APPLIED MICROBIOLOGY, Sept. 1971, p. 463-468Copyright © 1971 American Society for Microbiology

Vol. 22, No. 3Printed in U.S.A.

Characterization of Two Psychrophilic PseudomonasBacteriophages Isolated from Ground Beef'

P. A. WHITMAN2 AND R. T. MARSHALLDepartment ofFood Science and Nutrition, Untiversity of Missouri, Columbia, Missouri 65201

Received for publication 13 May 1971

Characterization studies were performed on two psychrophilic phages which wereisolated from ground beef samples. Phage inactivation by exposure to heat, low pH,osmotic shock conditions, and freezing showed that these two isolates were different.One-step growth experiments indicated that one isolate had a burst size five times aslarge (500) and a latent period two times as long (4 hr) as the other when tested at7 C. Nucleic acid type was 2-deoxyribonucleic acid for both. Electron micrographsshowed one to belong to Bradley's phage group A and the other to phage group C.

Spencer (16) reported the isolation of a bac-teriophage specific for a luminous marine bac-terium which formed plaques at or near 0 C.More than a decade later Olsen (12) also reportedthe isolation of phage of a psychrophilic Pseu-domonas. Olsen et al. (13) later characterized thisand other isolates obtained from sewage whichgrew on psychrophilic Pseudomonas hosts.

Bacteriophages capable of lysing psychrophilichosts have been shown to be normally presentwithin refrigerated food products (2, 7). The pur-pose of this study was to obtain basic informationon these two phage-host systems preliminary tostudies concerning phage-host interactions in re-frigerated food products.

MATERIALS AND METHODS

Bacteriophage isolation. Methods involved havebeen published in a companion paper (18). The twophages selected, wy and psi, were felt to be repre-sentative of those obtained from the refrigerated foodproducts examined.

Hosts. Both host bacteria were classified into groupII of Shewan et al. (15). They were tentatively clas-sified as P. fragi (18). Host WY was isolated fromhamburger, and PS, was isolated from pork sausage.Cultures were held at 4 C on slants of Trypticase soyagar (TSA; BBL).

Quantitation. The agar layer method described byAdams (1) was one of the two counting proceduresused (herein referred to as the double-layer method).During part of this research, the agar layer methodwas modified as described by Rizvi and Mora (14)by omitting the base layer of medium. The volume ofthe soft agar (TSA) layer was increased to 3.5 ml andthat of the host culture was increased to 0.25 ml. This

1 Contribution from the Missouri Agricultural ExperimentStation. Journal series no. 7099.

2 Present address: General Foods Corp., Battle Creek, Mich.

procedure was used only in the inactivation studies andwas referred to as the single-layer method.

Propagation. Pure stock phage was diluted to aconcentration that would produce overlapping plaqueson plates when mixed with indicator cells. Dilutedphage, 0.5 ml of host cells, and 7 ml of soft agarsupplemented with calcium were poured into 15-cmpetri plates and incubated for 12 to 15 hr at 21 C. Theagar was scraped into a sterile beaker, and the re-maining residue was rinsed from the plate into thebeaker with 6 ml of Trypticase soy broth (TSB; BBL)before incubation for 3 to 5 hr at 4 C. Centrifugationat 10,000 X g followed, after which the supernatantfluid was sterilized by membrane filtration (0.22 ,m).Sterile stock suspensions were stored at 4 C.

One-step and single-burst experiments. Host cellswere grown in TSB for 20 to 22 hr while the mediumwas agitated at 7 C. Cell concentration thereupon wasadjusted by optical density to 108 to 2 X 108/ml. Astoppered adsorption tube (10 by 75 mm), containing0.9 ml of this host suspension and 0.1 ml of phagesuspension [5 X 107 plaque-forming units (PFU) /ml],was agitated on a mechanical shaker (30 rev/min at7 C for 20 min). A 0.5-ml sample was withdrawn anddiluted 102 (dilution 1, D-1). It was further dilutedto 10-4 (growth tube 1, GT-1) and 10 (growth tube2, GT-2) in TSB at 7 C. Infected bacteria were re-moved from D-1 by membrane filtration (0.45 MAm),and the filtrate was plated to determine the number offree phage after 20 min of adsorption; this figure wasused to correct for free phage in GT-1. Samples weretaken from GT-1 and GT-2 at either 30-min or 1-hrintervals and plated by the double-layer method foruse in constructing the one-step growth curve.

