APuLED MICROBIOLOGY, Sept. 1969, p. 416-419Copyright @ 1969 American Society for Microbiology

Vol. 18, No. 3Printed in U.S.A.

Modified Two-Phase System for Partition ofBacillus macerans Spores

L. E. SACKS

Western Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture,Albany, California 94710

Received for publication 18 June 1969

An aqueous two-phase system made with polyethylene glycol (PEG) 1000 andpotassium phosphate gives much higher recovery of Bacillus macerans spores in the

upper phase (PEG rich) than does a similar system utilizing PEG 4000. The upperphase completely rejects vegetative cells, which collect at the interface. The systemmay be useful in purifying spores of other species. Scanning electron micrographsof B. macerans spores cleaned in this system show no obvious attached sporangialfragments. The micrographs show that the ridged coat may form polygonal struc-tures at the poles, as previously observed in Bacillus polymyxa spores.

An aqueous polymer two-phase system (1)containing potassium phosphate and polyethyleneglycol (PEG) 4000 (15) has found considerableuse in purifying bacterial spores. When an impurespore suspension is added to this system, thespores enter the upper phase; vegetative cells anddebris enter the lower phase and interface, respec-tively. However, spores of some species show suchan unfavorable distribution coefficient that manyrepeated extractions are required to collect rela-tively small amounts of spores. This unfavorablepartition coefficient may perhaps be caused byattached sporangial material (6), unusual periph-eral structures which are an integral part of thecoat (7), slimes, or capsules (16), or lipid materialsintimately associated with a peripheral spore layer(3). In such cases, separation of spores from vege-tative cells may still be possible if a different two-phase system is employed.

Bacillus macerans spores are of interest becauseof the unusual germination requirements of somestrains (14) and the ridged coats, characteristic ofthe polymyxa-macerans group (4, 8). These B.macerans spores, although apparently free ofattached sporangia, do not readily enter the upperphase of systemY (15).

This paper describes a modified two-phase sys-tem (W) in which PEG 1000 replaces PEG 4000,and which effectively separates B. macerans sporesfrom vegetative cells. The system has a largerupper-phase volume than system Y and may beuseful in purifying the spores of other species.

Scanning electron microscopy, used to verifythe cleanliness of these spores, reveals the charac-teristic ridged structure of the coat in dramatic

fashion, and also shows occasional polygonalstructures at the poles.

MATERIALS AND METHODSCultures. B. macerans NCA strain 7X1 was obtained

from the National Canners Association. The NorthernRegional Research Laboratory, U.S. Dept. of Agricul-ture, supplied B. macerans B-430 and B-70, B. subtilisB-572 and B-644, and B. megaterium B-938. B. cereusstrain T was obtained from H. 0. Halvorson, and B.subtilis ATCC 6633 was originally obtained from N.R. Smith.

Test suspensions. Cultures were sometimes grownon a liquid medium (14) but more commonly on asolid medium, similar to that of Powell and Strange(13). Potato extract was supplemented with 1% Cas-amino Acids (Difco), 1% yeast extract, and 2% agar.This medium was layered over a base layer containing1% CaCO, and 1.5% agar. This procedure eliminatedthe problem of removing insoluble CaC03 from theharvested cells. Petri plates were inoculated with 1 mlof rapidly growing culture (Trypticase Soy Broth) andincubated 16 hr (vegetative cells) or 3 days (spores) at35 C. Surface growth was washed off and freed fromsoluble material by three centrifugations in water.

Two-phase systems. System Y was prepared asdescribed previously (15). System W was preparedwith 27.5 g of PEG 1000 (Carbowax 1000; UnionCarbide, New York, N.Y.) and 22.6 ml of 3 M phos.phate buffer (15), made up to a final volume of 100 mlwith water or test suspension. The PEG was allowedto dissolve before adding the test suspension.

