DEOXYRIBONUCLEASE ACTIVITY OF MICROCOCCI FROM CLINICALSOURCES'

BARBARA G. WECKMAN2 AND B. WESLEY CATLINDepartment of Microbiology and Inmmunology, School of Medicine, Marquette University,

Milwaukee, Wisconsin

Received for publication December 4, 1956

Enzymes having the capacity to degrade highlypolymerized deoxyribonucleic acid have beendetected in numerous biological materials.Pancreatic deoxyribonuclease (DNase), whichhas been obtained in crystalline form (Kunitz,1950 a, b), is activated by magnesium, hasoptimum activity in the region pH 7, and is heatlabile (McCarty, 1946; Zamenhof and Chargaff,1949). Most of the bacterial DNases resemblepancreatic DNase in cation requirement and pHoptimum. Detailed study of several of the bacte-rial DNases has shown, however, that they can bedistinguished from pancreatic DNase or fromeach other on the basis of antigenic differences(McCarty, 1949; Warrack et al., 1951) or dif-ferences in response to specific DNase inhibitors(Bernheimer and Ruffier, 1951; Kozloff, 1953;Catlin, 1956).The DNase obtained from cultures of Micro-

coccus pyogenes var. aureus (Staphylococcusaureus) differs from other described DNases inrequiring calcium as a cation activator, in havinga high pH optimum (about 8.6) and a remarkableheat stability, and in bringing about a moreextensive degradation of deoxyribonucleic acidthan does pancreatic DNase (Cunningham et al.,1956). A correlation between DNase activityand positive coagulase reaction observed inpreliminary studies of stock culture collectionstrains suggested that DNase activity mightprove to be a taxonomic characteristic useful instudies of strains of the micrococcus-staphylococ-cus group.

1 This investigation was supported by researchgrant C-2405 from the National Institutes ofHealth, Public Health Service.

2 Part of this report is from a thesis submittedby B. Weckman in partial fulfillment of the re-quirements for the degree of Master of Science,Marquette University.

MATERIALS AND METHODS

Eighty-seven strains of micrococci werestudied. All were recently isolated from clinicalmaterial in three hospital laboratories.

Pigmentation. Cultures on nutrient agar (Difco)supplemented with 1 per cent yeast extract(Difco) were incubated at room temperature(20-26 C) for five days and were observed daily.

Coagulase reaction. Coagulase plasma (Difco)was used according to directions for the tubemethod. The reliability of this reagent (control430179) was verified initially by duplicate testingof twenty strains using fresh oxalated humanplasma.

Utilization of ammonium phosphate as the solesource of nitrogen. Cells from a young agarculture were emulsified in sterile water and usedas inoculum for the medium of Hucker (1948).Growth and acid production were noted during.incubation at 37 C for 7 days.

Nitrate reduction. Nitrate broth (Difco)cultures incubated at 37 C for 24 hr were testedfor nitrite using sulfanilic acid and at-naph-thylamine reagents (Conn, 1951).

Mannitol fermentation. A strain was regardedas having the capacity to ferment mannitol ifacidity was produced during incubation at 37 Cfor 48 hr in at least two of the following threemedia: mannitol salt agar (Difco) plates, slantsof phenol red agar base (Difco) with 1 per centmannitol, and peptone broth (1 per cent) with 1per cent mannitol and brom thymol blue indi-cator.

Gelatin liquefaction. Nutrient broth (Difco)with 12 per cent gelatin was inoculated induplicate. Tubes were incubated at 37 C and atroom temperature for two weeks.

Calcium activated deoxyribonuclease activity.The following media, inoculated with a DNase-producing strain, were tested after incubation bythe viscometric method to determine which

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provided greatest DNase activity; trypticasesoy broth (BBL), tryptose phosphate broth(Difco), heart infusion broth (Difco) alone andwith 1 per cent glucose added, brain heartinfusion (Difco). Each of these was used aloneand with single 1 per cent additions of glycerol,starch, and yeast extract (Difco).

