NUTRITION OF CELLULAR SLIME MOLDS

II. GROWTH OF POLYSPHONDYLIUM PALLIDUM IN AXENIC CULTURE

HANS-RUDOLF HOHL AND KENNETH B. RAPER

Departments of Bacteriology and Botany, University of Wisconsin, Madison, Wisconsin

Received for publication 15 August 1962

ABSTRACT

HOHL, HANS-RUDOLF (University of Wisconsin,Madison) AND KENNETH B. RAPER. Nutritionof cellular slime molds. II. Growth of Polysphon-dylium pallidum in axenic culture. J. Bacteriol.85:199-206. 1963.-Several strains of Polysphon-dylium pallidum were grown on a liquid solublemedium in axenic culture. The medium containedembryo extract, serum albumin, Tryptose, dex-trose, vitamins, and salts. The final cell yieldwas about 6-11 X 101 cells/ml, depending on thestrain. The generation time was usually about 5to 6 hr. The myxamoebae were grown for over125 generations on this soluble complex mediumwithout decrease in growth vigor or loss of theircapacity to form normal fructifications whenremoved to an appropriate surface (e.g., agar).Thus the whole life cycle of this species wascompleted in the absence of any bacteria orbacterial products. Other species of the Dictyo-steleaceae grew less well or failed to grow in theliquid medium described.

Many reports on the cellular slime molds,published within the past quarter-century, havedealt primarily with the latter phases of theirlife cycle, i.e., cell aggregation and fructification.Only a few studies have given special attention tothe growth and multiplication of the myxamoebae(Raper, 1937, 1939; Sussman and Bradley, 1954;Gerisch, 1959, 1960), and these have centeredalmost exclusively on Dictyostelium discoideumas a test organism. No method has been developedfor growing the cellular slime molds in the ab-sence of bacteria or bacterial products. It is agoal much to be desired.

Exploratory investigations in our laboratory,directed toward this objective, have included anumber of cellular slime molds, some of whichseemed to offer special promise for obtainingaxenic cultures. In the preceding paper (Hohland Raper, 1963) we described techniques for

growing these microorganisms on living and deadbacteria in liquid culture; in developing thesetechniques we explored various parameters thataffect the growth of the myxamoebae, includingsuch environmental factors as pH, temperature,osmotic pressure, and the presence of toxic sub-stances.

This report describes, for the first time, thesuccessful cultivation of one of the cellular slinmemolds, Polysphondylium pallidum, in a solublemedium free of any bacteria or bacterial products.Many nutrient combinations were investigatedin our efforts to develop such a medium, and thepresent paper records our progress toward thisgoal. To date, primary emphasis has been directedtoward achieving maximal vegetative growth ofthe myxamoebae. Studies on the morphologyand subsequent morphogenetic movements lead-ing to fructification by such axenically grownmyxamoebae are in progress and will be reportedlater.

MATERIALS AND METHODS

P. pallidum WS-320 was employed generallyas the test organism, and the results reportedherein relate to this culture unless otherwiseindicated. The following procedures were employedfor preparing the inoculum. Spores of the slimemold grown on agar plates with Escherichia coli(Raper, 1937, 1951) were transferred to tubescontaining 5 ml of an autoclaved suspension ofbacteria (E. coli strain B/r, 1010 cells/ml in0.016 M Sorensen's phosphate buffer at pH 6.0).The tube cultures were incubated at 25 C on arotary shaker at 250 rev/min. After 1 day, theywere checked by transferring samples to tubescontaining peptone-yeast extract nutrient broth;contaminated cultures were discarded. After 2.5days, the myxamoebae had usually consumed thebacteria, the suspension had cleared considerably,and a slightly yellow color had developed. Themyxamoebae were then centrifuged, washed inbuffer solution, counted, and properly diluted,

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usually to 5 X 105 cells/ml; 0.1 ml of this suspen-sion was inoculated into tubes containing 5 mlof the medium to be tested, thus giving a con-centrationof 104 cells/ml of medium. Stirred flasks(Hohl and Raper, 1963) were substituted for therotated tubes if larger quantities of myxamoebaewere required.The growth of the myxamoebae was measured

by duplicate hemacytometer cell counts. Turbid-ity measurements of the suspension proved to beinadequate for growth determinations, especiallyin the lower concentrations.