It is convenient to weigh the molten PEG (freezingpoint 37 to 40 C) into the desired vessel (normally, astoppered, graduated cylinder) using a single-pan,top-loading balance. System W has a larger upperphase (75 ml) than system Y (33 ml). The density ofthe upper phase ofW (1.09) is about the same as forY (1.08).

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PARTITION OF BACILLUS MACERANS SPORES

Separation of spores in system W is achieved bytechniques described previously (15). Centrifugationat 1,500 X g for 2 min at room temperature ensures

complete phase separation.Estimation of spore recovery in upper phase was

accomplished by diluting a sample of upper phasewith water (1:10) and determining the optical density(OD) at 625 nm in a 10-mm test tube. The totalamount of spores in the upper phase was estimated bymultiplying the volume of the upper phase by the ODcorrected for dilution. The amount of spores initiallyadded (test suspension) was found in the same way,allowing an estimate of the recovery efficiency. Innearly all cases, the test suspension was composedalmost entirely of either spores or vegetative cells.However, in one case (B. subtilis ATCC 6633) thespore preparation contained appreciable numbers ofvegetative cells. In such cases, for purposes of simplic-ity, the assumption is made that spores and vegetativecells, on an individual basis, contribute equally to OD.

Viscosity was measured at room temperature with a

Brookfield viscometer (Brookfield Engineering Lab-oratories, Inc., Stoughton, Mass.).

Scanning electron microscopy. Spores of B. maceransNCA 7X1 and B. macerans B-70 were grown on a

liquid medium (14), cleaned in system W, and washedthree to six times in water. Germinated spores were re-moved (11) in the course of these washings. After thefinal centrifugation, the packed, cold spores in a steelcentrifuge cup were placed on a cold Dural plate in avacuum desiccator and dried rapidly in vacuo. Dryspores were dusted onto a microscope slide which wasfreshly sprayed with acrylic lacquer. The slide wasgold-coated to a thickness of 50nm on a rotating stagewith its surface at an angle of 45 degrees to the source.

RESULTS

Recoveries of freshly harvested, washed B.macerans spores and B. subtilis spores in theupper phases of systems Y and W are shown inTable 1. For upper phases of B. macerans, re-coveries were 20 to 76 times greater in system Wthan in system Y. In the case of B. subtilis, re-

coveries of strains B-572 and B-644 in W wereabout the same as in Y, but in the case of B.subtilis ATCC 6633, upper-phase recovery was

approximately eight times as great as in system Y.In other experiments, B. cereus and B. megateriumB-938 showed approximately the same percentageof recovery in both systems.

Vegetative cells are rejected by the upper phaseand accumulate at the interface. An experimentin which vegetative cells of three B. macerans

strains and B. subtilis 6633 were introduced intointo system W showed that no detectable amountof vegetative cells remained in the upper phaseafter centrifugation. Two experiments involvingB. megateriwn indicated that a biphasic systememploying PEG 600 (10) could not be relied uponto reject vegetative cells.

Temperature did not affect the performance of

the system in the range 20 to 32 C. The cation"load" of B. macerans spores "stripped" in acid(2) or reloaded with sodium, potassium, or cal-cium in acetate buffers did not affect recovery ineither system Y or W.The upper phase of systemW has a considerable

capacity for spores. Upper-phase recovery of pre-

viously purified spores of B. megaterium increasedlinearly with the amount added to the system to a

level of about 23 mg of spores per ml of upper

phase.

TABLE 1. Recovery of spores in upper

phase of systems Y and W

Strain

Bacillus mace-rans NCA7X1..........

B. maceransB-70..........

B. maceransB-430.........

B. subtilisATCC 6633...

B. subtilis B-644.

B. subtilis B-572.

Avg. ODof testsuspen-siona

0.783

0.969

0.820

0.959

0.773

0.949

w

y

w

y

w

y

w

yw

yw

y

Upperphase

recoveryODC

0.1120.003

0.1450.021

0.1420.015

0.0660.0180.1070.1970.2610.447

Percentageof

recoveryd

53.8.69

56.23.71

65.23.13

26.23.25

52.342.1103.477.7

aA 1:10 dilution in water; volume test suspensionused, 2 ml.

