a temperate bacteriophage of clostridium perfringens

A temperate bacteriophage of Clostridium perfringens'

D. E. M . 4 ~ 0 ~ ~ 2 - 3 AND G. G. KALZ Department of Microbiology and Immunology, McGill University, Montreal, Quebec

Received November 22, 1967

A temperate bacteriophage was isolated from a lysogenic strain of Clostridium perfiingens. This lysogenic strain was inducible by ultraviolet light. Plaques produced on solid medium were small with turbid centers. Electron microscopy revealed that the phage has a polyhedral head, 80 X 70 mp, and a striated tail, 200 mp long.

One-step growth experiments revealed latent and release periods of 45 minutes each. Phage yields were variable but, in general, quite low. The adsorption rate and temperature and p H stability of the phage were determined.

The diphenylamine reaction and acridine orange staining suggested that the phage nucleic acid is deoxyribonucleic acid.

Canadian Journal of Microbiology, 14, 1085 (1968)

Introduction Reports from Russia during the early

1940's on bacteriophages active on C. perfringens dealt chiefly with the treatment of gas gangrene infections with bacteriophages (12, 18). A few additional phages, later isolated by French workers, were studied (7, 8, 9, 10, 13), but perhaps the largest survey of C.perfringens bacteriophages was conducted by Sames and McClung (15) and Sames (14), who described the isolation of 33 bacteriophages which could be divided into three serological groups and four plaque types.

Three electron microscopy studies of C. perfiingens phages have been reported (5, 6, 19), portraying differences in the morphology of the phages studied.

The above cited reports involved virulent phages of C. perfringens, i.e. those causing a lytic response in the host bacterium. In 1959 Smith (17) demonstrated lysogeny in this species and induced lysogenic strains by various agents; however, a study of the temperate phages carried by such strains was not made.

This paper describes the isolation and pre- liminary characterization of such a phage.

IResearch was supported by the Medical Research Council of Canada.

ZParts of this work were submitted in partial ful- fillment of the requirements for a Ph.D., Depart- ment of Microbiology and Immunology, McGill University, Montreal, Quebec.

,Present address: Department of Microbiology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia.

Materials and Methods Bacterial Strains and Their Maintenance

Strains of C. perjringens were obtained from Dr. V. Fredette, Institute of Microbiology and Hygiene, Laval-des-Rapides, P.Q.; Dr. L. S. McClung, Indiana State University, Bloomington, Indiana; the Depart- ment of Micro'ciology and Immunology and the Clinical Laboratory of the Royal Victoria Hospital, McGill University, Montreal, P.Q.; the American Type Culture Collection; and one isolation was made from waters surrounding Montreal. The strains were kept in Brewer's cooked meat medium (Robertson's meat mash in thioglycollate broth) at 4 ° C and subcultures were made when necessary. Stock strains were also preserved by lyophilization.

Media Lab Lemco agar, used as a basal nutrient layer

for plaque assays, consisted of 10 g proteose peptone (Difco), 4 g yeast extract (Difco), 8 g Lab Lemco beef (Oxoid), 20 ml stock salt solution (250 g NaCI, 20 g KCI, 10 g CaClz made up to 1000 ml with distilled water), and 15 g Bacto agar added to 1000 ml distilled water; final pH adjusted to 7.2.

Semisolid agar overlays were composed of 0.6% Noble agar dispensed in 2.5-ml amounts.

Dehydrated brain - heart infusion broth (Difco) was resuspended in distilled water as prescribed by the manufacturer and 0.1% sodium thioglycollate (w/v) was added. This medium will subsequently be referred to as "broth". The broth was always boiled and cooled immediately before inoculation to reduce the level of dissolved oxygen.

As.ray of Phage by the Agar Layer Method A modification of the method described by Adams

(1) was employed. Phage preparations were plated in 0.1-ml amounts onto agar plates. Approximately 0.8 ml of the indicator bacteria in late logarithmic growth was added to 2.5 ml of molten semisolid agar at 45 "C. The contents of the tube were mixed and poured on top of the applied phage sample. Mixing of the phage and bacteria was effected by gentle rocking of the plate, which caused an even distribution of fluid over the surface.

