensions employed in this study (Table 4). Onlyone bacterium was present for every 1.6 X 104spores at time-zero. After 5 hr of incubation,about 180 bacteria could have been engulfed bythe myxamoebae that emerged from the 89% ofthe spore population which germinated in thisexperiment. Assuming a Poisson distribution,each myxamoeba that ingested any bacteriaengulfed only one. In this case, then, there were1.9 X 104 myxamoebae which did not ingest anybacteria to every myxamoeba which did consumeone bacterium. From the above data, we wouldhardly expect that the consumed bacteria wouldchange the enzymatic pattern of the entiremyxamoebae population enough to be detectable.Emergent myxamoebae from washed spores

should constitute ideal material for investigatingmacromolecular syntheses. For example, the

problemOf cot inating bacterial ribonucleicacid (RNA) which can mask slime mold RNA,as reported by Inselburg and Sussman (5) formyxamoebae produced by vegetative growth,would not arise.

AcKNowLEDGMENTsThis investigation was supported by Public Health

Service Training Grant 5-TO1-GM-00686-07 from theNational Institute of General Medical Sciences andby research grant G-24953 from the National ScienceFoundation.

Dictyostelium discoideum strain B was obtainedfrom Maurice Sussman of Brandeis University,Waltham, Mass.

LiTERATURE CITED1. Adams, M. H. 1959. Bacteriophages. Interscience,

New York.2. Ceccarini, C., and A. Cohen. 1967. Germination

inhibitor from the cellular slime mould Dicty-ostelium discoideum. Nature 214:1345-1346.

3. Cohen, A., and C. Ceccarini. 1967. Inhibition ofspore germination in the cellular slime molds.Ann. Botany 31:479-487.

4. Cotter, D. A., and K. B. Raper. 1966. Spore ger-mination in Dictyostelium discoideum. Proc.Natl. Acad. Sci. U.S. 56:880-887.

5. Inselburg, J., and M. Sussman. 1967. Incorporationof 'H-uridine into RNA during cellular slimemould development. J. Gen. Microbiol.46:59-64.

6. Raper, K. B. 1939. Influence of culture conditionsupon the growth and development of Dicty-ostelium discoideum. J. Agr. Res. 58:157-198.

7. Snyder, H. M., III, and C. Ceccarini. 1966. Inter-specific spore inhibition in the cellular slimemoulds. Nature 209:1152.

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