J. Cell Sci. 12, 275-286 (1973) 275Printed in Great Britain

CELL MULTIPLICATION IN TETRAHYMENA

CULTURES AFTER ADDITION OF

PARTICULATE MATERIAL

L. RASMUSSENThe Biological Institute of the Carhberg Foundation, 16 Tagensvej, Copenhagen 2200 N.,Denmark

AND L. MODEWEG-HANSENDanish Institute for Biotechnical Research and Development, Venlighedsvej, DK 2970Horsholm, Denmark

SUMMARY

We have studied the effects of adding participate supplements to populations of Tetrahymenapyriformis in 2 % sterile-filtered proteose peptone broth which supports cell multiplicationpoorly (generation times in excess of 40 h). The tested compounds were: heat-sterilizedsuspensions of egg albumin, nutritionally inert particles of polystyrene, sulphopropyl andquarternary amino-ethyl substituted dextran (in concentrations of 4, 40 and 400 fig per ml).The particles had approximately the same size, but differed in their electric net charges.Particulate suspensions of 40 fig per ml or more greatly improved cell multiplication rates(generation times about 6 h). It is probable that the effect of the particles is to induce formationof food vacuoles without which cell multiplication and growth is very slow. The contributionof the food vacuole to nutrient uptake in Tetrahymena is discussed.

INTRODUCTION

Understanding of nutrient uptake in Tetrahymena pyriformis will eventually resultfrom a synthesis of studies representing several different fields. Among these are: themorphology of the feeding apparatus, the food vacuoles and their associated organelles(Miiller & Rohlich, i960; Nilsson & Williams, 1966; Levy & Elliott, 1968; Levy,Collon & Elliott, 1969; Nilsson, 1970 a, b); studies on the variety and the distributionof the digestive enzymes (Elliott, 1965; Miiller, Rohlich, Toth & Toro, 1969); studieson the induction of food vacuoles (Chapman-Andresen & Nilsson, 1968; Ricketts,igjia,b, 1972), on the uptake of specific metabolites (Hoffmann, Rasmussen &Zeuthen, 1970; Hoffmann & Rasmussen, 1972) and on rates of cell multiplicationunder different conditions (Rasmussen & Kludt, 1970). The present report deals withthe various multiplication rates observed in T. pyriformis after addition of particulatematerial of known electric net charges or of circumscribed chemical composition. Theconcept behind these experiments has been that fast multiplication rates reflect rapiduptake of nutrients. We hope that work along these lines finally will permit us toassess the contribution of the food vacuoles in uptake of nutrients.

Rasmussen & Kludt (1970) have reported that T. pyriformis needs particulatematerial for multiplication in sterile-filtered proteose peptone broth, probably because

18-2

2 7 6 L. Rasmussen and L. Modeweg-Hansen

Table i. Medium for growing T. pyriformis; weight for ioo ml of final medium

mg mg

DL-AlanineL-Arginine. HC1L-Asparagine. HC1L-Glutamic acidL-GlutamineGlycineL-Histidine.HCl.H2OL-IsoleucineL-LeucineL-Lysine.HClDL-MethionineDL-PhenylalanineL-ProlineDL-SerineDL-ThreonineL-TryptophanDL-ValineAdenosineCytidineGuanosineUridineGlucose

This medium is a slight modification of that proposed by Holz et al. (1962). It differs on thefollowing points: the 4 nucleosides have replaced uracil and guanylic acid, the phosphateconcentration is lowered to 50 % and the calcium salt of folinic acid has replaced folic acid.The original directions for preparation of the medium have been followed, except that thestock solutions were kept at — 20 °C, preservatives were omitted and the medium was sterilizedby filtration.

particulate material is a prerequisite to food vacuole formation in this organism. There-fore, we have considered it of interest to determine whether the electric net charges ofsuch particles were of any importance in this regard. Furthermore, we have found thatparticulate material can easily become a limiting factor determining both multiplicationrates and final cell yields. These results have lead us to suggest improvements of thegrowth potentials of some commonly used complex and chemically defined nutrientmedia.