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1086 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 14, 1968

Znclrbatiot~ Techniqrres Plate cultures were incubated for 18-20 hours

at 37 "C in an anaerobic incubator (National Appli- ance Company, Portland, Oregon). The chamber was evacuated to a pressure of 360 mm of mercury and then filled with hydrogen until a pressure of 737 mm of mercury was obtained. Six platinum cold catalysts were kept in the incubator to promote the reduction of the remaining oxygen to water. Brewer's anaerobic jars were used for small numbers of plates.

Isolntion o f the Plrnge Stationary phase cultures (8 hours) of C. perfringens

were washed twice by centrifugation and resuspension in 0.85% saline. The saline suspension of cells was irradiated for an arbitrary period of 100 seconds and then centrifuged to sediment the bacteria. The sediment was resuspended in broth for 4 hours of incubation. These cultures were centrifuged and the supernatant fluids were spotted onto semisolid agar seed layers prepared with 7%-hour cultures of the available C. perfiirtgens strains.

Growth Stltdies on the Indicator Strain Viable count and optical density growth curves

were performed on the indicator strain of C. per- ,fiinpens. Two optically matched tubes containing 15 ml of broth were inoculated with 0.6 ml of an overnight culture of the indicator strain. Uninoculated broth served as a blank. Both culture tubes were stoppered and incubated a t 37 "C; turbidity readings were made at 30-minute intervals with a Bausch and Lomb "Spectronic 20" at a wavelength of 660 milli- microns. From one of the tubes, 0.1-mI samples were taken every 30 minutes. Appropriate dilutions of these samples into chilled broth were plated in dupli- cate on blood agar plates. The remaining culture tube served as a control to determine whether the sam- pling procedure in the first tube interfered with the normal growth of the organism as determined by turbidity measurements.

Propngntion of'the Bncteriophn~e A 1% inoculum from an overnight culture of the

indicator strain was prepared in broth and incubated at 37°C for 1 hour. Bacteriophage was added at a multiplicity of infection (MOT) of 0.1 or less. After a further incubation for 2% hours, the bacteria were sedimented by centrifugation and the supernatant fluid was filtered under positive pressure through a Millipore 0.22-micron porosity membrane filter.

Electron ?+ficroscop.v Phage preparations, partially purified by centri-

fugation, were treated with neutralized (pH 6.8) phosphotungstic acid.

Lysoger~y Eight colonies of the lysogenic strain were sub-

cultured on blood agar a total of eight times. Eight colonies, representing each of the final subcultures, were inoculated into Brewer's cooked meat medium for overnight incubation. After a second such sub- culture, 0.4 ml of the cultures was added to 10 ml of broth and incubated for 3 hours. These cultures were

centrifuged and the supernatant fluids assayed for plaque-forming units (p.f.11.).

Various dilutions of phage were also plated on the lysogenic strain and on cultures derived from growth in turbid plaques on the indicator strain.

Indlrctior~ of' the Lysogenic Strain with Ultraviolet Light

Two 15-watt General Electric (GI5 T8) germicidal lamps were used at a distance of 38 cm from the plates to be irradiated. A mid logarithmic phase culture (2 hours) of the lysogenic strain was washed twice by centrifugation and resuspension in 0.85% saline. Five-milliliter volumes of these washed cells were placed into open Petri dishes, which were slowly rocked by hand beneath the light source. After irradiation, 0.1 ml of each suspension was diluted in 9.9 ml of broth, incubated for 150 minutes, centrifuged, and the supernatant fluids were assayed for plaque-forming units.

One-step Growth Crtrve An inoculum of 0.6 ml from an overnight cooked

meat culture of the indicator strain was added to 15 ml of broth and incubated for 3 hours. Then 0.2 ml of this culture was transferred to 17.8 ml of broth for an incubation period of 1 hour, at which time a viable count was performed on the culture.

A phage preparation in MI15 phosphate buffer containing 1.5 X 107 p.f.u. /ml was diluted 1 :I000 in broth and 2 ml was added to the 18-1111 culture with very gentle shaking. As incubation continued, samples were taken from this mixture and plated.