Deoxyribonucleic acid (DNA), which yieldshighly viscous solutions, was prepared from calfthymus by the method of Kay et al. (1952). A2 mg/ml solution in distilled water was used asthe substrate in both the qualitative and theviscometric tests. Stock sodium borate buffer wasprepared by adding NaOH to a solution of boricacid (1 M, analytical reagent) until pH 8.5 wasreached.

Reagents for a DNase test were combined in asmall tube in the following order immediatelybefore use: 0.175 ml of borate buffer solution(0.04 M), 0.175 ml of CaCI2 solution (0.04 M),0.35 ml of DNA solution, and 0.1 ml of culturefluid. The supernatant fluid of centrifuged(16,000 rpm) cultures was found to contain themajor portion of the DNase activity. For routinetests, however, whole broth cultures were used,either undiluted or diluted in a solution of sterilegelatin (0.1 per cent in distilled water; McCarty,1946). Some strains, especially coagulase positivemicrococci, showed greater DNase activity withincreased dilution. Accordingly, with cultureshaving more than 5-10 units/ml activity, dilu-tions were prepared (up to 1:10,000 for strainSA-B) and tested until one was found thatshowed activity of approximately 10-25 units.For the viscometric test, after the reaction

mixture was prepared, 0.7 ml was immediatelydrawn into a microviscometer, which then was

TABLE 1Effect on detectable deoxyribonuclease activity of

agitation-aeration during cultivation of coagulasepositive micrococci for 40 hr in brain heartinfusion

Culture Shaken Culture Not Shaken

StrainDilution DNase, units Dilution DNase,tested prml tested units per mnl

culture culture

J36 1:100 680 undil. 4J53 1:1000 14,100 1:10 52J161 1:100 3,000 undil. 2J605 1:1000 4,150 undil. 51SA-B 1:1000 20,600 1:100 278

placed in a 37 C water bath, and the flow timeswere determined at frequent intervals duringincubation. DNase activity was calculated asunits per ml (u/ml) of culture from the rate ofreduction of DNA viscosity (Laskowski andSeidel, 1945; Cunningham et al., 1956).

In the qualitative test (modified from McCarty,1949), the tube containing the reaction mixturewas incubated in a 37 C water bath and periodi-cally a drop was removed and allowed to fall into asolution of 90 per cent acetone in water. The"web" or fibrous precipitate, which is produced inacetone by undegraded DNA, does not formwhen depolymerized DNA is dropped in acetone.DNase activity was inversely proportional to thetime required for disappearance of web formation.

EXPERIMENTAL RESULTS

Composition of the culture medium, degree ofaeration, pH, temperature, and time of incuba-tion were found greatly to influence the DNaseactivity detected in culture of micrococci. In apreliminary study of various culture mediainoculated with a coagulase positive food poison-ing strain from the culture collection, the highestDNase activity was obtained in brain heartinfusion (5,500 u/ml). Supplementation of thismedium with glycerol, starch, or yeast extractdid not increase the activity. Levels of DNaseactivity detected in trypticase soy broth, tryptosephosphate broth, or heart infusion cultures(900, 900, and 1,600 u/ml, respectively) weresignificantly increased (to 4,300 u/ml in the caseof heart infusion) by supplementing these mediawith yeast extract.The effect of agitation-aeration (Finn, 1954)

was studied with five recently isolated coagulasepositive strains cultivated at room temperaturein paired flasks of brain heart infusion (100 mlin 300-ml capacity flasks). One of each pair wasagitated continuously on an Eberbach shaker(about 120 horizontal excursions per minute);the companion culture was unshaken. Aftercultivation for 40 hr, viscometric DNase testswere carried out on whole cultures or on dilutionsin gelatin solution (final concentration 0.01 percent). As shown in table 1, DNase activity wasone hundred to one thousand times greater withshaken than with unshaken cultures. In furthertests with strain SA-B, less efficient aeration (useof an aquarium air pump and Pasteur pipette"bubblers") resulted in lower DNase activity,whereas more efficient aeration (100 ml medium

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shaken in liter capacity flasks) resulted ingreatly increased activity.