Protein determinations were made by the Folinmethod. The myxamoebae were suspended in 4%trichloroacetic acid in 50% acetone, and theprecipitated protein was dissolved in 0.4 N sodiumhydroxide prior to the colorimetric measurements.Crystalline bovine serum albumin was used as astandard.

Hydroge-n-ion concentrations were measuredwith a Beckman model G pH meter at the begin-ning and at the end of the experiments, and withpHydrion papers during the periods of cellgrowth. All experiments were run in triplicate.The medium finally adopted contained the

following components: 2% bovine embryo ex-

tract, 4% bovine serum albumin, Eagle's vitaminmixture (Eagle, 1955) in double strength, 1%Tryptose, 1 % dextrose, and a modified Hank'ssalt solution (Paul, 1960). Before being incor-porated into the medium, each of the componentswas tested separately for toxicity by adding it tocultures of myxamoebae growing in a suspensionof dead bacteria in an appropriate buffer solution(Hohl and Raper, 1963). The detailed composi-tion of the medium is given in Table 1.The following stock solutions (sterile) were

kept in the refrigerator prior to use: Difco TCBovine embryo extract, 100%; Difco TC Bovineserum albumin, 5%; Difco TC Vitamins Eagle,100 X; modified Hank's salt solution, 10 X. Thedesiccated embryo extract was reconstituted byadding salt solution and was used within 10 days.To make 10 ml of medium, 100 mg of Tryptose

and 100 mg of dextrose were dissolved in 1 ml ofsalt solution (10 X) and 0.6 ml of distilled water.After autoclaving for 20 min at 121 C, 0.2 ml ofembryo extract (100%), 8 ml of serum albumin(5%), and 0.2 ml of Eagle's vitamin solution(100 X) were added aseptically prior to inocula-tion. The tubes were incubated at 25 C on a rotaryshaker at 250 rev/min.

TABLE 1. Composition of complex medium developedfor growing myxamoebae of Polysphondylium

pallidum in axenic culture

Constituent Amount

mg/100 ml

Bovine serum albumin ........... 4,000.0Bovine embryo extract (100%) ... 2.0 mlTryptose ........................ 1,000.0Dextrose*....... 1,000.0D-Biotin ....... 0.048Folic acid ....................... 0.088Niacinamide ..................... 0.024Ca-pantothenate ................. 0.048Pyridoxal-HCl (USP) ............ 0.040Thiamine-HCl ................... 0.068Riboflavine ...................... 0.008Choline chloride ................. 0.028NaCl .......................... 80.0KCl .......................... 40.0CaCl2 *2H20...................... 10.0MgSO4*7H20 .................... 10.0MgCl2*6H20 ..................... 10.0Na2HPO4*7H20 .................. 6.0KH2PO4 ......................... 6.0

* Whereas dextrose showed no stimulation ofgrowth in media yielding low cell populations, itwas recently observed that it has a slight butdefinite activity in combination with the com-plex medium finally adopted. It is now routinelyused, but all experiments reported in this paperrefer to the medium without dextrose.

If the medium was to be solidified, agar wasadded prior to autoclaving to give a final con-centration of 1%. The other components wereadded when the autoclaved solution was stillwarm but not hot enough to precipitate thealbumin. The mixture was poured into petridishes as a thin (2 mm) layer. Usually, 2% ratherthan 4% serum albumin was used for the solidi-fied medium.

RESULTS

Growth on complex medium and influence ofdifferent components. Typical growth curves forP. pallidum, strains WS-320 and Pan-17, on thesoluble complex medium are presented in Fig. 1.The final concentration of myxamoebae forstrain Pan-17 almost equaled the concentrationobtained with the controls (WS-320) grown onliving bacteria, whereas the final concentrationof amoebae in strain WS-320 was about one-halfto one-third that of the controls. The end con-centration of myxamoebae obtained varied con-

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Gw6 /0

E

E

M5.

0

8 16 24 32 40 48 56 64Incubation time (hrs.)