Adsorptior I Ezcperin~ents Two milliliters of an overnight cooked meat culture

of the indicator strain was added to 50 ml of broth and incubated for 3 hours when a viable count was made. One milliliter of phage !ysate containing 1 X 107 p.f.u. /ml was added to 9 ml of the 3-hour culture.

The phage-bacteria mixture was incubated at 37 OC in a water bath and 0.1-ml samples were removed and diluted in 9.9 ml of ice-chilled broth over a 15- minute period. Five-milliliter amounts of these dilutions were centrifuged to sediment the bacteria, and the supernatant fluids were assayed for un- adsorbed phage.

Sensitivity of the Bncteriophn~e to pH Broth was adjusted to various pH values ranging

from 3 to 9 by the addition of 1 and 10 N HCl or 1 and 10 N NaOH. Tenfold dilutions of a phage preparation containing 9 X 105 p.f.u./ml were made in broth adjusted to the desired pH. After 30 minutes of room temperature incubation, the preparations were assayed for surviving phage.

Tempernture Ser~sitivit.~ of the Bacteriophn~e The phage preparation used in these experiments

was produced by centrifuging a filtered broth lysate in a Sorvall RC-2 centrifuge at 37 000 g for 3 hours and resuspending the sediment to 1 130 of the original volume in MI15 phosphate buffer containing 2 X 10-3 M magnesium sulfate.

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MAHONY AND KALZ: BACTERIOPHAGE OF C. PERFRINGENS 1087

Two milliliters of this preparation was added to test tubes maintained in a constant temperature water bath. One unheated sample was placed in a chilled test tube and represented the unheated control. The heated tubes were removed at various times and chilled until assayed. Temperatures of 30, 40, 50, 60, and 70 "C were chosen for these tests.

N~rcleic Acid Detertninntiotz (a ) Prepnrntion of Plrage Filtered broth lysates were concentrated sixfold

by flash evaporation at a temperature ranging from 28 to 40°C and under an atmospheric pressure of 20-30 mm of mercury. Ribonuclease and deoxyri- bonuclease (Worthington Biochemical Corporation) were then added to a final concentration of 8.3 pg/ml of each enzyme. After thorough mixing, the lysate was incubated at 37 "C for 30 minutes.

Centrifugation of this lysate was done in a Spinco model L preparative ultracentrifuge at 38 457 g for 1 hour. The supernatant broth was removed and the sediment of each tube was resuspended in 1 ml of 0.85y0 saline and pooled for centrifugation at 12 000 g for 10 minutes. The supernatant fluid was removed for another cycle of differential centrifugation, the final concentration of phage being 70-fold.

(b ) N~rcleic Acid Extrnctiotz The nucleic acid of 9 ml of this phage preparation

containing a total of 8.3 X 1010 p.f.u. was extracted by a modification of the Schneider, Hogeboom, and Ross technique (16). Treatment with 5y0 perchloric acid (PCA) at 4 "C for 30 minutes was followed by centrifugation at 12 350 g for 10 minutes. The supernatant fluid was labelled cold PCA I. The sediment was washed twice with cold PCA, and all washings were kept for spectrophotometric study and (or) chemical analysis. These latter washings were labelled cold PCA I1 and IT1 respectively. Three milliliters of 5% PCA was added to the washed sediment and this was heated at 90 "C for 15 minutes. It was then centrifuged and the supernatant fluid containing the nucleic acid fraction was studied spectrophotometrically with a Zeiss PMQ IT spectrophotometer and chemically.

( c ) Chemical Tests Deoxyribonucleic acid was determined by the

Burton modification of the Dische method (4) and ribonucleic acid was determined by the Dische modification of Mejbaum's method (2). Standard curves were prepared for various concentrations of yeast ribonucleic acid (RNA) and fish sperm deoxyribonucleic acid (DNA) (Nutritional Biochemicals Corpora- tion).

(4 Acridine Omtlge Stnirririg Acridine orange was used to stain concentrated

phage particles on glass slides as described by Bradley (3). A 7- to 8-drop sample of the concentrated phage preparation containing 1 X 1010 p.f.u./ml was applied to a slide with a 2 0 4 capillary pipette, dried, fixed in cold methanol, and stained with acridine orange. After the slide was washed in McIlvaine's citric acid and phosphate buffer, and then in 0.15 M disodium hydrogen phosphate, the wet slide was ex-

amined under ultraviolet light at a wavelength of 2537 A.