Single-burst experiments were performed in thesame manner except that a further dilution was made(10-1) from GT-2 so that when 0.5-ml samples weredispensed into 50 individual sample tubes, each con-tained on the average 0.3 to 0.4 infected bacteria.These 50 tubes were incubated at 7 C until all burstshad occurred. Soft agar (3.5 ml) and the host (0.25

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ml) were added to each tube; the contents were platedand the plaques were counted.

Effect of temperature. The effect of time of exposureat various temperatures on plaque counts of platedphage was determined. Forty plates were preparedfrom a single dilution of phage wy at room tempera-ture (23 C). Four were placed immediately at 20 C,whereas two groups of 18 each were placed at 10 and29 C. At various time intervals up to 150 min, threeplates from each group (10 and 29 C) were placed at20 C. All plates were counted 18 hr after the start ofthe experiment.

Since it was known that psi was heat-labile, theeffect of plating temperature (45 C) on its survival wasdetermined. A phage dilution, which would result inabout 150 plaques per plate, was added to 12 tubes(10 by 75 mm) containing soft agar and these tubeswere held at 45 C. Samples were plated in duplicateat 2-min intervals up to 10 min, with the host beingadded 20 sec before plating.

Survival of phages wy and ps, to exposure at 60 Cover time periods up to 30 min was next determined. Asample of broth containing approximately 108 phageper ml was diluted 1:100 by addition to TSB whichwas being held at 60 C. Samples were withdrawn attime intervals of 1, 5, 10, and 30 min and then dilutedinto chilled TSB (4 C). Results of phage titrationswere compared to titers of controls.

Effect of pH. TSB was adjusted to pH levels rangingfrom 3.0 to 7.0, and 100-fold dilutions of the phagesuspension (approximately 108 per ml) were made intoeach. Samples were withdrawn after 1 hr, diluted withphosphate-buffered distilled water (pH 7.4), and thenplated.

Effect of osmotic shock. Suspensions of about 106phage per ml were exposed for 60 min to 2 M sucroseand 4 M NaCl solutions. Osmotic shock conditionswere producedby 100-fold dilution into distilled water.Surviving phage were enumerated by the single-layermethod with an additional 10-1 dilution being madein distilled water.

Effect of freezing. TSB, skim milk, and meat brothextract in 4.5-ml portions were inoculated with phageto obtain a final concentration of about 106 per ml.Preparation of the meat extract broth was as follows:100 g of ground beef was blended with 200 ml ofphosphate-buffered diluent, centrifuged at 5,000 X gfor 10 min, sterilized by filtration (0.45,um), and thenstored at 4 C. Skim milk was sterilized by heating at121 C for 10 min.As a control to the frozen samples, a tube of TSB

containing the same volume of phage stock was storedat 4 C. All samples were frozen within 20 min andstored at -20 C until sampled 24 hr later for survivingphage.

Effect of citrates. Citric acid was added at concen-trations of 0.1 and 1%o to soft agar unsupplementedwith calcium. The pH was adjusted to 7.0 with 0.1 NNaOH. Soft agar, supplemented with calcium chloride(0.001 M), was used as a control. The phage stock,diluted to produce 100 to 150 PFU per plate, wasplated with these media.

Electron microscopy. One-hundred milliliters ofsterile high-titer lysate was centrifuged for 120 min at

TABLE 1. Summary of results ofone-step growth andsingle-burst experiments with phages

psi and wy at 7 C

Determination psi wy

Per cent adsorption in 79 > 9920 min

Latent period (hr) 1.5 to 2 3.5 to 4Burst sizeOne-step growth 118 529Single burst 84 512

Single-burst experi- 6 to 115 203 to 1168ment (range of PFU/sample tube)

40,000 X g. The pellet was resuspended in 0.1 Mphosphate buffer to one-fifth its original volume andallowed to come back into suspension overnight. Thissuspension was centrifuged at 12,000 X g for 10 min.Supernatant fluid was recentrifuged at 40,000 X g for120 min. This pellet was allowed to come back intosuspension in 0.1 M ammonium acetate (pH 7.0) overan 8-hr period, after which it was filtered (0.45,m)and stored at 4 C. Specimens of phage were air-driedonto carbon grids. Negative staining was performed onboth phages by the method of Eisenstark (8).

