APPLIED MICROBIOLOGY, OCt. 1971, p. 706-715 Vol. 22, No. 4Copyright t 1971 American Society for Microbiology Printed in U.S.A.

Practical Procedures for the Purificationof Bacterial Viruses

URIEL BACHRACH AND ADAM FRIEDMANN

IMstitute of Microbiology, Hebrew University-Hadassah Medical School, Jerusalem, Israel

Received for publication 11 May 1971

The efficiencies of the various methods used for phage concentration have beencompared. The two-phase concentration method (with polyethylene glycol and dex-tran sulfate) gave maximal recoveries of infectivity for coliphages of the T-even andT-odd series and for ribonucleic acid phages and single-stranded deoxyribonucleicacid phages. Precipitation of phages by acid gave high yields when applied to T2 andT4 phages but not with T3 and T7 coliphages. Differential centrifugation was effi-cient when sedimented phages were gently dispersed before repeating the centrifuga-tion cycle. The efficiencies of the various methods have also been confirmed byelectron microscope studies, which also show that the two-phase concentrationmethod gave rise to intact phages. Zone centrifugations in sucrose gradients (12.5to 52.5%) indicated that coliphages of the T-even series sediment faster than T-oddcoliphages; they may thus be separated from each other and from empty ghosts bycentrifugation at 100,000 x g for 40 min. Equilibrium centrifugation in preformedcesium chloride gradients was also useful for phage concentration and purification.This study also deals with some optical properties of purified phages; optical crosssections and absorbance ratios (at 260 and 280 nm) of the various preparations aregiven.

For most purposes, it is desirable to use phagestocks which are free from bacteria, molds, andinsoluble debris. Further, many experimentsrequire the preparation of phages at large quan-tities and high titers. Earlier procedures for thepurification and concentration of phage werebased on centrifugation in a Sharples supercen-trifuge (13), concentration by removal of waterin vacuo (1), or by ultrafiltration through col-lodium membranes (14). The concentration ofphage by adsorption on diethylaminoethyl cellu-lose (33) or on hydroxyapatite (5, 18) has alsobeen reported.

Until recently, differential centrifugation andacid precipitation (10) were used extensively forthe purification and concentration of phage.During recent years, the newly developed two-phase separation method provided a useful toolfor the concentration of viruses. This procedure,first described by Albertsson (2), is based on theobservation that viruses tend to concentrate atthe liquid-liquid interface in a system consistingof dextran sulfate and polyethylene glycol. Thetwo-phase separation method with variousmodifications has been applied to coliphages ofthe T-even series (3, 6, 9, 19, 25), lambda phage(27, 32), filamentous deoxyribonucleic acid(DNA) phage fd, ribonucleic acid (RNA) phage

R17 (32), Q3 phage (8, 32), and single-stranded,X174 phage (22, 24).

Phages concentrated by the above mentionedprocedures can be further purified either by den-sity gradient centrifugation in sucrose or byequilibrium centrifugation in preformed cesiumchloride gradients. The latter method seems tobe most useful as it leads to the purification ofphage after relatively short centrifugation times.

In the present report, we compare the efficiencyof the various procedures employed for the con-centration of coliphages. It will be shown thatthe two-phase separation method seems to bethe most efficient procedure for the concentra-tion of phage from crude lysates. This study alsodeals with some optical properties of purifiedphages.

MATERIALS AND METHODSMaterials. Polyethylene glycol 6000 and dextran

sulfate were purchased from Pharmacia Fine Chemi-cals. Bacterial strains and phage stocks used werefrom the collection of this institute.

Preparation of phage lysates. Escherichia coli Bwas grown in M9 minimal medium (1) to a densityof 5 X 108 cells/ml and infected with coliphages ofthe T-series at a multiplicity of 0.1 to 0.5. Lysates ofOX174 were obtained by infecting exponentially grow-ing E. coli C cells in TPA medium (15). E. coli C 300

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was grown in TPA medium and infected with MS2phages. Lysates were treated with chloroform (5 mlper liter of medium) and left in a room at 4 C for 1to 24 hr. Phage titers were determined by the stand-ard double-layer technique (1). Ultraviolet spectrawere recorded on a Gilford model 2400 spectropho-tometer.