Controls for proper fluorescence were prepared using DNA and RNA.

Isolation of the Phage Of the 18 strains tested, only one lyso-

genic strain was detected and only one st-rain served as an indicator. These strains, obtained from Dr. Fredette, were respectively desig- nated T 12 and Lechien in his collection. A single plaque was picked and the phage was propagated with the indicator strain in broth.

The plaques produced by this phage were small and varied from pinpoint to 1.5 mm in diameter. Turbid centers were observable, especially in the large plaques. The edges of the plaques were irregular. Picking single plaques of contrasting size and propagating such examples before plating again resulted in the same variation in plaque size, suggest- ing that only one phage was present.

Grolvtlz Studies on tlze Indicator Strain The viable count reveals a lag phase of

about 1 hour and then a short logarithmic phase of 2 hours followed by a marked decline in viability (Fig. 1). At the end of the logarithmic phase, the pH of the broth was recorded at 6.5. The removal of samples from the culture tube did not interfere with the growth curve as determined by turbidity readings.

Propagation of the Bacteriophage The titers obtained in broth cultures never

exceeded 4 X 108 p.f.u./ml, with the maximum yield occurring 2 hours after infection of the culture. No lysis of the bacterial culture could be detected. Propagation of the phage by producing confluent lysis of the bacterial growth on agar plates with subse- quent freezing and thawing did not improve the yield.

Electron Microscopy The electron micrographs (Figs. 2 and 3)

revealed that the bacteriophage has a very regular head, hexagonal in shape, measuring 80 m p long and 70 m,u wide. The tail of the phage is about 200 m p long and 8 m p wide and possesses a number of striations at 4 mp intervals. There was no evidence of an end- plate, tail fibers, or contractile sheath.

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CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 14, 1968

T ~ M E i~ M ~ N U T E S

FIG. 1. Viable count and optical density growth curves of the indicator strain of C. perfringens.

Lysogeny It was found that 0.1-ml volumes of the

supernatant fluids of each culture grown from serially subcultured colonies of the lysogenic strain contained sufficient plaque-forming units to produce semiconfluent lysis of the indicator strain on agar plates. This showed that each bacterium (as represented by a colony) bore the potential of releasing bacteriophage.

The phage released by the lysogenic strain never produced plaques on the lysogenic strain, and growth picked from the center of turbid plaques on the indicator strain was unable to support plaque formation, suggest- ing lysogenization of the indicator strain.

Induction of the Lysogenic Strain with Ultra- violet Light

Thirty seconds of ultraviolet light was the optimal inducing dose for this strain as shown in Fig. 4, but the marked decrease in plaque-forming units brought about by a further 10 seconds of irradiation suggested that 30 seconds of irradiation was very close to some critical point either in prophage induction or bacterial sensitivity to ultraviolet light. The maximum induction in this experiment yielded a 108-fold increment in p.f.u./ml over the control titer. Without dilution of the irradiated cells, however, induction could not be detected.

A modified one-step growth curve of an

FIG. 2. Electron micrograph of the C. perfringens bacteriophage negatively stained by phosphotungstic acid (199 500 X). Bar represents 100 mp.

FIG. 3. Electron micrograph of the C. perfringens bacteriophage negatively stained by phosphotungstic acid (228 000 X). Bar represents 100 mp. Note the striation of the tail.

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Can. J. M~crobiol.-Mahony and Kalz

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T ~ M E i~ SECONDS

FIG. 4. Induction of the lysogenic strain with ultraviolet light.

induced culture revealed a latent period of 60 minutes followed by a release period lasting 90 minutes.

