Department Pennsylvania - Journal of Bacteriologyjb.asm.org/content/62/4/395.full.pdfDepartment ofMicrobiology, ... Total pigment wasmeasured as cyanmethemoglobin, ... Standard sacks

STUDIES OF THE ACTION OF STREPTOCOCCI ON BLOOD WITHTHEIR APPLICATION TO METABOLISM AND

VARIATION OF STREPTOCOCCI

WARREN R. STINEBRING AND HARRY E. MORTON

Department of Microbiology, University of PennsylvaniaSchool of Medicine, Philadelphia, Pennsylvania

Received for publication May 28, 1951

During investigations of hemolysis produced by streptococci, we observedthat the substance or substances responsible for the greening reaction in alphahemolysis were capable of passing through a dialysis membrane which couldhold back the specific protein hemolysins produced by Group A beta hemolyticstreptococci. Utilizing this method of separation we demonstrated that evenGroup A beta hemolytic streptococci could produce the greening reaction. Thisdiscovery is of value both in the study of the mechanism of alpha hemolysisand of alpha hemolytic variants from Group A beta hemolytic streptococci.Many papers have been published concerning variation in the hemolytic

character of streptococci. Brown (1919) in his monumental work on the classifi-cation of the streptococci cites many references dealing with this problem. Someof the more recent papers are those of Grinnell (1928) who observed variantsgiving the greening reaction produced from single cell cultures of beta hemolyticstreptococci; Todd (1928) who was able to isolate green-producing variantsafter mouse passage of beta hemolytic streptococci; and Fry (1933) who ob-served streptococci which gave the greening reaction upon aerobic incubationbut produced beta hemolysis upon anaerobic incubation. Later Coburn andPauli (1941) followed an epidemic caused by an unusual Group A type 12 strep-tococcus which frequently produced nonhemolytic and green-producing vari-ants. Colebrook et al. (1942) described another streptococcus serologicallyidentified as Group A type 12 which was capable of producing greening on horseblood agar. Isaacs (1947) has studied the conditions which appear to be mostfavorable for the selection of alpha hemolytic variants and has described hismethod in detail. From the preceding references one can only conclude that thistype of variation does occur among the streptococci and that it is not an in-frequent phenomenon.

Herbert and Todd (1944) showed that in the case of Group A beta hemolyticstreptococci a protein material designated by them as streptolysin S was re-sponsible for beta hemolysis on the surface of blood agar while in some casesanother distinctly separate serological entity, streptolysin 0, also protein innature, caused beta hemolysis surrounding deep colonies in blood agar. Usewas made of the fact that the hemolysins of beta hemolytic streptococci ofGroup A are protein in nature in setting up the following experiments to demon-strate that beta hemolytic streptococci and alpha hemolytic streptococci, aswell as pneumococci, can produce the same reaction on blood agar.

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Since the substance causing greening of blood agar was able to pass throughthe dialysis membrane, it was also decided to determine whether or not thesubstance or substances responsible for the conversion of oxyhemoglobin tomethemoglobin could do likewise. This was of theoretical interest in relatingthe greening reaction to methemoglobin formation. The production of methemo-globin from oxyhemoglobin by streptococci has been studied in great detail.Griuter (1909) studied the production of methemoglobin spectroscopically asdid Butterfield and Peabody (1913). The works of primary importance alongthese lines are those of Avery and Morgan (1924), Avery and Neill (1924a,b),Neill and Avery (1924), Morgan and Neill (1924), and Neil (1925a,b,c,d). Theseauthors established that in the case of pneumococci an intracellular, heat labilecomponent plus a substance supplied by infusion broth or yeast extract wasneeded to produce the change of oxyhemoglobin to methemoglobin with cellfree extracts. The preparation of the cell free extracts and conditions necesaryfor the reaction are discussed in the papers previously mentioned. It was alsoshown that the substance directly responsible for the reaction was unaffectedby blood catalase and was probably a peroxide of some type other than hydrogenperoxide.

EXPERIMENTAL METHODS AND RESULTS

Culture medium. The culture medium used was extract blood agar which hadthe following composition: beef extract, &.0 g; peptone (Parke-Davis Com-pany), 10.0 g; agar, 20.0 g; NaCI, 5.0 g; and distilled water, 1 liter. The reactionwas approximately pH 7.2. To this was added enough sterile defibrinated normalhorse blood to give a final concentration of 10 per cent and, where indicated,enough glucose to give a 1 per cent concentration. The blood and glucose wereadded after the melted agar had cooled to about 45 C.