Aeration of cultures is known to produceimportant physiological effects, some of whichare reflected in differences of pH and populationsize. Shaken cultures of strain SA-B in brainheart infusion (which contains 0.2 per centglucose) commonly exceeded pH 8 and theaverage population size was greater than that ofcompanion cultures that were not shaken, wherethe pH usually dropped below 6.5. Numbers ofviable cells were difficult to determine reliably,however, because of cluster formation by micro-cocci. Cellular aggregation was minimized inshaken brain heart infusion cultures, but wasgreatly increased by slime formation (Catlin,1956) in unshaken cultures, or some kinds ofshaken cultures. Experiments, though pre-liminary, indicate that shaken cultures producedless DNase activity per colony forming cell inbrain heart infusion buffered with succinate topH 6.1 than in brain heart infusion (withoutsuccinate), initially pH 7.4.The relation between culture growth and

DNase activity was studied in detail with threerecently isolated coagulase positive strains(SA-B, J824, J707). Cultures in brain heartinfusion (100 ml in 500-ml capacity flasks) werecontinuously shaken at room temperature. Thegeneration time (36-40 min during the logarith-mic growth phase; calculated from semilogarith-mic plots of numbers of colonies formed by cellsor cell-aggregates) and the maximum number ofcolony forming cellular units (1-2 X 1010 per ml)were essentially the same for each of the threestrains, but maximum DNase activities differedmarkedly, as can be seen in figure 1. Activitiesdetected in cultures of the three strains after36 and 80 hr of incubation, respectively, were:for SA-B 52,680 and 57,500 u/ml; for J824 ap-proximately 6,000 and 7,000 u/ml; for J707 1,630and 1,480 u/ml. DNase was not detected untilthe populations reached about 2 X 108 colonyforming cells/ml (sixth to seventh hour of incuba-tion). DNase activities increased at logarithmicrates during the next few hours and attainedmaximum levels before the populations signif-icantlv decreased in numbers of cells determinedby colony counts.From the foregoing studies it was concluded

that cultures in brain heart infusion (volumesnot greater than one-fifth of the flask capacity)incubated with continuous shaking at room

temperature (24 to 30 C on the shaker platform)for 40 to 72 hr were adequate for tests of calciumactivated DNase. Cultures grown under theseconditions were studied by the viscometricmethod (52 strains) or by the qualitative method(87 strains). To avoid excessive tabulation, the87 strains were subdivided (groups A, B, C, D) onthe basis of four reactions: Production of nitritefrom nitrate, utilization of ammonium phosphateas the sole nitrogen source, capacity to coagulateoxalated plasma, and DNase activity.As shown in table 2, the coagulase positive

strains (group A) comprised a relatively homo-geneous group having golden to cream coloredcolonies, capacity to ferment mannitol, and allbut 4 strains liquefied gelatin within 2 weeks ofincubation. Qualitative DNase tests showedstrong activity for all 34 strains. Further testsof 20 strains showed the following levels ofactivity, in terms of viscometric units/ml: Two

Figure 1. Results of tests for deoxyribonucleaseactivity and number of colony forming cells/mlon samples of brain heart infusion cultures ofthree coagulase positive strains of micrococci.Curves plotted from data of two experiments withstrains SA-B (open and filled circles) and J707(open and filled squares) and one with strainJ824 (open triangles).

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TABLE 2Physiological reactions of 87 strains of micrococci recently isolated from clinical material

Nitrate NH4H2PO4 Coagulase Ca++ DNase Gelatin Mannitol ColonialReduction Utiliz- Reaction (Qualitative)t Lique- Fermen- PigmentationtGroup ation faction tation

Pos. Neg. Pos. Neg. Pos. Neg. I II III IV Pos. Neg. Pos. Neg. G C W X Y Z

A 34* 34 34 34 30 4 34 29 5

5 5 5 5 32 23 1 1 1 221 21 21 21 10 11 9 12 1 10 9 113 13 13 13 1 12 13 4 1 8

o 4 4 4 4 3 1 1 3 3 1

D 1 1 1 1 1 1 14 4 4 4 1 3 1 3 1 2 14 4 4 4 4 2 2 1 1 1 1

* Arabic numerals show number of strains grouped in the category.t I, positive within 15 min incubation; II, positive after 2, 3, or 4 hr (negative for first 1/ hr);