Differential centrifugation. Bacterial debris wasremoved by centrifugation at 4,000 X g for 20 minin a high-speed centrifuge (model 18; Measuring &Scientific Equipment, Ltd.). Phages were next sedi-mented by centrifugation at 20,000 X g for 30 to 60min, and the supernatant fluid was removed by suc-tion with a pipette connected to a suction flask. Thepacked, sedimented phage particles were then sus-pended in a small volume of borate buffer (sodiumtetraborate-1.14% in 10-5 M ethylenediaminetetra-acetic, pH 9.0) and left in the cold for 12 to 24 hr tofacilitate dispersion of aggregated phages. Phageswere then further diluted in borate buffer and centri-fuged again at 4,000 X g for 20 min. These cycles oflow- and high-speed centrifugation were repeatedseveral times to remove undesired impurities.

Acid precipitation. Bacterial debris was removed bycentrifugation at 4,000 X g for 20 min as above, andphages were precipitated from the clear supernatantby a dropwise addition of 1 N hydrochloric acid untila final pH of 3.9 was reached. (Changes of pH weremonitored during the addition of the acid by a pHmeter.) The resulting turbid lysate was then pouredinto a 2,000-ml cylinder and placed in the cold room.After 24 hr, the clear upper fluid was removed bysuction and the turbid residue was centrifuged at4,000 X g for 30 min. The sedimented phages werefinally suspended in borate buffer as above.

Two-phase separation. Lysates were transferredinto a conical separatory funnel which was placed ina cold room. After 12 to 24 hr, the chloroform layerand most of the bacterial debris were removed fromthe bottom of the funnel. Thereafter, the followingchemicals were added to 2-liter quantities of phagelysates: 130 g of polyethylene glycol 6000, 4.0 g ofpulverized dextran sulfate, and 35.0 g of sodiumchloride. The separatory funnel was thoroughlyshaken and placed in the cold for 12 to 18 hr. Theheavy turbid bottom layer (of approximately 25 ml)was then collected in a 50 ml-centrifuge tube and cen-trifuged at 3,000 X g for 15 min. The resulting inter-face ("cake"), containing the phages, was then re-covered after removing the clear bottom and topphases by means of a pipette. This interface, whichremained in the tube, was then suspended in 20 ml,of borate buffer. To precipitate the dextran sulfate,2.0 ml of 3 M potassium chloride was added, and themixture was kept in the cold for 2 hr. The precipitate,containing dextran sulfate and residual bacterialdebris, was finally removed by centrifugation at 3,000X g for 10 min. Viruses in the supernatant fluid werefurther concentrated by centrifugation at 20,000 X gfor 30 to 60 min and dialvzed against borate buffer ifnecessary.Zone centrifugation in sucrose gradients. Portions

(1 ml) of phage suspensions were layered on the topof a 30-ml sucrose gradient (12.5% in the top and

52.5% in the bottom) in 0.05 M tris(hydroxymethyl)-aminomethane-hydrochloride plus 0.01 M sodiumphosphate buffer (pH 7.2) in 0.05 M sodium chloride.After centrifugation in a Spinco SW25 swingingbucket rotor for 40 min at 100,000 X g (Spincomodel L ultracentrifuge), phages, concentrated at thevisible band, were either withdrawn by means of acapillary pipette or collected after puncturing thebottom of the tube with a needle.

Equilibrium centrifugation in cesium chloride. Thisprocedure, which is based on the technique ofThomasand Abelson (28), involves the following steps. Avolume of 1.3 ml of each of the three cesium chloridesolutions (densities of 1.3 g/ml-0.45 g of CsCl plus1.0 ml of water; density of 1.5 g/ml-0.830 g of CsClplus 1.0 ml of water, and density of 1.7 g/ml-1.280 gof CsCl plus 1.0 ml of water) was pipetted sequen-tially with a capillary pipette into the bottom of a5.0-ml cellulose nitrate centrifuge tube. Phage sus-pensions (up to 1.0 ml) were then layered on the topof the gradients, and tubes were centrifuged at 100,000X g for 60 min in a Spinco SW39 swinging bucketrotor. During this time, a roughly linear gradient ofcesium chloride was formed in the tubes. Phages werefinally collected as above. The density of the cesiumchloride solution can be determined by means of apycnometer (31) or by reading the refractive index(29).

Electron microscopy. Phages were dialyzed for 12hr against 0.3 M ammonium acetate buffer (pH 7.0)and mounted on a carbon-coated collodion film,supported by a 400 mesh copper grid. Phages werenegatively stained with 1% ammonium molybdate(pH 7.0) and examined by a Philips model 300 elec-tron microscope.