Cellophane membrane preparation. Eight ml of melted extract blood agar werespread over the bottom of a standard 90 mm petri dish. This was allowed tosolidify, and a round piece of sterile cellophane about the size of the petri dishwas placed on top of the layer of blood agar. The edges of the cellophane wereringed with a small quantity of melted blood agar. The inoculum was then spreadon the cellophane membrane with a platinum loop, and 10 ml of melted bloodwere poured over the membrane.

Cellophane sack preparation. A relatively pure hemoglobin solution was pre-pared as follows: normal, sterile, defibrinated horse blood was centrifuged andthe red blood cells washed three times in saline. The cells were then lysed witha volume of distilled water equal to the volume of the serum removed andcentrifuged again to remove debris. This preparation, if stored at all, was storedat 10 C in an atmosphere of 90 per cent nitrogen and 10 per cent carbon dioxide.A cellophane sack was prepared as follows and filled with 5 ml of a 1:10 dilu-

tion of the above hemoglobin solution. The sacks were made by cutting suitablelengths of "nojax" dialysis casings ("visking" cellulose sausage casings 36/32).One end was folded over three or four times and sealed by means of a smallmetal clip. These clips are those used in sealing ordinary collapsible tubes as

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ACTION OP STREPTOCOCCI ON BLOOD

used for tooth paste and ointment. The sacks were then sterilized by autoclav-ing. The hemoglobin solution was then added, and the other end of the sackwas sealed with a sterile clip as previously described.

Eight ml of melted extract blood agar containing 1 per cent glucose were addedto the bottom of a petri dish and allowed to solidify. The surface was then inocu-lated with the organism to be studied and the cellophane sack was then placedupon the inoculated area. The upper surface of the cellophane sack was inocu-lated with organisms, and another layer of blood agar plus glucose was layeredover the top. Thus, the cellophane sack was imbedded in the agar which con-tained the organisms growing in a state simulating pour plate conditions. Aftervarious periods of time the contents of the sacks were collected, stained for thepresence of organisms, and examined spectrophotometrically in the BeckmanModel DU spectrophotometer. In no case were organisms found growing in aproperly prepared sack.

Spectrophotometric studies. Spectrophotometric studies were made as follows:The contents of the sacks were diluted 1:10 in M/150 phosphate buffer pH 6.7.This material was then centrifuged in an angle centrifuge to throw down anyprecipitate. Total pigment was measured as cyanmethemoglobin, and then themillimolecular extinction coefficients of the mixture were determined at wavelengths of 630, 578, 562, 542 myA as recommended for mixtures of methemo-globin and oxyhemoglobin by Drabkin (1950). By use of the method of Austinand Drabkin (1935), the per cent methemoglobin for each mixture was obtained.The identification of the pigment, resulting from the action of the organisms

upon the solution of oxyhemoglobin, as methemoglobin was accomplished inthe following manner. Standard sacks were prepared using each of the threeorganisms and an uninoculated preparation as a control. These preparationswere incubated for fifteen hours at 37 C. The contents of the sacks were thencollected, diluted in M/15 phosphate buffer pH 6.5, then examined spectro-scopically in the General Electric recording spectrophotometer.1 The pigmentfrom the inoculated preparations was examined first. Since the absorption pat-tern was that of acid methemoglobin, potassium cyanide was then added toeach of the preparations and examined for the typical absorption spectrum ofcyanmethemoglobin. Sodium dithionite was then added to a second portionof each sample and the material examined for the. absorption spectrum of reduced(deoxygenated) hemoglobin. Material from the uninoculated preparation wasexamined last for a control on the amount of methemoglobin produced spon-taneously in the preparations.

Cultures. The cultures used were Diplococcus pneumoniae, type 1 (strainP27), Streptococcus pyogenes, Group A, strain C203M, (P221A), and Strepto-coccus, sp., viridans group (strain G). The last-named organism was a typicalalpha hemolytic streptococcus of the viridans group isolated from a case of

1 The authors are greatly indebted to Dr. D. L. Drabkin, Department of PhysiologicalChemistry, Graduate School of Medicine, University of Pennsylvania, for making thesedeterminations with the General Electric recording spectrophotometer and interpretingthe results, as well as for valuable assistance given during the course of the investigation.