Type of nucleic acid. The fluorescent stainingmethod of Bradley (4) was modified slightly as followsto determine nucleic acid type. Purified phage stockinstead of phage lysate was digested for 2 hr at 37 Cin a solution containing 30 jg/ml each of ribonucleaseand deoxyribonuclease. To obtain the desired fluores-cence, five to six drops of purified solution were neededper spot.

RESULTS AND DISCUSSION

Quantitation. The major part of this researchwas performed by using the double-layer method(1). However, since the modified method of Rizviand Mora (14) omitted the foundation layer, thusproviding economics in time and expense, thismethod was tested. No differences were observedin plaque counts (PFU/ml) when these twomethods were compared by using phage-host sys-tems psi-PS1 and wy-WY (variations in plaquecounts were within 4- 10%).

Propagation. When using broth methods inpropagation with the two phages, the broth nevercompletely cleared, and poor titers resulted. Muchhigher titers were routinely obtained by the platemethod; therefore, this method was used through-out the research for the propagation of high-titerphage stocks.

One-step growth and single-burst experiments.The results of the one-step growth and single-burst experiments are shown in Table 1. The twophages differed greatly in their growth kinetics at7 C. Phage wy had a latent period twice that of

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TABLE 2. Summary of the responses of phages psiand wy to stressful conditions

Percentagereduction ascompared to

Treatment control

pSi WY

Heating at 60 C1 min......................... >99 1430 min.........................>99 39

pH level6.0......................... 0 05.0......................... 41 114.0......................... >99 303.0......................... >99 99

Osmotic shock4MNaCl...................... >99 262 M Sucrose.................... 50 48

Freezing inTrypticase soy broth.......... 7 0Skim milk.................... 70 6Gound beef extract............ 0 0

Exposure to citrate (0.1%) 30 30

phage psi and a burst size about five times greater.Preliminary one-step growth experiments, per-formed at 20 C, showed larger burst sizes andshorter latent periods for both phages than werelater observed at 7 C. At 7 C, the latent period forpsi was approximately three times longer than at20 C, whereas with phage wy it was twice as long.The rise period at 7 C was extended when com-

pared to that at 20 C. Burst sizes at 20 C showedvery large variations from experiment to experi-ment, although conditions were held constant inall five experiments.

Olsen et al. (13) worked with psychrophilicphages of Pseudomonas at a lower temperature(3.5 C). Of five phages, none had a burst size ofover 25 or a latent period shorter than 6 hr,whereas at 25 C the burst sizes were much largerand the latent periods were shorter. Also, Olsen(12) observed that the burst size depended uponthe temperature used for growing the host cells.Burst sizes were 300 to 350 when cells were grownat 3.5 C as compared to 10 to 40 when they were

grown at 25 C. Temperature of infection was 3.5C. In our experiments, host cells were grown andinfected at 7 C, and we observed an unexpect-edly large burst size for phage wy (Table 1).

Because of these large burst sizes, a single-burstexperiment was used to validate the findings of theone-step growth experiments. This experimentwas performed only once for each phage, butvalues averaged over all tubes in which burstsoccurred agreed well with those of the one-stepgrowth experiments for both phages.

Effect of temperature. Both phage psi and wyproduced plaques when plates were incubated at2, 7, and 21 C. Both hosts grew at 32 C, butplaques were not formed. Neither host grew at37 C in TSA. Diameter of plaques increased asincubation temperature was decreased.

Unexpected variations in plaque counts wereobserved with phage wy when plates were left atroom temperature (25 to 27 C) for more than 1 hrbefore incubation at 20 C. Plaques were not ob-served if plates were incubatedat 32-C. Therefore,an incubation temperature of 29 C was selected,and the time interval at this temperature wasvaried. The mean plaque count of the control wassignificantly higher (P < 0.05) than the mean forthe treatment at 29 C, but means for the control,placed immediately at 20 C, and the treatment at10 C were not significantly different. Significantdifferences were also shown between plaque countmeans of the treatments at 10 and 29 C. Platesincubated at 29 C for 1 hr or more showed sig-nificantly fewer plaques than untreated controls orthose held for only 30 min at 29 C (P < 0.05).Plaques failed to develop when plates were heldfor 150 min at this temperature. Incubation for150 min at 10 C had no effect on the plaque countas compared to the control (20 C).