Recent reviews on these topics have been written by Holz (1964, 1972), Conner(1967) and Miiller (1967).

30

302 0

4 0

1 0

402 0

2 0

2 0

2 0

30

302 0

304 0

152 0

2

2

2

2

IOOO

K2HPO4KH2PO4MgSO4.7H2OCaCl2Citric acid

Nicotinic acidd-Pantothenate. CaThiamine.HClRiboflavin-s'-phosphate. NaPyridoxamine. 2HCIPyridoxal.HClBiotinDL-6-Thioctic acidFolinic acid, calcium salt

Trace metals:Fe (NH4)2(SO4)2.6H2OZnSO4.7H,OMnSO4.4H2OCuSO4.5H2OCo(NO3)2.6H2O(NH4)6Mo,O24.4H2O

252550

1

60

m90

75S°45

550

1

1

1400

45°160

3°5°1 0

METHODS

Cultures

Tetrahymena pyriformis, strain GL, was cultivated axenically in 10 ml of nutrient culturemedium contained in 125-ml conical flasks with screw caps. Three different types of culturemedium were used in this study: (a) 2 % proteose peptone (Difco) broth, sterilized for 30 minat 120 °C in a pressure cooker, (6) 2% proteose peptone, sterile-filtered (Millipore membrane

Tetrahymena and paniculate material 277

oZ

1 35

'Diameter' of particles, «m

Fig. 1. Size distribution of the 3 differently charged particles used in this study. We useda modified Coulter counter equipped with a 3O-/tm orifice and polystyrene latex beadsof known volumes as standards. The diameters shown have been calculated on theassumption that the particles are spheres. O, QAE-Sephadex; # , SP-Sephadex; • ,polystyrene.

filters, pore size 0-22 Jim) and (c) a chemically defined medium (see Table 1) also sterilized byfiltration. Rasmussen & Kludt (1970) have previously described the details of the procedure.These 3 preparations were supplemented or otherwise modified as indicated for individualexperiments reported in the Results section.

The stock cultures were maintained in heat-sterilized proteose peptone broth containingundissolved material (Holst-Sorensen & Rasmussen, 1971). This material could have promotedcell multiplication if it, upon inoculation, had been carried over in sufficiently high concen-trations. Consequently, its concentration was reduced by transfer of i-ml samples of mediumand cells from a 2-day-old stock culture to 5 ml of sterile-filtered deionized water 24 h prior toinoculation of the experimental flasks. All cultures were kept at 280 C and the initial concen-trations were about I X I O S cells per ml.

Synthetic particles

Three kinds of beads were tested for their ability to stimulate growth; one had negative, onehad positive and one had neutral electric net charge. The beads were broken into fragments in aBraun ball mill. Fig. 1 shows the size distributions for each type of particle. They are rather

278 L. Rasmussen and L. Modeweg-Hansen

uniform in size from one kind of particle to the other. In order to determine their net charge theparticles were suspended in 2 % proteose peptone and subjected to electric fields of 10 V cm"1.The positive quarternary aminoethyl-substituted dextran particles (QAE-Sephadex, PharmaciaFine Chemicals) moved towards the cathode, whereas the negative sulphopropyl-substituteddextran particles (SP-Sephadex, Pharmacia) moved towards the anode. The uncharged XAD-ipolystyrene particles ('Amberlite', British Drug Houses) did not move. Before they were addedto the cultures, all particles were suspended in distilled water and autoclaved.

Addition of egg albumin

We have compared the effects on cell multiplication of addition of solutions and suspensionsof egg albumin to proteose peptone broth. We began these experiments by making sterile-filtered stock solutions of egg albumin (10 times the desired concentration). Samples of thissolution could then be added to the experimental flasks directly, or they could be heat-coagulated(boiled for 10 min) before they were added to the cultures. We want to emphasize that the dryweights of the egg albumin in the solutions and in the suspensions are identical in our experi-ments.