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398 WARREN R. STINEBRING AND HARRY E. MORTON [VOL. 62

subacute bacterial endocarditis and obtained from Dr. E. H. Spalding, TempleUniversity, Philadelphia. All cultures used were approximately 18 to 24 hoursold and had been transferred several times from the stock culture. Blood brothcultures kept at 4 to 8 C were used as stock cultures. Incubation of all testcultures and preparations was conducted at 37 C.

0.7CONTROL0.5_>0

650 600 500 450

0.7 D. PNEUMONIAE

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650 600 500 450

0.7 -

< a STREPTOCOCCUS

2o.5O [650 600 500 450

0.7S. PYOGENES0.5-

650 600 500 450WAVELENGTH IN MILLIMICRONS

Figure 1. Absorption spectra identifying the pigment which is produced inside of thecellophane sacks, spontaneously and by the action of the cultures studied, as methemo-globin.

pigment before treating with reagents.- --- pigment after the addition of potassium cyanide...... ..pigment after the addition of sodium dithionite.

RESULTS

The pigment produced within the cellophane sacks in the case of the culturesstudied was found to be acid methemoglobin. After the addition of potassiumcyanide the absorption spectrum of cyanmethemoglobin was obtained. Aftertreatment of the material from the sacks with sodium dithionite the absorptionspectrum of reduced (deoxygenated) hemoglobin was obtained. The controlpreparations showed the same reactions, but, of course, the total amount ofmethemoglobin produced was rather small. Figure 1 shows the results obtainedwith the four preparations. As may be seen, these data prove that the pigmentproduced in the sacks is methemoglobin.

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1951] ACTION OF STREPTOCOCCI ON BLOOD 399

Table 1 gives the results of an experiment using the cellophane membranepreparations. The preparations were incubated 24 hours. Although the greeningproduced below the membrane in the case of the beta hemolytic streptococcus(P221A) with no glucose was slight, it was definite. As has been known for sometime, Ruediger (1906), Davis (1917), Brown (1919), and many others, the addi-

TABLE 1The results obtained with Streptococcus pyogenes, Diplococcus pneumoniae, type 1, and a

streptococcus of the viridans type employing cellophane membrane preparations

CULTURESLOCATION OF

MEDIUM EXAMINED . .S. fyogenes D. pmumonac VrCdans

No glucose in medium Above mem- Lysis complete. No Greening Greeningbrane greening observed.

Below mem- No lysis. Slight Greening Greeningbrane amount of greening.

Glucose in medium Above mem- Lysis present, but in- Greening Greeningbrane hibited. Greening.

Below mem- No lysis. Greening. Greening Greeningbrane

100 £

am soccSTREPTOCOCCUS~8 / D. PNEUMONIAEE 60 \S.PYOGENESw

w 40 I

20- CULTURE MEDIUM

5 10 15 20 25 30

HOURS OF INCUBATIONFigure 2. Graphs obtained by plotting against time the per cent methemoglobin pro-

duced by Diplococcus pneumoniae, Streptococcus pyogenes, and an alpha hemolytic strepto-coccus using cellophane sack technic.

tion of glucose to the medium suppresses lysis in the case of the beta hemolyticstreptococcus but increases greening in all the organisms studied. The cellophanemembrane technic demonstrates the fact that even without glucose in the me-dium and with conditions optimal for the production of a specific protein hemoly-sin, beta hemolytic streptococci may produce a greening effect on blood agar.The results obtained with the cellophane sack technic are shown in figure 2.

The organism in this case was the viridans streptococcus described earlier. As

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WARREN R. STINEBRING ANI' HARRY E. MORTON

may be seen from the graph, if one plots per cent methemoglobin in the cello-phane sack against time, a sigmoid type of curve results. The control shown wasprepared by setting up a cellophane sack preparation in the manner describedbut omitting the organisms. The control showed a significant conversion ofoxyhemoglobin to methemoglobin, but this conversion took place at a constantrate. During the period of incubation of from two to nine hours, the eonversionof oxyhemoglobin to methemoglobin took place at a logarithmic rate as shownby the plot of the log per cent of methemoglobin against time for this period(figure 3). Similar types of curves were obtained for Diplococcus pneumoniae,