FIG. 1. Typical growth curves of two strains ofPolysphondylium pallidum on the soluble complexmedium (curve B: WS-320; curve C: Pan-17). Forcomparison, the growth of strain WS-320 on livingbacteria is also included (curve A).

siderably. For strain WS-320, this ranged between4 and 8 X 106 cells/ml; for strain Pan-17, between8 and 14 X 106 cells/ml. The comparison isbased on the number of cells produced. We shallsubsequently see that cells grown on the complexmedium are, on the average, larger than myxa-

moebae grown on bacteria. Growth of the myxa-

moebae, measured as increase in protein, thereforeusually equals or even exceeds the amount foundin bacteria-grown cultures. The pH rises from 6.0to about 7.2 during the 3-day growth period.

There was a difference in the rate of growthbetween cultures grown on bacteria and in thecomplex medium. During the phase of logarithmicgrowth, the myxamoebae of P. pallidum WS-320had a generation time of 2.5 hr when grown on

living bacteria, 3.5 hr on dead bacteria (Hohland Raper, 1963), and 5 to 6 hr in the complexmedium. Figure 1 also demonstrates the com-

paratively long period of transition from the endof the log phase to the termination of growth.When we consider the influence of each of the

main components of the medium, it becomesapparent (Table 2) that no component alone is anabsolute requirement (except for serial transfers),but that a combination of all of them gives thehighest cell yield. The most important factor isthe embryo extract, since it not only allows some

growth in the absence of the other components,but it also limits the growth to a large extent whenomitted from the whole medium. The embryoextract seems to act as a growth factor (Fig. 5)or as a macronutrient. This latter possibilityappears reasonable from the fact that embryo

TABLE 2. Influence of different components ongrowth of Polysphondylium pallidum WS-320

St Tryp- Vita- Serum Embryo No. of Finalltose mins albumin extract genera- concntions (cells/mi)

+ + 6.3 7.8 X 105+ + + + 7.0 1.2 X 106+ + + + + 9.4 6.6 X 106+ + + + 8.9 4.5 X 106+ + + 8.4 3.3 X 106+ + + + 5.4 4.3 X 105

Concentrotion Embryo Extroct (%)

FIG. 2. Influence of embryo extract on the growthof Polysphondylium pallidum WS-320 on a mediumwith 4% serum albumin (curve A) and on a mediumwith 2% serum albumin (curve B), measured after3 days of growth.

extract contains about 0.6 mg of protein per ml(in the 2% solution used), compared with about1 to 2 mg of protein in the myxamoebae that are

able to grow in 1 ml of medium. It can be furtherdemonstrated by experiments (Fig. 2) in whichthe concentration of the embryo extract is varied.When the normal medium with 4% serum

albumin is used, the yield increases rapidly at lowconcentrations and then tends to level off. Theembryo extract acts here mainly as a growthfactor. If the amount of serum albumin is reduced,or if it is omitted completely, the cell yield in-

creases proportionally to the amount of addedembryo extract, which demonstrates that it actsas a macronutrient in this latter case.

In the early experiments, when it was stilluncertain whether the myxamoebae would growin the absence of particulate food, a suspension of

autoclaved lactalbumin (Difco, 3 to 10%) was

used. Later, when it was shown that this nutrient

supported equally good growth after passagethrough a Seitz filter, lactalbumin was replaced

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Percent Serum Albumin

FIG. 3. Influence of different concentrations ofserum albumin from two different sources on thegrowth of two strains of Polysphondylium pallidum:strain Pan-17 on serum albumin from Calbiochem(curve A) and from Difco (curve C), and strainWS-320 on serum albumin from Difco (curve B)and from Calbiochem (curve D).

by the more desirable soluble product, bovineserum albumin. In other experiments, bovine,rabbit, and chicken sera, instead of serum al-bumin, were included in the medium at concen-

trations between 5 and 20%. In the absence ofembryo extract, rabbit serum allowed an increaseof the myxamoebae up to about 1.5 X 106 cells/ml, whereas with bovine serum the cell concen-

tration reached only 1.1 X 105 cells/ml. In thepresence of embryo extract, concentrations of3.5 X 101 cells/ml were obtained with eitherserum. Comparative tests of bovine serum al-bumin and bovine serum ultrafiltrate (Difco)showed that the activity was entirely in thealbumin fraction. Consequently, this was sub-stituted for serum, and at proper concentrations(4%) gave yields twice as high as 20% serum.