III, positive after 20 hr (negative for first 5 hr); IV, negative after 20 hr incubation.$ G, golden; C, cream colored; W, white; X, translucent, grayish; Y, yellow; Z, opaque, white colonies

during first 2 days, becoming golden yellow.

strains about 200 (172, 226); fifteen strainsranged from 1,300 to 7,000; two strains in thevicinity of 12,000 (11,500 and 14,100); onestrain (SA-B) above 50,000.Of the group B coagulase negative strains,

only one (strain Sll) showed a fast positivequalitative DNase test; viscometric tests of twoindependent cultures (tested after 48 and 65 hrincubation) showed 76 and 102 u/ml activity,respectively. Gelatin liquefaction and mannitolfermentation suggested that this strain, whichwas isolated from a lung at autopsy, was amember of M. pyogenes var. aureus (Staphylococ-cus aureus) that had lost its capacity to coagulateplasma. Of the remaining 39 strains in group B,5 showed intermediate DNase activity (visco-metric tests of additional cultures showed levelsranging from 3-12 u/ml) and 34 showed slight ornegative activity (categories III and IV).

In evaluating the results of the qualitativeDNase test it should be realized that DNA thathas been partially depolymerized by DNaseaction (revealed by reduction in viscosity) maystill form a web when dropped in acetone. Disap-pearance of web formation by DNA, like failureof gelatin to solidify when chilled, apparentlyrequires the cleavage of a large proportion of themolecules. Thus, a so-called negative result withthe qualitative test does not necessarily imply

that the culture lacked detectable DNase. Thiswas found with further cultures of group B strainstested by the viscometric method. Eight strainswere selected at random from DNase categoryIII and 7 from category IV. (The remaining 6strains in category IV were not investigatedfurther, but probably were not different fromthe others.) In all cases some DNase activitywas detected, levels ranging from less than 1 toabout 4 viscometric u/ml.Although differences between DNase categories

III and IV were not regarded as significant,results were separately tabulated because of thepossibly significant differences in gelatin andmannitol reactions. Only one of the 13 DNaseIV strains of group B liquefied gelatin (duringthe second week), and none fermented mannitol.However, among the 21 DNase category IIIstrains, only 7 were negative in both gelatin andmannitol; 4 were gelatin negative and mannitolpositive, 5 were gelatin positive and mannitolnegative, 5 were positive in both gelatin andmannitol. As frequently found (Greer, 1956),some coagulase negative strains exhibited acombination of characteristics that did notcorrespond exactly with any description in thesixth edition of Bergey's Manual (Hucker, 1948).Even disregarding differences in colonial pig-

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mentation (Cowan, 1956), all strains could notbe named with certainty.The 4 strains in group C were obtained from

urine specimens. Three were tentatively identifiedas Micrococcus conglomeratubs and one as Micro-coccus varians. Cultures of all 4 showed DNaseactivity ranging from 1.5-2.7 u/ml.The strains of group D most closely resembled

descriptions of Micrococcus flavus (2 strains) andMicrococcus candidus (7 strains). One gave apositive qualitative DNase test after incubationfor 3 hr; a viscometric test revealed 17 u/mlactivity. Cultures of the 8 other strains hadsignificantly lower DNase activity; viscometrictests showed activity in all, ranging from a traceto 2 u/ml.

Several of these strains of micrococci wereselected for further study of DNase activity,specifically directed toward two questions: (1)does a DNase inhibitor contribute to differencesin DNase activity between aerated and non-aerated cultures of coagulase positive strains onthe one hand, or, on the other, between aeratedcultures of the coagulase positive and most of thecoagulase negative strains; (2) do various kindsof micrococci produce the same kind of DNase orcan micrococci be subdivided on the basis ofdifferences in the characteristics of DNasesproduced by the cells? The finding (Cunninghamet al., 1956) that whole broth cultures or thesupernatant fluid of centrifuged cultures couldbe boiled for 15 min or longer without undue lossof DNase activity provided a technique fordetecting a possible heat-labile DNase inhibitor(Zamenhof and Chargaff, 1949) or, alternately,for revealing the presence of a heat-labile typeof DNase. A second technique for seeking evi-dence of a DNase inhibitor was to carry outviscometric tests using a culture having lowDNase activity, dilutions of a culture having highactivity, and a mixture of the two. If the lowactivity was due to the influence of a DNaseinhibitor, the activity of the mixture should belower than the total activity of the two culturestested separately. Evidence that more than onekind of DNase was present in cultures of themicrococci was sought by investigating not onlyheat stability, but also cation requirement andpH optimum for DNase activity of wholecultures, supernatant fluid, or of Mickle disinte-grated cellular material sampled after variousperiods of culture incubation.