Because of the heat lability of phage psi at60 C, it was suspected that plaque counts wouldbe decreased when plates were poured in the usualmanner with the medium tempered at 45 C. Ourexperiments showed that phage survival wasmarkedly decreased by holding the broth suspen-sions at 45 C for at least 2 min. Decreases inplaques of 15 and 72% occurred when exposuretimes were 2 and 10 min, respectively. However,this would normally be of little consequence be-cause phages were exposed to this temperature foronly a few seconds during the plating procedure.These data and the observations of Olsen et al.(13), which showed that most psychrophilicphages are heat-labile, indicated that this stepshould be performed with dispatch. Phage psi wasvery heat-labile when exposed to 60 C, whereasphage wy was relatively heat-insensitive (Table 2).Olsen et al. (13) concluded that thermal sensi-tivity correlated well with phage psychrophily.This appeared to be true with phage psi but notwith phage wy.

Effect of pH. Phage psi was more susceptible toacid inactivation than was phage wy. The effectof low pH was exerted at pH 5 and below forboth phages (Table 2). These values agree wellwith those of other workers (11, 13).

Effect of osmotic shock. Phage psi showed 99%Oreduction when osmotically shocked by 100-folddilution into distilled water from 4 M NaCl,

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FIG. 1. Electroti micrographs ofphage ps, (a) X 79,000; (b, c, d) X 112,000.

whereas when diluted from 2 M sucrose only 50%of the phage population was inactivated (Table 2).Similar results (48%- reduction) were observed forphage wy when it was diluted from sucrose, butdilution from NaCl resulted in only 26 ,% reduc-tion.

Olsen et al. (13) found only one of five psychro-philic phages was osmotically sensitive when di-luted from 4 M NaCl, but all five were sensitivewhen diluted from 2 M sucrose.

Effect of freezing. Nearly 100%' of the phagessurvived frozen storage at -20 C for 24 hr inTSB and in ground beef extract (Table 2). Phagewy survived well in skim milk, but a 70%l- loss inviability occurred when ps, was stored in skimmilk. At first it was thought that the phages wereinactivated by some constituent of the skim milk,as has been reported to occur when phages ofstreptococci and staphylococci are titered fromskim milk (6, 17). This hypothesis was disprovedby comparing titers of psi obtained from skimmilk and TSB, which had not been frozen afterthey received identical inocula of the phage. This

procedure was replicated twice with the titersobserved from each suspending mentruum differ-ing by less than 5%.

Effect of citrate. Titers of both phages were re-duced by 30%>/, when suspensions were plated inagar containing 0.1 > citrate. Growth of the hostwas inhibited by 1.0%,to citrate.

Olsen et al. (13) reported that the reduction inplating efficiency for five psychrophilic phagesranged from 69 to 100%, when media contained1.0% citrate, whereas at 0.1 %'the range was 14to 100%' for three phages. The other two wereunaffected.

Electron microscopy. Electron micrographs ofphage specimens were prepared by negative stain-ing with phosphotungstic acid. Phage psi (Fig. 1)appeared to have a hexagonal shape, especiallythose phage particles which were devoid of nucleicacid (Fig. la). Infectious intact phage particles(Fig. lb) were also observed to have a hexagonaloutline. Short tails appeared to be present (Fig.ic, d), although resolution was poor.Phage wy possessed a contractile tail (Fig. 2,

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FIG. 2. Electron micrograph ofphage wy. X 178,000.

TABLE 3. Morphology ofphages psi and wy

Phage Description Dimen- B radley'saPhage Description sions (nm) classification

pSI Short tail 18 Group CHexagonal head 52

WY Contractile tail 93 Group AHexagonal head 59

a See reference 5.

arrow) attached to a hexagonal head. There ap-peared to be a base plate structure, visible on mostof the noncontracted phage particles.The morphological characteristics of phages

ps, and wy are summarized in Table 3.Pseudomonas phage wy was morphologically

similar to three Pseudomonas phages reported byOlsen et al. (13) but was somewhat smaller thanthe Pseudomonas phage studied by Kropinski andWarren (9). It was much smaller than T2 ofEscherichia coli B. Bradley (3), Olsen et al. (13),and Lee and Boezi (10) have described group C

Pseudomonas phages, which are almost identicalin size to phage psi .