Several factors oppose the simple assumptionthat the serum albumin serves as a main energy

source. To obtain optimal results, 4% albuminmust be added to the medium (Fig. 3), i.e., 40mg of protein/ml of medium compared with only1 to 2 mg of protein/ml contained in themyxamoebae grown therein. Two of many pos-

sible explanations are (i) that the favorableaction of serum albumin results from its physicalproperties (adsorption of toxic metabolites,changes in the viscosity of the medium) or (ii)that possibly only one specific part of the wholemolecule is used (e.g., a single amino acid or a

peptide). However, these two possibilities do notexplain the fact that bovine serum albuminsfrom different sources have different activities.Whereas bovine serum albumin from Difco

Laboratories possessed excellent growth activityfor all strains of P. pallidum tested (e.g., WS-320and Pan-17 in Fig. 3), a bovine serum albuminfrom Calbiochem (25% in Tyrode's solution) hadalmost no activity for any strain except Pan-17,in which organism growth was considerably im-proved (Fig. 3, curve A). At the moment it seemsthat impurities may be partly responsible forthe growth response, although it must be addedthat a twice-crystallized preparation of plasmaalbumin (Calbiochem) supported growth ofstrain WS-320 up to 3.5 X 106 cells/ml.Serum and serum albumin tend to decrease the

rate of growth, compared with controls withoutthese substances. In other words, addition ofserum or serum albumin increases the final yieldsubstantially, but at the same time also increasesthe generation time of the myxamoebae (fromabout 4 hr to 5 to 6 hr with serum albumin).The serum albumin is also responsible for theincrease in the average size of the myxamoebae,as revealed by comparative measurements ofcells in the presence and absence of this com-ponent. The same effect is exerted by wholeserum, as one might anticipate.Many compounds and combinations of media.

have been tested for improvement of growth.Among them, a mixture of vitamins (Difco-Eagle) and 1% Tryptose were chosen and in-cluded in the final medium, since they had someadditional positive effect (Table 2). Media usedfor animal tissue cultures, such as Eagle's.Medium L, Medium NCTC 107, and Medium199 (all from Difco Laboratories), had to bediluted too much (because of their high sodiumchloride concentration) to give appreciable cellyields, even in the presence of lactalbumin orserum. Other components tested included yeast-extract, peptone, casein hydrolysate, malt ex-tract, beef extract, Casamino Acids, neopeptone,and trace elements. No combinations better thanthat listed in Table 1 were found.

Size of inoculum. The influence of the size ofthe inoculum on the growth of the myxamoebaeis demonstrated in Fig. 4. There seem to be twomajor influences. First, it may be seen that re-duction of the number of cells in the inoculumresults in an improved generation time; e.g., withan inoculum of 103 cells/ml the generation timewas about 3.5 hr, as compared with about 10 hrwith 106 cells/ml. Second, the final concentrationof myxamoebae attains a substantially higherlevel where the heavier inoculum is used: about

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7

12 24 36 48

Incubation time (hrs.)FIG. 4. Influence of size of inoculum o0

of Polysphondylium pallidum WS-320 icomplex medium.

107 cells/ml, in contrast with 2.5 X 1where the starting population was 1CThese differences in growth rate haviserved in media with serum alllactalbumin. In media containing 2(serum, the situation is different. Thererate is the same with all amounts oftested, and the generation time for all7.5 hr.