Brain heart infusion cultures, incubated with

aeration for 53 hr, of one coagulase positive andthree coagulase negative group B strains (S11,the strain in DNase category I; S4 and G301,representatives of categories II and III) weretested by the qualitative method for DNaseactivity in the presence of various added cations,using two different buffers: tris (hydroxymethyl)aminomethane hydrochloride, pH 7.5 and suc-cinate, pH 5.5. The cations, listed in their ap-proximate order of activities, were: CaCl2, (finalconcentration 10-2 M); K2Cr2O7, (2.5 X 10-5 M);FeSO4, (5 X 10-4 M); CuSO4, (5 X 10-4 M);Co(C2H302)2, (2.5 X 104 M); MgCI2, (1.25 X10-2 M); Ni(NO3)2, (2.5 X 10-3 m); SrNO3,(2.5 X 10-8 M). In addition, dichromate andmagnesium were used in viscometric tests with anumber of cultures, including a representative ofgroup D. In none of these tests did activity exceedthat obtained using calcium at pH 8.5. Thatsome DNase activity was found using othercations was not surprising in view of the factthat traces of culture medium were present; also,it has been shown repeatedly that more than onecation may function as DNase activator (Kunitz,1950b).

Results with a 96-hr brain heart infusionshaken culture of S4 were illustrative of mixingexperiments carried out with 4 different strains.DNase activity was determined viscometricallyin the presence of Ca++ at pH 8.5. A 1:2 dilutionin gelatin of S4 showed 4.7 u (9.4 u/ml totalactivity when multiplied by the dilution factor).A 1:10,000 dilution in gelatin of a 28-hr cultureof strain SA-B showed 2.4 u (total activity24,000 u/ml culture). Equal volumes of undilutedS4 and 1:5,000 dilution of SA-B showed 7.7 u.Thus, no evidence of a DNase inhibitor wasobtained. Tests of the effect of boiling undilutedcultures for 15 min likewise failed to reveal thepresence of either heat-labile DNase or inhibitor.

DISCUSSION

Micrococci recently isolated from clinicalmaterial were found to produce a calciumactivated, heat stable DNase. Coagulase positivestrains evidenced very much higher DNaseactivities than did coagulase negative strains,with one exception. This one strain, wvhich hadbeen designated as a coagulase negative S.aureus in the hospital laboratory where it was

isolated, exhibited fairly high DNase activity.Capacity of micrococci to coagulate oxalated

or citrated blood plasma is generally regarded as

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a more reliable criterion of potential pathogenicitythan other physiological reactions commonlyexamined, such as fermentation of mannitol,liquefaction of gelatin, hemolysis of erythrocytes,or production of golden pigment. In view of thepossibility that capacity to coagulate plasma,like many other characteristics of bacterialpopulations, may be subject to loss by mutation,the close correlation observed between coagulaseand DNase activities is of practical interest. Asdetermined either by a simple qualitative test orby a more intricate but quantitative viscometricmethod, strong activity of this kind of DNaseappears to be a useful characteristic for distin-guishing strains that are potentially pathogenic(S. aureus) from those that are less commonlypathogenic. Phosphatase also has been foundto be closely correlated with the coagulasereaction (Barber and Kuper, 1951).