One-step Growth Curve The results of one of three such ex~eri-

ments are depicted in Fig. 5. There was a latent period of 45 minutes after infection followed by a rapid rise in titer which reached its maximum in 40 minutes. The increase in p.f.u./ml was 17-fold. In this particular experiment, a second burst was shown to follow the first, beginning 100 minutes after the initial phage infection. I t was essential to use a very low MOI in these experiments. Two other experiments revealed identical latent and burst periods, although the actual increment in phage titer was variable.

decreased rate until 70-757, of the particles were adsorbed by 20 minutes. Two other experiments employing lower numbers of bacteria and a higher MOI failed to demon- strate any definite pattern of adsorption.

pH Sensitivity A pH of 3 completely inactivated the

phage and there was some inactivation a t pH 4. The phage appeared stable to pH values ranging between 5 and 9.

Tenzpernture Sensitivity Temperatures of 60 and 70 "C inactivated

virtually all of the phage in 30-60 minutes. At 50°C, however, there was as much as a 10-fold increase in p.f.u./ml within 5-15 minutes, followed by a steady rate of falling phage titer. Heating at 40 OC caused a rapid activation of phage during the first 15 minutes followed by a slower rate until a maximum titer was obtained at 90 minutes. No inactivation occurred at this temperature. There was little change in phage titer at 30 "C (Fig. 7).

This phenomenon was readily repeated with this phage preparation, but no heat activation was ever observed with crude broth lysates. A number of experiments performed in an attempt to explain these results were unsuccessful. Phage purified by a gel adsorption method, not involving centrifugation, also demonstrated heat activation, but to a lesser extent.

Nucleic Acid Determination The determination of the nature of the

phage nucleic acid was difficult because of the relatively low titers attainable in propagation cycles and the apparent ease with which the plaque-forming units were inactivated by high speed centrifugation.

The ultraviolet absorption spectra of the PCA extracts are presented in Fig. 8. These results suggest that most of the ultraviolet- absorbing materials have been removed by the second PCA washing. The hot PCA treatment succeeded in extracting a fraction which had been insoluble in cold PCA and this

Adsorption Experiments fraction had an ultraviolet absorption spec- There was a rapid adsorption of approxi- trum typical of nucleic acids.

mately 507, of the plaque-forming units Orcinol and diphenylamine tests were per- within 2 to 4 minutes as shown in two experi- formed on the cold PCA I11 and hot PCA ments in Fig. 6. Adsorption continued at a extracts and the hot PCA insoluble fraction

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FIG. 5. One-step growth experiment. Bacterial viable count, 4.3 X 106/rnl; MOI, 0.0008.

TABLE I Determination of nucleic acids in perchloric acid

extracts of phage

Extract pg DNA* pg RNA*

Cold PCA 111 0 Hot PCA 35 Hot PCA insoluble 0

*As determined from standard curves of D N A and RNA. l o \ (Table I). The hot PCA extract had both orcinol- and diphenylamine-reacting material, although the diphenylamine-reacting material was associated only with the hot PCA fraction, while orcinol-reacting material was detected in all fractions, including the hot - 0 l-,--- PCA insoluble fraction. Since this latter o 10 20 fraction would not be expected to contain T I M E I N MINUTES

nucleic acid, a positive orcinol test may FIG. 6. Adsorption of bacteriophage to the indi- indicate that substances other than RNA cator strain. Bacterial viable count, 3.6 X 10a/ml;

pentose are giving a positive test. The MOI, 0.0026.

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MAHONY AND KALZ: BACTERIOPHAGE O F C . PERFRINGENS 1091

FIG. 7. Temperature sensitivity of the C. pevfvingens phage.

amount of DNA, 35 pg, as determined by the diphenylamine reaction, was very close to the total amount of nucleic acid estimated to be present, 39 pg, by the absorptioil value at 265 mp.

Acridine orange staining of the concentrated phage preparation revealed a bright greenish-yellow fluorescence similar to that of a control DNA preparation; no red color was observable. The control RNA fluoresced a flame red color under ultraviolet light.

Discussion This paper describes the isolation and

characterization of a temperate bacteriophage of C. perfiingens. At first it was not realized that C. petfiingens grew so rapidly and that the release of phage from a lysogenic strain would diminish after 3 hours of growth. It would, therefore, be advisable to look for temperate phages of these organisms in very young cultures, since the super- nates of older cultures possessed very few plaque-forming units by comparison.