Continuous subculture. To test thesoluble complex media to supportgrowth of the slime mold, the myxamobeing washed, were serially transferremedium. Figure 5 shows the result obt

a medium containing lactalbumin,vitamins, and amino acids. This mediilated with 104 cells, supported growtl1.5 X 106 cells/ml in the first cultu:without added embryo extract, butsence of embryo extract it lost thirapidly and the myxamoebae failed toabout 20 generations. If embryo e)present, the growth rate even improithe experiment, as indicated by a decgeneration time from 7.5 to ca. 5.0 hsequence of seven transfers. The me

serum albumin, as finally adopted, shovcharacteristics. The medium with emb:has now supported growth for over 1tions without decrease in growth vigorcapacity. Without the embryo extra

continued through only two subculttotal of 14 generations and then ceasectherefore, that for prolonged cultival

____ myxamoebae in the absence of bacteria or bac-O.0- terial products a growth factor is necessary, which

may be supplied by adding embryo extract.Serial transfers may also be made in petri dishes

on a solidified medium (see below) by transferringspores to fresh plates, that is, by following thesame procedure usually employed when the myx-amoebae are grown together with bacteria.

Growth and fructification on solidified medium.Three methods of obtaining fruiting structuresfrom myxamoebae grown on the complex mediumwere employed. In the first method, myxamoebae

. were grown in liquid medium, then washed and60 72 transferred to plates containing water-agar

s the growth (1.5%) or thin hay agar (Raper, 1937). Care hasyn a soluble to be taken to avoid too long a cultivation (more

than 4 to 5 days) of the myxamoebae in the liq-uid medium, since they tend increasingly to round

06 cells/ml up and become incapable of fruiting. The plated) cells/ml. cells start to aggregate after 15 to 20 hr and fruite been ob- normally. It may be mentioned that the fruitingbumin or structures of strain WS-320 are symmetrical)% bovine (Hohl and Raper, 1963, Fig. 3), with regularlythe growth spaced whorls of side branches, whereas those off inoculum strain Pan-17 are often irregular in pattern, withcultures is twisted sorophores. These differences, however,

represent strain characteristics, for the most

ability of part, since they recur independently of the typeindefinite of culture employed.

)ebae, after In the second method, the myxamoebae ared to fresh transferred to roller tubes, as described byained with Gerisch (1960). Within a few minutes, the cells

Tryptose, clump together and form very small agglutinates,um, inocu- which constantly increase in size during the fol-h to about lowing hours. If, after 8 to 16 hr, these agglu-re with or tinates are put on an agar surface, they initiate

in the ab-s capacity 12

grow after A o

Ktract was '°0ved during E

line in the 88 or during a ',dium with A 6wed similar 0

ryo extract 425 genera-

or fruiting,ct, growthiures for a

i. It seems,

tion of the

2 3 4 5 6 7Number of Times Transferred

FIG. 5. Continuous subculture of Polysphondy-lium pallidum WS-320 in the presence (B) and

absence (A) of embryo extract.

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fructification within a few hours. Dependingupon the size of the agglutinate, one or severalsorocarps may develop. No detailed investiga-tions have yet been made to determine the exacttime relationships between fructification by nor-mal aggregation and fructification by inducedagglutination under controlled conditions.

In a third method, the myxamoebae are cul-tivated directly on the solidified medium. Theamount of growth desired can be regulated by theamount of medium supplied. If too much nutrientis available, the myxamoebae cannot exhaust it,waste products presumably reach a concentrationinhibitory to cell aggregation, and only micro-cysts are formed. Therefore, the plates should bepoured thinly to a depth of about 2 mm. Withan inoculum of five cells/mm2, sparse fructifica-tion is obtained on the medium after about 2days, even in the complete absence of serumalbumin. With 2% serum albumin, the numberof fruiting structures is much increased, but theirformation is somewhat retarded and starts afterabout 3 days. This is partly due to the longergrowth period, but perhaps also to the slightdecrease in growth rate in the presence of serumalbumin, as shown in the liquid cultures. Theagar itself may have some stimulatory effect uponthe growth rate. If a small amount (0.1 %) ofagar is added to the liquid medium and the cul-tures are grown on the rotary shaker, the slopeof the growth curve is steeper than that of thecontrol without added agar. However, the finalconcentration of myxamoebae remains un-changed, showing that the agar affects only therate of cell growth. The myxamoebae grown onthe surface of the agar-solidified complex mediumare more uniform in size and smaller than themyxamoebae grown in liquid culture, where theyreach unusually large dimensions.