Sampling and counting

Cell concentrations in the experimental cultures were measured at preselected time points.Prior to sampling the contents of each culture flask were mixed thoroughly by gentle shaking toensure withdrawal of representative aliquots. Cell number was determined as follows: sampleswere removed aseptically from the cultures, the cells were fixed with glutardialdehyde andcounted with a 'Celloscope' as described previously (Rasmussen & Kludt, 1970).

RESULTS

Some new experiments which extend earlier findings will be described first.Rasmussen & Kludt (1970) have shown that iron or aluminium salts forming in-soluble hydroxides in aqueous solutions can stimulate multiplication rates of Tetra-hymena in sterile-filtered proteose peptone broth. Fig. 2 shows the number of cellgenerations as a function of time in 4 different nutrient media. Curve 1 represents theslow growth rate of cells suspended in sterile-filtered proteose peptone broth. Curve 4shows in contrast the much faster rates obtained when the same medium is supple-mented with o-i% egg albumin heat-coagulated into an insoluble, flocculent pre-cipitate. Sterile-filtered egg albumin stimulates multiplication only slightly at the sameconcentration (curve 2). It appears that particulate egg albumin - like the insolublemetal hydroxides - promotes growth, whereas dissolved egg albumin does not. Torule out the possibility that sterile-filtered native egg albumin inhibits cell proliferation,it was added to cells multiplying rapidly in heat-sterilized proteose peptone broth(curve 3). No adverse effects on cell multiplication were observed. Thus egg albuminsuspensions increase multiplication rates under conditions in which egg albuminsolutions are nearly inert. This result corroborates the idea that particulate material isrequired for rapid multiplication of Tetrahymena when grown in sterile-filtered pro-teose peptone (Rasmussen & Kludt, 1970).

We have tried to assess whether the electric net surface charge of growth-inducingparticles is an important feature for stimulation of growth of Tetrahymena. We addedsynthetic particles of rather uniform size and differing charges to cells suspended insterile-filtered proteose peptone. The particles were employed at 3 different concen-

Tetrahymena and paniculate material 279

oa

Fig. 2. Average number of cell doublings (i.e. cell generations) as functions of time.PPS and PPa are abbreviations of 2 % proteose peptone, sterilized by filtration andautoclaving, respectively. Egg albumin (final concentration o-i %) was added to thesterile-filtered broth either as sterile-filtered solutions (EAE) or as precipitates formedupon autoclaving of the sterile-filtered solutions (EAa). The points represent the meanvalues of 8 experiments. 1, PP.; 2, PPS + EA8; 3, PPa; 4, PP8 + EAa.

trations, 4, 40 and 400 /tg per ml (see Fig. 3) and cell multiplication was followedduring 3 days of growth. Parallel controls to which no particles were added werecarried out with sterile-filtered and heat-sterilized proteose peptone broth. In theformer case the growth was poor (left blank columns) and in the latter case good (rightshaded columns). It may be seen in Fig. 3 that stimulation is slight with the lowestconcentration of particles during the first 2 days: however, in the 2 higher concen-trations it is high. More importantly, it may be noted that the stimulation within eachof the 9 groups shown in Fig. 3 appears to be fairly independent of the charge of theparticles.

In addition, we have attempted to rule out the possibility that the observed growthstimulation arose from contamination of the particles with traces of chemicals whichremain after their manufacture. As a first approximation we have observed that thefluid obtained upon filtration of the most concentrated particle suspension employed

28o L. Rasmussen and L. Modeweg-Hansen

O

Q

48Time, h

72

Fig. 3. Average numbers of cell doublings as a function of time and concentrationof Sephadex and polystyrene particles. The columns in each group represent growthwith the following: 2% sterile-filtered proteose peptone broth, • ; sterile-filteredbroth containing particles of positive (•), negative (E3), and neutral (U01), netcharges; and heat-sterilized 2% proteose peptone, IHL A, B, C, 4, 40 and 400 fig ofparticles/ml, respectively. The results are the mean values of 13 experiments.

did not stimulate growth in the sterile-filtered proteose peptone broth. Therefore thegrowth stimulation seems to be specific for the particles.