RESULTSEfficiency of the various methods. A compari-

son of the various methods showed (Table 1)that the two-phase separation method gave thehighest recoveries and that the yield of T-coli-phages ranged from 85 to 100%, whereas theRNA phage MS2 and the single-stranded tX174phage gave recoveries of 36 and 6%, respectively.Acid precipitation, on the other hand, gavesatisfactory results with T2 and T4 only, whereasT7 and T3 phages were inactivated at low pHvalues (Table 1). The efficiency of differentialcentrifugation varied from one experiment tothe other; recoveries were highest when sedi-mented phages were kept in borate buffer forseveral hours before repeating the sedimentationcycle. It appears that aggregated phage cosedi-mented with bacterial debris, thus reducingyields. In general, differential centrifugation gaverecoveries below 50% (Table 1).

Optical properties of phages. The purity of thephages in the various preparations was nextdetermined by comparing their optical proper-ties, such as optical cross section or absorbanceratio (A26o/A2so). The optical cross section of a

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TABLE 1. Efficiency of the various methods used for phage conzcentration

Recovery (%)Method

T2 T4 T3 TS T7 AIS2 4X174

Polyethylene glycol-dex-tran sulfate ............ 95 100 89 100 85 36 6

Acid precipitation........ 53 45 0.1 19 <0.1Differential centrifuga-

tion ................... 42 30 1.0 28 34

TABLE 2. Optical cross sections of conicenitrated phages

Prepn T2 T4 T3 T5 T7 MS2 1X174

Differential centrifugation ....... 0.51a 0.8 16.1 1.5 0.3Acid precipitation............... 0.81 0.42 116.1 0.53 45.0Polyethylene glycol-dextran sul-

fate......................... 0.61 0.15 0.26 0.28 0.13 0.03 2.05Sucrose gradient centrifugation. 0.33 0.42 0.34 0.36 0.27Cesium chloride centrifugation. 0.46 0.37 0.45 0.4 0.53 0.043

a Optical cross sections are expressed in square centimeters/PFU, multiplied by 1011.

TABLE 3. Absorbance ratios of different phages

Prepn T2 T4 T3 T5 T7 MS2 OX174

Differential centrifugation ....... 1. 28a 1.28 1.34 1.28 1.39Acid precipitation ............... 1.42 1.24 1.07 1.20 1.19Polyethylene glycol-dextran sul-

fate......................... 1.38 1.38 1.31 1.21 1.24 1.33 1.38Sucrose gradient centrifugation. 1 .32 1.30 1.32 1 .37 1.48Cesium chloride centrifugation. 1 .34 1.33 1.43 1.48 1.43 1 .36

a Absorbance ratios are expressed as the ratio of absorbancies at 260 and 280 nm.

phage (11), which is defined as absorbance perphage [absorbance per centimeter at 260 nm,divided by number of plaque-forming units(PFU) per milliliter], is similar for phages pre-pared under identical conditions and permittedthe estimation of the number of phage particlesin a suspension without performing the tedioustitration on agar plates.

Optical cross section. The optical cross sectionobtained for a phage suspension is a relativemeasure of the extent to which the phage havebeen purified, lower values indicating greaterpurity. The lowest values were obtained withphages purified by zone centrifugation in sucrosegradients, equilibrium centrifugation in cesiumchloride, or after concentration by the two-phase(polyethylene glycol-dextran sulfate) methods(Table 2). In all of these experiments, the opticalcross sections for coliphages of the T-series werebetween 0.13 X 10-11 to 0.61 X 10-11 cm2/PFU. Differential centrifugation, on the otherhand, gave rise to phages of lower purity, as re-

flected by relatively high optical cross sections(Table 2). Acid precipitation gave poor resultswhen applied to T3 or T7 coliphages. This iseasily explained by the susceptibility of thesephages to acid.The cross section of the RNA phage MS2, con-

centrated by the two-phase method, was rela-tively low (0.03 x 10-11 cm2/PFU), unlike thatof the single-stranded phage 4,X174 (Table 2).Absorbance ratio. Contamination of phages

either by foreign proteins or by DNA may easilybe detected by determining their absorbanceratios at 260/280 nm. Pure phage give ratios ofapproximately 1.30, whereas pure DNA orphages disrupted by osmotic shock have anabsorbance ratio of approximately 1.8 (4, 7, 17).Phages purified by differential centrifugationgave absorbance ratios of approximately 1.30(Table 3). On the other hand, the absorbanceratios for T3 and T7 phages, after precipitationwith acid, were somewhat lower (Table 3). Thiscould be explained by the disruption of phages