Upper phase volumes: W, 7.5 ml; Y, 3.3 ml.A 1:10 dilution in water.

d Percentage of recovery, volume upper X OD up-per/volume test suspension X OD test suspension.

TABLE 2. Influence of centrifugation time onsedimentation of spores in upper phases

of systems W and Y

Test suspesonI' Upper phaseMin at recovery

1,500 X gStrain ODb ODb

Y WBacillus subtilisB-644.......... 0.831 1 0.151 0.075

10 0.124 0.060

B. subtilis B-572 ... 1.046 1 0.263 0.15310 0.249 0.129

a A 1 ml per 10 ml system.b A 1:10 dilution in water.

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APPL. MICROBIOL.

FIG. 1. Scanning electron micrographs of B. macerans after cleaning in system W and removal of germinatedspores (11); (a) X 17,000, (b) strain B-70, X 8,500; (c, d, e), strain 7X1, X 9,200. Note polygonal structuresformed by ridges at poles (arrows).

Although the upper phase of system W is lessviscous (11.0 cycles/sec) than that of system Y(17.1 cycles/sec) at room temperature, centrifuga-tion time is not critical. That is, prolonged cen-trifugation does not greatly diminish spore recov-ery (see Table 2). This may merely reflect thegreater average distance to be traveled by sporesin system W with its larger upper phase. In the

experiment summarized in Table 2, spores of twostrains of B. subtilis showing approximately equalaffinities for the upper phases of both systemswere centrifuged for 1 and 10 min in the completesystems. Upper-phase recovery was not much lessafter 10 min for W than for Y, and in neither casewas it excessive.

Spores cleaned in system W show no evidence

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PARTITION OF BACILLUS MACERANS SPORES

of attached sporangial material in scanning elec-tron micrographs (Fig. 1). The scanning electronmicrographs clearly show the ridged structure andthe polygonal structures at the poles previouslyobserved in B. polymyxa spores (12, 17).

DISCUSSIONVarious modifications of system Y (15) have

appeared in the literature (5, 9, 10, 18). They havebeen devised for various purposes, such as separa-tion of spores from crystalline inclusions (5) or toincrease the relative volume of upper phase tolower phase (9). Evidently, none of these systemshas been formulated in order to isolate sporeswhich show an unfavorable partition coefficient insystem Y. System W was developed primarily toseparate B. macerans spores from vegetative cells.

Preliminary results with B. subtilis ATCC 6633suggests the system may find similar use in thepurification of spores of other species. Even incases where spore recovery in Y and W are equal,it is possible that its large upper phase, low viscos-ity, and low phosphate content may occasionallymake W the system of choice. On the other hand,the accumulation of vegetative cells in the lowerphase ofY may be desirable in some cases. More-over, Y may be more selective, in the sense of re-jecting spores with atypical surfaces. The inabilityof a system made with PEG 600 (10) to rejectvegetative cells suggests that decreasing molecularweight ofPEG may be accompanied by decreasingselectivity.The scanning electron micrographs of spores

cleaned in system W indicate the absence of at-tached vegetative cell material. The ridged coatcharacteristic of spores of B. polymyxa (4) andB. macerans (4) is strikingly apparent, and sincespecimen preparation for scanning electronmicroscopy is relatively simple, it confirms theusefulness of this technique in determining thesurface morphology of bacterial spores (12).

Polygonal structures formed by the coat ridgesat the poles of B. polymyxa spores were firstdescribed by van den Hooff and Aninga (17).Franklin and Bradley (4) were unable to verifytheir existence, but very recently, scanning elec-tron microscopy and carbon replicas have con-clusively demonstrated such structures in B.polymyxa spores (12). Their presence in B. ma-cerans spores is here demonstrated for the firsttime (Fig. 1).Whether the ridged coat of B. macerans is in

any way associated with its poor recovery in sys-tem Y is not certain at this time.