The next series of experiments was carried out in order to substantiate the idea thatstimulation of cell multiplication in sterile-filtered proteose peptone by addition ofparticles is correlated with induction of food vacuoles. First we have to point out thatthe average number of food vacuoles per cell is very low (3-4) in populations suspendedin sterile-filtered proteose peptone as shown by Rasmussen & Kludt (1970). In thepresent experiments, we failed to obtain any precise vacuole counts after addition ofparticles because it was difficult to observe the borderlines between the individual foodvacuoles. However, we saw that irrespective of which of the 3 kinds of charged particleswe had added, food vacuoles were formed readily and that most of the cells becamefilled-up with particles in 1-2 h. Consequently, we conclude that particles induce foodvacuoles, and that the net charge of the particles is not decisive for their capacity toinduce food vacuole formation in this medium.

The experiments of the growth stimulation were continued with cells multiplyingin the chemically defined nutrient medium. The cells grew well in this medium (D,Fig. 4, curve 1) but failed to grow if the Trace Metals stock solution (TM) was with-held (D minus TM, curve 2). Addition of sterile-filtered proteose peptone (0-05 %) to

Tetrahymena and paniculate material 281

oQ

Fig. 4. Average numbers of cell doublings in the chemically defined sterile filterednutrient medium. Curve 1, the complete medium (see Table 1); 2, as curve 1 exceptthat the trace metals stock solution has been withheld. Sterile-filtered proteosepeptone (005 %)-PP8-is present in the experiments depicted in curves 3-5. Further-more, the negatively charged SP-Sephadex particles have been added in the experi-ments of curve 4 and the neutral polystyrene particles have been added in theexperiments of curve 5. (1, D; 2, D - TM; 3, D - TM + PP8; 4, D - TM + PPS + SP;5, D —TM + PPB + XAD.) Each point is the average of 8 experiments.

this medium (D minus TM plus PPS) only supported growth slightly (compare curves2 and 3). However, we obtained rapid and reproducible multiplication rates when thismedium was supplemented with 40 /ig per ml of the SP-Sephadex particles (D minusTM plus ¥Psplus SP), see curve 4. Also the uncharged polystyrene particles (XAD-i)supported growth, under these conditions, although not as well as the SP-Sephadexparticles did (curve 5). We also tested the positively charged QAE-Sephadex particlesin this system. They often killed the cells in the applied concentrations, perhaps dueto reactions between the surface of the cells and the beads.

In some experiments we followed cell multiplication in the D minus TM plus PPS

plus SP-Sephadex (40 /tg per ml) medium during a period of 10 days reinoculatingnew cultures every other day. After each transfer cell multiplication ceased after about

282 L. Rasmussen and L. Modeweg-Hansen

6 doublings (at about 6 x io4 cells per ml). These 6 doublings were achieved duringthe first 48 h after inoculation, i.e. the initial growth rates compared well with thegrowth rates obtained in the complete chemically defined medium and shown in Fig. 4curve 2. Control experiments have shown that if either proteose peptone or particlesare withheld from this medium cell multiplication ceases. We conclude that the com-pound effect of 0-05 % sterile-filtered proteose peptone and SP-Sephadex particlescan substitute for the missing trace metals.

DISCUSSION

Previous experiments have lead to the suggestion that food vacuole formation is aprerequisite for rapid cell multiplication of T. pyriformis in sterile-filtered proteosepeptone broth (Rasmussen & Kludt, 1970). These authors showed that particulatematerials strongly stimulate growth although in themselves they have little or nonutritional value. The present report confirms these results and extends the list ofgrowth-promoting materials to include heat-coagulated egg albumin and particles ofpolystyrene and dextran. Particles carrying positive, negative and neutral electric netcharges seem to be equally efficient in inducing growth of T. pyriformis in sterile-filtered proteose peptone broth. However, in the chemically defined nutrient mediumthe negative and neutral particles stimulated cell multiplication whereas the positiveparticles often killed the cells. We think that the protective agent in the broth may benegatively charged macromolecules partially covering the particle surfaces. Thusstatements of the effects of the particles should include mention of the suspensionmedium used.