In addition to the use of high DNase activityas a determinative characteristic, the type ofDNase produced by cells may be a useful taxo-nomic characteristic. The calcium activated,high optimum pH, heat stable type has not beenreported in other biological materials, althougha wider distribution may be found if a search isundertaken with the objective of identifying anddistinguishing between various DNases. Studyof bacterial DNases may elucidate naturalrelationships. Among the various mass forminggram positive cocci, taxonomic relations havenot yet been clearly established. As requested bythe International Judicial Commission onBacteriological Nomenclature (Editorial Board,1954), a number of proposals have been maderecently concerning the advisability of separatingthe genera Micrococcus and Staphylococcus (forexample, Van Eseltine, 1955; Evans et al., 1955;Breed, 1956). Although DNase activity offers anatural basis for separating coagulase positivefrom coagulase negative micrococci, the occur-rence of "micrococcal type" DNase in cultures ofvarious members of the micrococcus-staphylo-coccus group suggests close evolutionary rela-tions, and does not appear to indicate a naturalbasis for subdividing the coagulase negativemembers.

ACKNOWLEDGMENTS

We are indebted to Dr. James E. Greer,Division of Dermatology, Henry Ford Hospital,Detroit, for generously providing 22 strains of

micrococci and to Miss Grace Jessel, MilwaukeeCounty Hospital, for 27 strains. Thymus DNAwas provided by Dr. Lew Cunningham, Mar-quette University School of Medicine, to whomwe are grateful also for helpful advice.

SUMMARY

Cultures of Micrococcus pyogenes var. aureus(Staphylococcus aureus) yield a deoxyribonucleasethat differs from other described DNases inseveral properties, including pH optimum (8.6),cation activator (calcium), and heat stability.The effect of certain culture conditions on DNaseactivity was studied. High DNase activity wasfound in brain heart infusion cultures incubatedwith continuous shaking at room temperaturefor 40-72 hr.These conditions were employed in investigat-

ing DNase activity of 87 strains of micrococcirecently isolated from clinical material. Eachstrain was examined also for coagulase, nitratereduction, utilization of ammonium phosphate assole source of nitrogen, gelatin liquefaction,mannitol fermentation, and colonial pigmenta-tion. The 34 coagulase positive strains of thegroup all exhibited high DNase activity. Signif-icantly lower DNase activities were produced byall coagulase negative strains except one; thisstrain exhibited the characteristics of S. aureusexcept for the coagulase reaction. Thus, highDNase activity may be a useful determinativecharacteristic, supplementing coagulase and phos-phatase reactions, in identifying potentiallypathogenic micrococci. In addition, the possibilityis discussed that the kind of DNase produced bycells may be a taxonomic characteristic useful instudies of natural relations among bacteria.

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fication of Staphylococcus pyogenes by thephosphatase reaction. J. Pathol. Bacteriol.,63, 65-68.

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BREED, R. S. 1956 Staphylococcus pyogenesRosenbach. Internat. Bull. Bacteriol. No-menclature and Taxonomy, 6, 35-42.

CATLIN, B. W. 1956 Extracellular deoxyribo-nucleic acid of bacteria and a deoxyribonu-clease inhibitor. Science, 124, 441-442.

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FINN, R. K. 1954 Agitation-aeration in the lab-oratory and in industry. Bacteriol. Revs.,18, 254-274.

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HUCKER, G. J. 1948 In Bergey's Manual of De-terminative Bacteriology, 6th ed. p. 235. Editedby Breed, R. S., Murray, E. G. D., andHitchens, A. P. The Williams & Wilkins Co.,Baltimore.

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A. L. 1952 An improved preparation ofsodium desoxyribonucleate. J. Am. Chem.Soc., 74, 1724-1726.

KOZLOFF, L. M. 1953 Origin and fate of bac-teriophage material. Cold Spring HarborSymposia Quant. Biol., 18, 209-220.

KUNITZ, M. 1950a Crystalline desoxyribonu-clease. I. Isolation and general properties.J. Gen. Physiol., 33, 349-362.

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WARRACK, G. H., BIDWELL, E., AND OAKLEY, C.L. 1951 The beta-toxin (deoxyribonuclease)of Cl. septicum. J. Pathol. Bacteriol., 63,293-302.

ZAMENHOF, S. AND CHARGAFF, E. 1949 Studieson the desoxypentose nuclease of yeast andits specific cellular regulation. J. Biol.Chem., 180, 727-740.

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