Size of the myxamoebae. During the course ofthese studies, it became clear that the averagesize and the behavior of the myxamoebae mayvary depending on the kind of medium used. Tomeasure the relative size of the cells, they weresuspended in a saturated solution of ethylene-diaminetetraacetic acid (EDTA) to round themup, and their diameters were determined.

In our studies of slime mold growth in liquidcultures, three main types of myxamoeba popula-tions were observed (Fig. 6). The first, which wemay consider normal, is obtained by growing theslime mold on dead bacteria and is characterized

6 8 10 12 14Diometer of Myxamoeboe (,u)

FIG. 6. Size distribution of cells of Polysphondy-lium pallidum grown on different media. Number ofcells measured: (A) 117, (B) 133, (C) 169.

by the uniform size of its cells (curve A), whichflatten easily when transferred to a surface, e.g.,agar.The second type consists of cells that are, on

the average, smaller than normal myxamoebae(curve B); it is found in media giving low cellyields, that contain no serum albumin or serum.The cells tend to round up, especially towards theend of the growth period, and do not flatten whenplated on a surface. Often, small parts of suchcells are separated from the main bodies by con-striction, and these may or may not adhere tothe parent cells.The third type is typical for the complex me-

dium described in this paper. The myxamoebaeare, on the average, larger than normal cells andhave a marked tendency to become giant. Yetthere are not two different cell types-giant andnormal-but a continuous transition betweenthe smallest and the largest cells (curve C).

Phase-contrast microscopy reveals that someof the larger myxamoebae are multinucleate.Other cells seem to have several arms, each con-taining one nucleus; but here it is often verydifficult to decide whether this complex consistsof a single cell or of several cells clumped tightlytogether, or of cells connected by anastomoses(Huffman, Kahn, and Olive, 1962; Olive, Dutta,and Stoianovitch, 1961). By adding EDTA to thesurrounding fluid, the myxamoebae are forcedto round up and thus reveal their individuality,This whole problem of cell size and cell behavior,as influenced by nutrition and other conditionsof culture, will be further investigated in thehope that it may be resolved.

Growth of different strains and species. To testthe ability of the complex medium to support

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TABLE 3. Comparative growth of various species and strains of Acrasieae on the complex medium

No. ofSpecies Strain Final concn (cells/ml) Increase (X) generations

Polysphondylium pallidum Pan-17 1.1 X 107 1,100 10.1Salvador 8.4 X 106 840 9.8WS-320 6.9 X 106 690 9.5Campbell 6.0 X 106 600 9.3FR-47 4.2 X 106 420 8.7

P. violaceum Shaffer's "Founder" 8.0 X 104 8 3.0V-6, P-6, V-9 1.0 X 104 0 0

Dictyostelium mucoroides S-2 8.2 X 104 8.2 3.0Singh 6.0 X 104 6.0 2.6WS-47 5.5 X 104 5.5 2.5WS-278 3.5 X 104 3.5 1.8

D. purpureum WS-321 7.7 X 104 7.7 3.0V-1 2.0 X 104 2.0 1.03645 1.0 X 104 0 0

D. sphaerocephalum (?) FR-14, WS-331 1.0 X 104 0 0D. lacteum Purdue-7a 1.0 X 104 0 0D. discoideum V-12 2.3 X 104 2.3 1.2

NC-4 (type), Gerisch'sV-12/Ml 1.0 X 104 0 0

the growth of different strains of P. pallidum,and of other cellular slime molds as well, a seriesof experiments was run that included about 25strains of our stock cultures. Some of the resultsare summarized in Table 3. To date, the resultsshow that all strains of P. pallidum grow well on

this medium, but that all other species either failto grow or grow only to a very limited degree.It is thus clear that the nutritional requirementsfor the cellular slime molds vary from species tospecies, and that additional factors (nutritionaland environmental) must be considered as we

attempt to devise optimal liquid media for thecultivation of a wide variety of species andgenera.