Is nutrient uptake limited to food vacuoles in T. pyriformis} This does not seemlikely in as much as Hoffmann & Rasmussen (1972) showed that the measured uptakeof L-methionine did not increase after stimulation of the formation of food vacuoles.Moreover, Chapman-Andresen & Nilsson (1968) have shown that the rate of foodvacule formation is reduced shortly before cell division, but Crockett, Dunham &Rasmussen (1965) found that this is not reflected in the reduced rate of incorporationof radioactive amino acids into the TCA-precipitable cell fraction. Furthermore,Seaman (1955) compared rates of acetate and glucose consumption with rates of for-mation of food vacuoles and their volumes, and concluded that only a small fraction(2-3 %) of the consumed acetate and glucose had entered into the cells through thefood vacuoles. Recently, Nilsson (1971, 1972) has drawn our attention to a possiblesource of error in considerations of this type in which an investigator fails to take intoaccount the possibility of concentration of nutrients in the extracellular mucous layerprior to uptake. None the less, on the basis of the presented evidence it seems reason-able to suggest that nutrient uptake in T. pyriformis is not limited to food vacuoles.This statement leads to the question whether the cells can grow and multiply in anutrient medium in the absence of food vacuole formation. We cannot yet give a final,general answer to this question. However, Rasmussen & Kludt (1970) have shownthat T. pyriformis multiply very slowly in 2 % sterile-filtered proteose peptone broth,that it is impossible to propagate cultures in this medium and that the cells maintained

Tetrahymena and particulate material 283

under these conditions contain very few food vacuoles. Thus it seems justified toconclude that the food vacuoles are required in order to support rapid multiplicationin this medium.

The characteristics of growth of the cell populations depend on the concentrationof the particles of the nutrient medium. Heat-sterilized proteose peptone has oftenserved as a reference medium (Fig. 2, curve 3: several curves in a previous report(Rasmussen & Kludt, 1970)). This medium seems to be suboptimal with respect to itsparticle content. Thus the cell concentration in stationary phase of growth (4 days afterinoculation) in autoclaved proteose peptone, ranged from 3 to 5-15 x io5 cells per ml(average 4-09 x io5 cells per ml, n = 14) compared to the cell concentration in auto-claved proteose peptone fortified with 70 /IM ferric hydroxide, where we obtainedfrom 8-92 x io5 to 1-92 x io6 cells per ml with an average of 1-27 x io6 cells per ml,n = 14. The cells probably do not suffer from iron shortage in the unsupplementedproteose peptone broth. We have determined that this medium prior to inoculationcontains 0-7 /(g iron per ml. This concentration is also found in the extracellular fluidafter growth up to 3 x io5 cells per ml. Conner & Cline (1964) observed luxuriantgrowth of T. pyriformis in proteose peptone broth after addition of glucose and iron.They interpreted their results as indicating a shortage of iron in the broth. We cannow reinterpret their results to indicate that growth conditions are improved, not byovercoming a shortage of iron needed for cell metabolism, but rather by overcominga shortage of particulate material preventing full utilization of the nutrient mediumby the cells. It is easy to understand that the growth-promoting potential of a nutrientmedium is a function of its particle concentration. One reason for this is that the cellsremove them from the medium by collection in the food vacuoles, surround them bymaterial of 'gel'-like consistency, and defaecate them as such. Therefore, as theymultiply, the cells create aggregate masses of particles too large to be re-ingested, and,when the supply of particles of ' suitable sizes' becomes limiting, growth stops. (Thesuitable sizes have not been determined experimentally, but the diameter of a foodvacuole is about 5 /im so we suspect that only particles smaller than this may be usefulfor the cells.)