I t was interesting to note the very marked drop in the viability of the indicator strain at the end of the logarithmic growth phase. Since the pH of the culture had only dropped to 6.5 at this time, it would not seem prob- able that pH was a factor involved in such rapid death of the bacteria, nor would it seem likely that the nutrient value of the medium had been exhausted in so short a time. Because phage propagation in such a culture ceased in this period of declining viability, it was apparent that a logarithmic phase bacterial culture must be employed for propagation purposes.

Three papers have appeared dealing with electron microscopy of C. peifiingens phages. The first of these studies was performed on two phages by Elford, GuClin, Hotchin, and Challice (6). One phage has a head diameter of 35 mp and the other a diameter of 60 mp. Both phages have tails 120 X 15 mp. Bychkov (5) described another phage which possesses a spherical head 50-60 mp in diameter, and a short tail 15-20 m p long. A more recent study by Vieu, Guelin, and Dauguet (19)

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0 COLD PCA I A COLD P C A I1 A COLD P C A I I I

HOT P C A

WAVELENGTH i N M ~LL.~M~CRONS

FIG. 8. Perchloric acid extraction of nucleic acid.

described a phage, 70 mp long, with a poly- hedric head, 40 mp in diameter, and a short tail, 30 mp long. The tail portion is unique in that it possesses a contracted end-plate, 8 mp long and 34 mp in diameter, containing four to seven distinct structures.

All these phages differ from the temperate phage of C. perfringens reported in this paper. The tail of the temperate phage (200 mp long) is longer than that of the other isolates and the head structure is somewhat larger. No tail fibers or sheaths could be observed. Of interest is the fact that this phage is almost identical, morphologically, with a temperate bacteriophage isolated from a lysogenic strain of C. histoIyticum by GuClin, Beerens, and Petitprez in 1966 (11).

Lysogeny was demonstrated by the fact that after several subcultures of isolated colonies of the lysogenic strain on blood agar plates, the colonies chosen for study were capable of releasing bacteriophage spon- taneously when grown in broth. The phage

did not produce plaques on this strain nor did it produce plaques on growth taken from plaques produced on the sensitive strain of C. perfringens.

The lysogenic strain could be induced by exposure of washed cells to ultraviolet light. Without dilution of the bacteria after irradiation, no induction could be observed. The adsorption of released phage particles might be a factor in this failure to detect released phage, although it seemed unlikely that adsorption could completely mask the induction burst in undiluted cultures.

The one-step growth curve of this phage demonstrated a characteristic latent and release period. The latent period, 45 minutes, was the same as that observed by GAspAr and Tolnai (8) using one of GuClin's phages, although the phage yield was much lower with the temperate phage.

It is not known why the rate of adsorption decreased after the original adsorption velo- city observed during the first 2-4 minutes of

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the experiment. The proposal that there 3. BRADLEY, D. E. 1966. The fluorescent staining of bacteriophage nucleic acids. J. Gen. Microbiol. might be a limited number of adsorption 44: 383-391.

sites on the bacteria is unlikely in view of the 4. BURTON, K. 1955. A study of the conditions and low MOI employed. q-he fact that it was mechanism of the diphenylamine reaction for

the color~metric estimation of deoxyribonucleic necessary to employ a very low MOI in acid. Biochem. J, 62: 315-322, one-step growth experiments might suggest 5. BYCHKOV, K. YA. 1964. Electron microscopic

the presence of some inhibiting in study of Cl. perfr'fgetzs phagolysis. J . Microbiol. Epidemiol. Immunobiol. 41(3) : 39-41.

the lysate. 6. ELFORD, W. J. , G U ~ L I N , A. M., HOTCHIN, J. E., Although the experimental data of such and CHALLICE, C. E. 1953. Le phenomene de la

bacteriophage chez les anatrobies (Wplclrin per- experiments are not presented in this paper, fii!~ge~rs). Ann. Inst. Pasteur, 84: 319-327. the factors involved in the heat activation 7. GASPAR, G. 1960. Studies of a CI. petfirtigens

phenolnenon were not identified. A somewhat phage. 11. The latent period of its growth curve. Acta Microbiol. Acad. Sci. Hung. 7: 269-276.

silnilar response was observed by Wallace, 8. GASPAR, G. and TOLNAI, G. 1959. Studies on a Smith, and Melniclc in 1964 (20), involving CI. perfiingens phage. Acta. Microbiol. Acad.

reovirus activation by heat. Sci. Hung. 6 : 275-281. 9. GUELIN, A. 1949. Comportment d'un bacterio-

In the nucleic acid experiments, diphenyl- phage actif sur Cl. ,velchii. Ann. Inst. Pasteur, amine-reacting material was only associated 77: 40-46.