DIscussIoN

This study demonstrates that at least one

species of the Acrasieae, or cellular slime molds,is capable of completing its whole life cycle (vege-tative growth and multiplication, aggregation,and fructification) in the absence of any bacteriaor bacterial products. Cell populations kept forover 125 generations in their vegetative state by:serial transfers are still able to fruit when re-

moved to an appropriate environment, thus con-

firming quantitatively the results obtained earlierby Raper (1940) for Dictyostelium discoideum andother species grown on living bacteria in con-

ventional agar-plate cultures.

It is further shown that the myxamoebae donot have to feed on particulate food, but cangrow and multiply in soluble liquid media. Themechanism of food intake seems to be similar ineither case: particles or liquid droplets are en-gulfed and the contents of vacuoles so formed aredigested. Whereas the process of ingestion anddigestion of bacteria has been studied (Raper,1937; Mercer and Shaffer, 1960; Gezelius, 1961),the process of pinocytosis has yet to be analyzed.The growth of P. pallidum on media containing

embryo extract and serum, or serum albumin,suggests that the nutrition of these cells is relatedto that of animal cells, as in tissue cultures, ratherthan to the nutrition of plant cells. Yet the highincrease in cell numbers (up to about 1,000 X,or ten generations, within 3 days) makes neces-sary higher concentrations of food material thanare employed for animal tissue cultures, wherethe increase in cell numbers is normally muchless. However, the salt concentration has to belowered relative to media used in animal tissueculture, because of the sensitivity of the myx-amoebae to osmotic pressure (Hohl and Raper,1963).Although both P. pallidum WS-320 and Phy-

sarum polycephalum, a plasmodium-forming slimemold, need embryo extract for their continuousgrowth, the active factors hemin or hemoglobin,which can replace embryo extract for the growth

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of Physarum (Daniel, Kelley, and Rusch, 1962),are not sufficient to replace embryo extract inour medium.

It is to be hoped that further analysis of eachcomponent of the complex soluble medium now

in use may eventually lead to a completely de-fined medium for P. pallidum, and that the pres-

ent medium may serve as a basis for the axeniccultivation of other species of the cellular slimemolds which seem to require additional factors.Investigations directed toward these ends are

now in progress.

ACKNOWLEDGMENTS

We wish to thank Marianne Hohl for invalu-able technical assistance. The work was sup-

ported in part by the Research Committee of theGraduate School from funds provided by theWisconsin Alumni Research Foundation, and inpart by research grants from the National ScienceFoundation (G-3375) and the National Institutesof Health (C-2119), U.S. Public Health Service.

LITERATURE CITED

DANIEL, J. W., J. KELLEY, AND H. P. RUSCH.1962. Hematin-requiring plasmodial myxomy-

cete. J. Bacteriol. 84:1104-1110.EAGLE, H. 1955. Nutrition needs of mammalian

cells in tissue culture. Science 122:501-504.GERISCH, G. 1959. Ein Submerskulturverfahren

fur entwicklungsphysiologische Untersuchun-gen an Dictyosteliur discoideum. Naturwiss-enschaften 46:654-656.

GERISCH, G. 1960. Zellfunktionen und Zellfunk-tionswechsel in der Entwicklung von Dictyo-stelium discoideumn. I. Zellagglutination und

Induktion der Fruchtkorperpolaritat. Arch.Entwicklungsmech. 152:632-654.

GEZELIUS, K. 1961. Further studies in the ultra-structure of Acrasieae. Exptl. Cell Res. 23:300-310.

HOHL, H. R., AND K. B. RAPER. 1963. Nutritionof cellular slime molds. I. Growth on livingand dead bacteria. J. Bacteriol. 85:191-198.

HUFFMAN, D. M., A. J. KAHN, AND L. S. OLIVE.1962. Anastomosis and cell fusion in Dictyo-stelium. Proc. Natl. Acad. Sci. U.S. 48:1160-1164.

MERCER, E. H., AND B. M. SHAFFER. 1960. Elec-tron microscopy of solitary and aggregatedslime mould cells. J. Biophys. Biochem.Cytol. 7:353-356.

OLIVE, L. S., S. K. DUTTA, AND C. STOIANOVITCH.1961. Variation in the cellular slime moldAcrasis rosea. J. Protozool. 8:467-472.

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