The chemically defined nutrient medium has some important features. Its tendencyto form insoluble compounds has been reduced by addition of the chelating agentcitric acid which greatly delays formation of precipitates in the medium, by reductionof the phosphate concentration to 50 % of that in the original recipe by Holz, Erwin,Rosenbaum & Aaronson (1962); by substitution of slightly soluble compounds (e.g.uracil) by their more soluble counterparts (e.g. uridine). These modifications of themedia of Kidder & Dewey (1951) and Holz et al. (1962) have been essential to makeparticulate material a limiting factor in cell multiplication. One drawback of thismedium is the rather long average generation times of about 8 h (Fig. 4, curve 1), butit can easily be overcome if the medium is supplemented with ferric hydroxide par-ticles. In some of our experiments the iron content was 3 times as high as the valueshown in Table 1. The excess iron was added from a suspension of ferric hydroxidemade by adding potassium hydroxide to an aqueous solution of ferrous ammoniumsulphate until a pH value of 9 was obtained. This supplement stimulated cell multi-

284 L. Rasmussen and L. Modeweg-Hansen

plication. We obtained the very satisfactory average generation times of 4-2 + 0-8 h(n = io), i.e. about half of the control generation times, and final cell densities inexcess of 5 x io5 cells per ml.

The trace metals solution of the denned medium has at least 2 functions. First, cellmultiplication will cease if it is omitted from the medium, obviously because the cellsare lacking essential elementary compounds. However, this requirement can be metwith a low concentration of sterile-filtered proteose peptone, provided a source ofparticulate material is also added. SP-Sephadex, XAD-i polystyrene particles andprecipitates of ferric and aluminium hydroxides will all suffice. Thus a second functionof the trace metals solution probably is to provide insoluble compounds to the finalmedium.

Reilly (1964) reported that axenic growth of Paramecium caudatum is enhanced byaddition of particles such as corn starch, rice starch or activated charcoal and thataddition of a synthetic magnesium silicate (Celkate) permitted growth in a chemicallydefined medium. The author interpreted these results to mean that P. caudatum needsthe presence of adsorbents in the nutrient medium. It is possible that the observedstimulation of growth was due to stimulation of food vacuole formation induced bythe presence of particles.

The growth-stimulating effect of particulate material is not limited only to ciliates.Indeed Holst-Sorensen & Rasmussen (1971) and Agrell (1971) found improvedgrowth rates — and thus improved rates of nutrient uptake - of Acanthamoebafollowing the addition of iron salts to a medium which previously supported poorgrowth. Extending these thoughts one step further it is conceivable that there is anutrient uptake stimulating effect of particulate material in addition to a metaboliceffect of iron at the base of Birch & Pirt's (1970) finding that the growth rate fora suspension of mouse cells (strain LS) was greatly improved by the addition of ironsalts to a serum-free medium.

We do not know the mechanism by which the particulate material induces foodvacuole formation in Tetrahymena. Using the phase-contrast microscope we have notobserved forming food vacuoles in this organism from particle free, sterile-filteredproteose peptone broth. However, upon addition of particles to these cells, foodvacuoles are formed within minutes. Thus it is tempting to speculate that mechanicalstimulation caused by the presence of solids in the posterior part of the buccal cavityregion (the cytostome region) is required for initiation of food vacuole formation inTetrahymena.

We thank most heartily Professor Erik Zeuthen for his great interest in the present study andfor reading the manuscript. We are also indebted to Dr Howard E. Buhse for illuminatingdiscussions and helpful advice throughout the preparation of the manuscript. Jesper Zeuthenhas kindly carried out the size distribution determinations, and Bjorn Andersen Nexo hassuggested some of the modifications of the chemically defined medium. Miss Diana Moenbohas skilfully assisted in this investigation.

Tetrahymena and particulate material 285

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286 L. Rasmussen and L. Modeweg-Hansen

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[Received 1 May 1972)