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Ec

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Bottom TopFIG. 1. Zone sedimenztatiot: oJ phages in2 sucrose

gradienzts. Phages were layered on a 12.5 to 52.5%sucrose gradient and centrifuged at 100,000 X g for40 min. Absorbanicies of the various fractionts weremonitored by a Gilford model 2400 spectrophotometer.

of the T-odd series by the acid added. Theabsorbance ratios of phage concentrated by thetwo-phase method ranged from 1.21 to 1.38 andwere similar to those obtained for phages con-

centrated by differential centrifugation, zonecentrifugation in sucrose gradients, and after

FIG. 2. Separationz of phages by equilibrium celi-trifugationI in cesium chloride. Phages were layered on apreformed cesium chloride gradient and cenztrifuged at100,000 X g for 60 miii. Absorbancies of tlhe variousfractions were montitored by a Gilford model 2400spectrophotiometer.

equilibrium centrifugation in cesium chloride.The absorbance ratios of concentrated MS2 and4X174 were practically the same (Table 3).

Concentration of phages by ultracentrifugation-zonal centrifugation in sucrose gradients. Thisrapid centrifugation procedure may be appliedto lysates or concentrated phage suspensions.Figure 1 shows that phages sedimented in arelatively narrow band and that coliphages ofthe T-even series differed from T-odd phages in

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TABLE 4. Biu.vyant densit l of dliffierenlt phages-

Phage Density (g/ml)

T4...... 1.5350T5 1 .5775T7 1.5150T2 ghosts... 1.3240dX 1 74 .... 1.459)

a Phages were centrifuged at 100,000 X g for 60min in a preformed cesium chloride gradient, andthe density of the phage-containing band wasdetermined by means of a pycnometer.

their sedimentation properties. The sedimentationratio (distance of sedimented particles from thetop of the gradient, divided by the height of thegradient) for T-even phages was 0.48 to 0.5,whereas T3, T5, and T7 had ratios of 0.69 to0.71. It should be noted that ghosts of T2 phagescould be separated from intact phages by zonecentrifugation; the former had a sedimentationratio of 0.6. It thus appears that coliphages of theT-even series may be separated from either ghostsor phages of the T-odd series by zonal centrifu-gation under the above described conditions.

Equilibrium centrifugation in cesium chloride.Equilibrium centrifugation in preformed CsClgradients provides another useful method for thepurification and concentration of phages. Thismethod, too, could be used for the purificationof phages from lysates or applied to phages con-centrated by the above mentioned procedures.Because of differences in density, T5 coliphageswere separated from T2 phages by equilibriumcentrifugation (Fig. 2). Bacterial debris, phageghosts, and internal proteins were recovered fromzones of different densities. Table 4 illustratesthe results of some experiments and gives thebuoyant densities of various phages, as deter-mined by measuring the density of the CsClsolution in the corresponding phage-containingbands.

Ultraviolet spectra of purified phages. Datapresented in Table 3 indicated that the purity ofphages could be determined by measuring theirabsorbance ratio at 260 and 280 nm. Additionalinformation for the absence of ultraviolet-ab-sorbing impurities may be gained by recordingthe absorption spectra of the different phagepreparations. Figure 3 shows that T2 and T5phages, after sucrose gradient centrifugation,exhibited similar optical properties, with a maxi-mal absorption peak at 260 nm.

Electron microscopy. Electron microscopicstudies were next undertaken to determinewhether the different preparations containednoninfective phage particles or were contaminated

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FIG. 3. Ultraviolet spectra of phages. Pliages werepurified by zoiial cenitrifugationt in slucrose graidienits,antd their absorption spectra were recorded.

with bacterial debris. Acid precipitation gave riseto relatively pure T2 phages (Fig. 4). Most ofthese phages contained DNA, as demonstratedby the negative staining technique. On the otherhand, treatment with acid had an adverse effecton T3 phages which lost most of their DNAduring acid precipitation. This finding is in linewith the results depicted in Table 1, which illus-trated low recoveries of T7 and T3 phages afterexposure to acid. Negative staining also demon-strated that approximately 50%7O of the T2 phagespurified by differential centrifugation did notcontain DNA and that the preparation was alsocontaminated with detached phage tails (Fig. 5).As expected, the two-phase concentration methodwas fairly efficient. The suspensions contained

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FIG. 4. Electront microscopy ofphlages. Phlages were concentrated by acid precipitation and negatively stainedwith ammonium molybdate. (a) T2 phiages; (b) T3 pliages. Bars represent 0.1 m.