ACKNOWLEDGMENTS

I thank P. A. Thompson for technical assistance and W. H.Ward and T. L. Hayes, Lawrence Radiation Laboratory, U.S.Atomic Energy Commission, for the scanning electron micro-graphs.

LITERATURE CITED

1. Albertsson, P. A. 1958. Particle fractionation in liquid two-phase systems. The composition of some phase systems andthe behaviour of some model particles in them. Applicationto the isolation of cell walls from micro-organisms. Biochim.Biophys. Acta 27:378-395.

2. Alderton, G., P. T. Thompson, and N. Snell. 1964. Heatadaptation and ion exchange in Bacillus megaterium spores.Science 143:141-143.

3. Bertsch, L. L., P. P. M. Bonsen, and A. Kornberg. 1969.Biochemical studies of bacterial sporulation and germina-tion. XIV. Phospholipids in Bacillus megaterium. J. Bac-teriol. 98:75-81.

4. Franklin, J. G., and D. E. Bradley. 1957. A further study ofthe spores of species of the genus Bacillus in the electronmicroscope using carbon replicas and some preliminaryobservations on Clostridium welchii. J. Appl. Bacteriol.20:467-472.

5. Goodman, N. S., R. J. Gottfried, and M. H. Rogoff. 1967.Biphasic system for separation of spores and crystals ofBacillus thuringiensis. J. Bacteriol. 94:485.

6. Grecz, N., A. Anellis, and M. D. Schneider. 1962. Procedurefor cleaning of Clostridium botulinum spores. J. Bacteriol.84:552-558.

7. Hodgkiss, W., Z. J. Ordal, and D. C. Cann. 1967. Themorphology and ultrastructure of the spore and exosporiumof some Clostridium species. J. Gen. Microbiol. 47:213-225.

8. Holbert, P. E. 1960. An effective method of preparing sectionsof Bacillus polymyxa sporangia and spores for electronmicroscopy. J. Biophys. Biochem. Cytol. 7:373-376.

9. Irie, K., N. Yano, and H. Kembo. 1968. Kinetic analysis ofthe UV-resistant transient stage in spore germination ofBacillus subtilis. J. Gen. Appl. Microbiol. 14:279-293.

10. Lawrence, N. L., and Y.-C. Tsan. 1962. Reactions of sporesand cells of Bacillus cereus with pyrimidine nibosides. J.Bacteriol. 83:228-233.

11. Long, S. K., and 0. B. Williams. 1958. Method for removal ofvegetative cells from bacterial spore preparations. J.Bacteriol. 76:332.

12. Murphy, J. A., and L. L. Campbell. 1969. Surface features ofBacillus polymyxa spores as revealed by scanning electronmicroscopy. J. Bacteriol. 98:737-743.

13. Powell, J. F., and R. E. Strange. 1957. as-Diamino-pimelicacid metabolism and sporulation in Bacillus sphaericus.Biochem. J. 65:700-708.

14. Sacks, L. E. 1967. Adenine and 2,6-diaminopurine as germi-nants for Bacillus macerans spores. J. Bacteriol. 94:1789-1790.

15. Sacks, L. E., and G. Alderton. 1961. Behavior of bacterialspores in aqueous polymer two-phase systems. J. Bacteriol.82:331-341.

16. Tomcsik, J., and J. B. Baumann-Grace. 1959. Specific exo-sporium reaction of B. megaterium. J. Gen. Microbiol.21:666-675.

17. van den Hooff, A., and S. Aninga. 1956. An electron micro-scope study on the shape of the spores of Bacillus polymyxa.Antonie van Leeuwenhoek. J. Microbiol. Serol. 22:327-330.

18. v. Hofsten, B., and G. D. Baird. 1962. Fractionation of cellconstituents of Bacillus megaterium in a polymer two-phasesystem. Biotechnol. Bioeng. 4:403-410.

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