100

o STREPTOCOCCUS60J-

D. PNEUMONIAE

30 _

2

0 3 3

0-j002 lo0

HOURS OF INCUBATION

Figure S. Graphs obtained by plotting the log per cent of methemoglobin against timeon semilog scale. These show that Diplococcus pneumoniae, Streptococcus pyogenes, and aviridans streptococcus have identical rates of conversion of oxyhemoglobin to methemo-globin when using the cellophane sack technic.

type 1, and Streptococcus pyogenes, C203M. The rate of conversion during thelogarithmic period was the same for all three organisms. However, the.-betahemolytic streptococcus showed a lag of about t-wo hours in obtai'ni'ng the maxi-mum rate of conversion as compared to the other organisms.

DI[CUSSION

The results of the experiments with the cellophane membrane technic pointout a possible explanation of the mutation from beta type of hemolysis to thealpha hemolytic or greening type of reaction in streptococci. That certain fac-tors in the medium can cause beta hemolytic streptococci to produce a greeinmgreaction on blood agar has been demonstrated.by Fuller and Maxted (1939).Thus, one is led to believe that under certain conditions beta hemolysis is irnter-

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fered with and a substance or substances capable of producing the greening re-action diffuse into the medium surrounding the colonies of streptococci. However,by use of the methods of Fuller and Maxted (1939), one could demonstrateonly one type of reaction to the exclusion of the other. The cellophane membranetechnic now shows that it is possible for beta hemolytic streptococci, underconditions favoring the production of protein hemolysins and in an undissociatedstate, to produce a greening reaction on blood agar. Thus, if a mutation hadoccurred which could interfere with the production of either streptolysin S orstreptolysin 0, then the organism might produce a characteristic greening re-action on blood agar. Furthermore, any ingredient of the medium which mighttend to inhibit production of the protein hemolysins would thus favor the ap-pearance of a greening reaction. This is especially true of a substance like glucosewhich not only tends to inhibit beta hemolysis but actually tends to increasegreening as well. Thus, one may see that in each individual case of the productionof green-producing variants from beta hemolytic streptococci many factors maybe involved and must be taken into account.The relationship of the greening reaction to the production of methemoglobin

is not definitely proved by these studies. However, the experiments with thecellophane membrane technic and the cellophane sack technic suggest that thesame substances or substance may be responsible for both reactions. The con-cept that greening and methemoglobin formation are the same process has beenboth accepted and denied by many authors (Cole, 1914; Rother, 1925; Bar-nard and Gowen, 1932; and others). Neill and Avery (1924) showed that sterilereduced extracts of the pneumococcus caused a change in blood agar similarto that caused by living pneumococci. These same extracts were those studiedwhich produced methemoglobin from oxyhemoglobin and were capable of form-ing H202 if exposed to the air under certain conditions. These authors also statedthat further oxidation products of methemoglobin were formed from the ex-tracts. Hart and Anderson (1933) and Anderson and Hart (1934) were able todemonstrate under certain conditions the formation of a green pigment whichthey believed to be the cause of the greenish discoloration produced by pneumo-cocci and certain streptococci on blood agar. They believed this substance tobe a product of oxidation-reduction reactions of hemoglobin. One need not takethe view that methemoglobin formation is unrelated to the greening reactionsince the foregoing studies and our results suggest that the same system may beinvolved in both methemoglobin formation and in the production of greeningon blood agar. Of interest is the fact that in the cellophane sack technic described,one is able to demonstrate the production of an insoluble precipitate in the sackafter the conversion of the oxyhemoglobin to methemoglobin. This problem isunder further study and may give some insight into the events after productionof methemoglobin.The cellophane sack technic also allows the demonstration of the fact that

the conversion of oxyhemoglobin to methemoglobin under normal conditionsof growth follows a sigmoid type curve. This further substantiates earlier im-pressions (Morton and Stinebring, 1950) that the substance or substances re-

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sponsible for the change are produced in greatest amount during the period ofmaximum metabolic activity of the organisms. Of further interest is the findingthat the rate of conversion of oxyhemoglobin to methemoglobin is the same forall organisms studied during the period of maximum conversion.