::t

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FIG. 5. Electron microscopy of phlages. T2 plhages were concentrated by differential cenitrifugation anld nlega-tively stained. Bar represents 0.1 um.

mainly intact phages with only a few contaminat-ing structural elements. It is of interest that thetwo-phase concentration method did not elimi-nate bacterial pili from a suspension containingRNA phages (Fig. 6, 7). Banding of phages on

preformed CsCI gradients gave satisfactory re-

sults, and almost all of the recovered T2 phageswere intact (Fig. 8a). The same was also true forT2, T3, and T5 phages purified by zone centrifu-gation in sucrose gradients (Fig. 8). By this proce-

dure, bacterial debris, empty ghosts, or frag-mented phages were completely removed fromthe intact phages.

DISCUSSIONExperiments described in this paper confirm

previously reported findings that the two-phaseconcentration method gives highest recoveries ofphages and that this concentration method isapplicable to coliphages of the T-even and -odd

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b

FIG. 6. Co,,cenitrationt oJfphages by the two-plhase techniiqute. (a) T2; (b) T3. Bars represent 0.1 ,un.

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L.22, 1971 PURIFICATION OF BACTERIAL VIRUSES 713

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series as well as to RNA and single-strandedDNA phages. Acid precipitation gave reasonableyields only for the concentration of T-even coli-phages, whereas T3 and T7 phages were inacti-vated by the acid added. This was demonstratedby electron microscope studies, determination ofoptical cross sections, and absorbance ratios.Differential centrifugation should be carried outwith great care, and sedimented phages shouldbe gently dispersed, otherwise a considerableproportion of the sedimented phages may be lostbecause of aggregation. Electron microscopicstudies indicated that even careful treatment andgentle dispersions of phages lead to considerableinactivation.Data depicted in Table 2 suggest that the

approximate number of phage particles in a sus-

pension may be estimated by measuring theirabsorbance at 260 nm. In many cases, this simpleprocedure can replace the tedious titration,mainly when only a rough estimation of phagetiters is required. In general, the values of opticalcross sections of the various phages is in goodagreement with the results obtained by otherauthors. Thus, the optical cross sections of T2phages were shown to be 0.7 X 10-11 to 10-1"cm2/PFU (11, 16, 30) and those of T6 and T4phage ot be 0.7 x 10-11 to 1.0 x 10-11 cm2/PFU(30). Purified T3 phages had cross sections of0.2 x 10-11 to 0.3 X 10-11 cm2/PFU (20) andT5 phages had cross sections of 0.3 X 10-11 to0.6 x 10-1" (21, 30), compared to 0.2 x 10-11 to0.6 X 10-11 cm2/PFU for T7 coliphages (7, 20, 21,26). The reported cross section of the RNA phage

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FIG. 8. Electronz microscopy ofpuirified phlages. T2 phages were purified by equilibrium sedimenitationz in cesiumchloride (a) or bv zonie cenitrifitgationi in sucrose gradienits (b) T2, (c) T3, (d) T5. Bars represenit 0.1 Am.

R17 was 0.06 X 10-11 (32), and that of fd was0.05 X 10-11 cm2/PFU (12). On the other hand,the reported optical cross section of the single-stranded DNA phage 4X174 was 0.013 x 10-11cm2/PFU (23).

Centrifugation experiments described in thispaper permit the separation of T-even from T-oddcoliphages. This finding is not surprising as thesephages are known to differ in the mass of theirnucleic acids. It is also understandable that

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empty phages (ghosts) differ in their sedimenta-tion from intact phages. This too is easily ex-plained by differences in masses. Centrifugationin preformed cesium chloride gradients also gave

good separation between the various phages andpermitted the determination of their buoyantdensities after relatively short centrifugationtimes.

LITERATURE CITED

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2. Albertsson, P. A. 1960. Partition of cell particles and macro-

molecules. John Wiley & Sons, New York.3. Albertsson, P. A. 1967. Two-phase separation of viruses, p.