10. GUELIN, A. 1955. Isolement et etude des bacterio- with the nucleic acid fraction extracted by phages actifs sur les anaerobies, Leur r61e dans hot PCA, whereas a considerable qi~antity of l'evolution du phlegmon experimental. Ann. orcinol-reacting material was associated with ~ $ ~ ~ ~ ~ r 6 ~ ~ ~ ~ ~ ~ ~ ~ a n d PrnTPREz, A. 1966. all fractions of the procedure, making a con- Un bacteriophage des anaerobies actif sur elusive result on RNA content impossible Clostri~liriart~ histolyticrlm. Ann. Inst. Pasteur, 111:

141-148. with this test. The need for a better nucleic 12. HAuDuRoy, P, 1946. Travaux rCcents sur le acid preparation is apparent. bacteriophage dlHerelle. Presse Med. 54: 703-

Acridine orange staining of the phage 704. 13. KREGULR, A., GU~LIN, A., and LE BRIS, J. 1947. articles indicated that DNA was the phage 1~01ement d . ~ n bacteriophage actif sur le Clostri-

nucleic acid. diltm perfii11get1.s type A. Ann. Inst. Pasteur, 73: 1038-1039.

14. SAMLS, R. W. 1956. Studies on the bacteriophages of Clostridi~ittr perfringetrs. Ph.D. Thesis,

Acknowlledgrnents Indiana State University.

The thank D ~ . V. ~~~d~~~~ and D ~ , 15. SAME% R. W. and MCCLUNG, L. S. 1953. Pre- liminary studies of the bacteriophagy of Clostri-

L. S. McClung for their generous donations ~ ~ L I I I I perfihrger~s. Bacterial. Proc. G52, 40. of C.perfiingens strains, which were employed 16. SCHNEIDER, W. C., HOGEBOOM, G. H., and Ross,

H. E. 1950. Intracellular distribution of enzymes. in the greatly VII. The distribution of nucleic acids and ade- appreciated was the electron microscopy per- nosine triphosphatase in normal mouse liver and formed on the C. pei'fiinger2s bacteriophage mouse hepatoma. J. Natl. Cancer Inst. 10: 977-

982. by Dr. R. G . E. Murray at the University 17. SMITH, H. W. 1959. The bacteriophages of of Western Ontario. Clostrirliiri~~ perfriilget~s. J. Gen. Microbiol. 21:

622-630. The assistallce of Research 18. SPENCER, M. C. 1953. Gas gangrene and gas

Council of Canada is acknowledged and the gangrene organisms 1940-1952. An annotated Council than]& for the studentship awarded bibliography of the Russian literature, 1940-

1952, and the non-Russian literature for 1952. to one of the authors (D.E.M.). Armed Forces Medical Library Reference Divis-

ion, Washington, D.C. Titles: 38, 39, 40, 41, 42, 158, 162, 210, 265.

19. VIEU, J. F., GuiLIN, A., and DAUGUET, C. 1965. 1. ADAMS, M. H. 1959. Bacteriophages. Interscience Morphologie du bacteriophage 80 de Welcliin

Publishers, Inc., New York. perfiitrgens. Ann. Inst. Pasteur, 109: 157-160. 2. ASHWELL, G. 1957. Colorimetric analysis of 20. WALLIS, G., SMITH,. K. O., and MELNICK, J. L.

sugars. h~ Methods in enzymology. Vol. 111. 1964. Reovirus act~vation by heating and in- Edited by S. P. Colowick and N. 0. Kaplan. activation by cooling in MgClz solutions. Virol- Academic Press, Inc., New York. pp. 87-90. ogy, 22: 608-619.

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