Nucleic acid type. Color reactions, produced byfluorescent dye added to nuclear materials onglass slides, indicated the presence of 2-deoxyribo-nucleic acid (DNA) in both phages. Confirmingevidence was obtained when spots of the phageswere digested with deoxyribonuclease and thenfailed to take the stain when exposed to thefluorescent dye.Only 2-DNA has been found within phages of

groups A and C. These results give further evi-dence, therefore, that phages psi and wy shouldbe classified within groups C and A, respectively.

Thus, the two phages were distinctly different,but their characteristics were similar to those ofother phages of Pseudomonas. Burst sizes, espe-cially that of wy, were higher than expected andshow that proliferation of these phages can pro-ceed very rapidly even at 7 C.

LITERATURE CITED

1. Adams, M. H. 1959. Bacteriophages. Interscience Publishers,Inc., New York.

2. Billing, E. 1963. The value of phage sensitivity tests for the

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identification of phytopathogenic Pseudomizonias spp. J.Appl. Bacteriol. 26:193-210.

3. Bradley, D. E. 1963. The structure of some Staphylococcusand Pseudomonas phages. J. Ultrastruct. Res. 8:552-565.

4. Bradley, D. E. 1966. The fluorescent staining of bacteriophagenucleic acids. J. Gen. Microbiol. 44:383-391.

5. Bradley, D. E. 1967. Ultrastructure of bacteriophages andbacteriocins. Bacteriol. Rev. 31:230-314.

6. Das, N. K., and R. T. Marshall. 1967. Adsorption of staphy-lococcal bacteriophage by milk proteins. Appl. Microbiol.15:1095-1098.

7. Delisle, A. L., and R. E. Levin. 1969. Bacteriophages of psy-chrophilic Pseudomonas. I. Host range of phage poolsactive against fish spoilage and fish-pathogenic pseudo-monads. Antonie Van Leeuwenhoek J. Microbiol. Serol.35:307.

8. Eisenstark, A. 1967. Bacteriophage techniques, p. 517. In K.Maramorosch and H. Koprowski (ed.), Methods in virol-ogy, vol. I. Academic Press Inc., New York.

9. Kropenski, A. B., and R. J. Warren. 1970. Isolation andproperties of a Pseudomontas acidovoranis bacteriophage.J. Gen. Virol. 6:85-89.

10. Lee, L. F., and J. A. Boezi. 1966. Characterization of bac-teriophage gh-1 for Pseudonzontas putida. J. Bacteriol. 92:1821-1827.

APPL. MICROBIOL.

11. O'Callaghan, R. J., W. O'Mara, and J. B. Grogan. 1969.Physical stability and biological and physicochemicalproperties of twelve Pseudontonias aeruiginiosa bacterio-phages. Virology 37:642-648.

12. Olsen, R. H. 1967. Isolation and growth of psychrophilicbacteriophage. Appl. Microbiol. 15:198.

13. Olsen, R. H., E. S. Metcalf, and J. K. Todd. 1968. Charac-teristics of bacteriophages attacking psychrophilic andmesophilic pseudomonads. J. Virol. 4:357-364.

14. Rizvi, S., and P. D. Mora. 1963. Bacteriophage plaque-countassay and confluent lysis on plates without bottom agarlayer. Nature (London) 200:1324.

15. Shewan, J. M., G. Hobbs, and W. Hodgkiss. 1960. A deter-minative scheme for the identification of certain gramnegative bacteria with special reference to the Pseudo-monadaceae. J. Appl. Bacteriol. 23:379-399.

16. Spencer, R. 1955. A marine bacteriophage. Nature (London)175:690-691.

17. Whitman, P. A., and R. T. Marshall. 1969. Interactionbetween streptococcal bacteriophage and milk. J. DairySci. 52:1368-1371.

18. Whitman, P. A., and R. T. Marshall. 1971. Isolation of psy-chrophilic bacteriophage-host systems from refrigeratedfood products. AppI. Microbiol. 22-220-223.

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