303-321. In K. Maramorosch and H. Koprowski (ed.),Methods in virology, vol. 2. Academic Press Inc., NewYork.

4. Bachrach, U., R. Levin, and A. Friedmann. 1970. Studies on

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5. Bernardi, G. 1965. Chromatography of nucleic acids on

hydroxyapatite. Nature (London) 206:779-783.6. Brody, E. N., H. Diggelmann, and E. P. Geiduschek. 1970.

Transcription of the bacteriophage T4 template. Obligatesynthesis of T4 prereplicative RNA in vitro. Biochemistry9:1289-1299.

7. Davison, P. F., and D. Freifelder. 1962. The physical prop-

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erase, p. 530-540. In L. Grossman and K. Moldave(ed.), Methods in enzymology, vol. 12B. Academic PressInc., New York.

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E. coli. Z. Naturforsch. 18b:876-883.13. Hook, A. E., D. Beard, A. R. Taylor, D. G. Sharp, J. W.

Beard. 1946. Isolation and characterization of the T2bacteriophage of Escherichia coli. J. Biol. Chem. 165:241-257.

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viral specific chloramphenicol-resistant protein in 4X174-infected cells. J. Mol. Biol. 39:655-668.

16. MacHattie, L. A., D. A. Ritchie, C. A. Thomas, Jr., andC. C. Richardson. 1967. Terminal repetition in permutedT2 bacteriophage DNA molecules. J. Mol. Biol. 23: 355-363.

17. Misra, D. N., R. K. Sinha, and N. N. Das Gupta. 1969.Molecular weight of DNA fromn coliphage T7 by electronmicroscopy. Virology 39:183-193.

18. Miyazawa, Y., and C. A. Thomas, Jr. 1965. Nucleotidecomposition of short segments of DNA molecules. J. Mol.Biol. 11:223-237.

19. Philipson, L., P. A. Albertsson, and G. Frick. 1960. Thepurification and concentration of viruses by aqueous

polymer phase systems. Virology 11:553-571.20. Ritchie, D. A., C. A. Thomas, Jr., L. A. MacHattie, and

P. C. Wensink. 1967. Terminal repetition in non-permutedT3 and T7 bacteriophage DNA molecules. J. Mol. Biol.23:365-376.

21. Rubenstein, I. 1968. Heat-stable mutants of T5 phage. 1. Thephysical properties of the phage and their DNA molecules.Virology 36:356-376.

22. Sedat, J., and R. L. Sinsheimer. 1964. Structure of the DNAof bacteriophage X174. V. Purine sequences. J. Mol.Biol. 9:489-497.

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24. Sinsheimer R. L. 1966. Culture and purification of bacterio-phage q5Xl74, p. 569-576. In G. L. Cantoni and D. R.Davies (ed.), Procedures in nucleic acid research. Harper& Row, New York.

25. Stone, K. R., and D. J. Cummings. 1970. Isolation andcharacterization of two basic internal proteins from the T-even bacteriophages. J. Virol. 6:445-454.

26. Studier, F. W. 1969. The genetics and physiology of bacterio-phage T7. Virology 39:562-574.

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metric transcription of coliphage T7 in vivo: correlationwith poly G binding sites. Virology 34:9-16.

28. Thomas, C. A., Jr., and J. Abelson. 1966. The isolation andcharacterization of DNA from bacteriophage, p. 553-561.In G. L. Cantoni and D. R. Davies (ed.), Procedures innucleic acid research. Harper & Row, New York.

29. Thomas, C. A., Jr., and K. I. Bems. 1961. The physicalcharacterization of DNA molecules released from T2 andT4 bacteriophage. J. Mol. Biol. 3:277-288.

30. Thomas, C. A., Jr., and T. C. Pinkerton. 1962. Sedimentationequilibrium studies on intact and fragmented bacteriophageDNA. J. Mol. Biol. 5:356-372.

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Kaplan (ed.), Methods in enzymology, vol. 6. AcademicPress Inc., New York.

32. Yamamoto, K. R., B. M. Alberts, R. Benzinger, L. Lawhorne,and G. Treiber. 1970. Rapid bacteriophage sedimentationin the presence of polyethylene glycol and its applicationto laige-scale virus purification. Virology 40:734-744.

33. Zarybnicky, V., and P. Horacek. 1968. Influence of ionicstrength on the stability of phage T2r to somotic shock.Folia Microbiol. 13:391-400.

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