SUMMARY

New technics for the study of the action of streptococci and pneumococci onhemoglobin are described. The principle of the technics is maintaining theseparation of microorganisms and hemoglobin at all times by means of a cello-phane membrane.By use of the new technics it was possible to demonstrate that the substance

or substances responsible for the greening of blood agar and for the productionof methemoglobin from oxyhemoglobin, as exhibited by a streptococci and pneu-mococci, are able to pass through a cellophane membrane which is capable ofholding back the protein hemolysins produced by certain beta hemolytic strep-tococci. By maintaining a solution of hemoglobin within a cellophane sack duringthe growth of cultures, it is possible to study the quantitative conversion ofoxyhemoglobin to methemoglobin. From such quantitative data it has beenshown that representative strains of Streptococcus pyogenes, Diplococcu.s pneu-moniae, and a viridans streptococcus act in much the same way on solutions ofoxyhemoglobin.Evidence is presented which shows that the substance or substances responsi-

ble for the conversion of oxyhemoglobin to methemoglobin are similar in actionfor the three above-mentioned strains under the conditions studied. Thesesubstances appear to be produced during the period of greatest metabolic ac-tivity of the cultures.

REFERENCES

ANDERSON, A. B., AND HART, P. D. 1934 Viridans effect of the streptococci and theproduction of the green pigment from haemoglobin by other reducing systems. J.Path. Bact., 39, 465-479.

AUSTIN, J. H., AND DRABKIN, D. L. 1935 Spectrophotometric studies. III. Methemo-globin. J. Biol. Chem., 112, 67-88.

AVERY, 0. T., AND MORGAN, H. J. 1924 The occurrence of peroxide in cultures of pneu-mococcus. J. Exptl. Med., 39, 275-287.

AVERY, 0. T., AND NEILL, J. M. 1924a Studies on oxidation and reduction by pneumo-coccus. I. Production of peroxide by anaerobic cultures of pneumococcus on exposureto air under conditions not permitting active growth. J. Exptl. Med., 39, 347-355.

AVERY, 0. T., AND NEILL, J. M. 1924b Studies on oxidation-reduction by pneumococcus.II. The production of peroxide by sterile extracts of pneumococcus. J. Exptl. Med.,39, 357-366.

BARNARD, R. D., AND GOWEN, G. H. 1932 The greenish discoloration produced on bloodagar by the growth of the pneumococcus. Proc. Soc. Exptl. Biol. Med., 29, 521-524.

BROWN, J. H. 1919 The use of blood agar for the study of streptococci. MonographNo. 9 of the Rockefeller Institute for Medical Research.

BUTTERFIELD, E. E., AND PEABODY, F. W. 1913 The action of pneumococcus on blood.J. Exptl. Med., 17, 587-592.

COBURN, A. F., AND PAULI, R. H. 1941 The interaction of host and bacterium in the de-velopment of communicability by Streptococcus haemolyticus. J. Exptl. Med., 73, 551-570.

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COLE, R. I. 1914 The production of methemoglobin by pneumococci. J. Exptl. Med.,20, 363-378.

COLEBROOK, L., ELLIOT, S. D., MAXTED, W. R., MORLEY, C. W., AND MORTELL, M. 1942Infection by non-hemolytic group A streptococci. Lancet, II, 30-31.

DAVIS, D. J. 1917 Observations on the growth of streptococci in blood-carbohydratemedium. J. Infectious Diseases, 21, 308-313.

DRABKIN, D. L. 1950 Spectroscopy: Photometry and spectrophotometry. Medicalphysics. 0. Glasser, editor. Year Book Publishing, Inc., Chicago, Illinois.

FRY, R. N. 1933 Anaerobic methods for the identification of haemolytic streptococci.J. Path. Bact., 37, 337-340.

FULLER, A. T., AND MAXTED, W. R. 1939 The production of haemolysin and peroxideby haemolytic streptococci in relation to the non-haemolytic variants of Group A.J. Path. Bact., 49, 83-94.

GRINNELL, F. B. 1928 Streptococcus studies. I. Streptococcus viridans. J. Bact., 16,117-121.

GRUTER, W. 1909 Die Methiamoglobinbildung in bluthaltigen Nahrboden durch Strepto-kokken. Centralbl. f. Bakteriol. Abt. I, Orig., 60, 241-261.

HART, P. D., AND ANDERSON, A. B. 1933 Formation of green pigment from haemoglobinby the pneumococcus. J. Path. Bact., 37, 91-105.

HERBERT, D., AND TODD, E. W. 1944 The oxygen-stable haemolysin of group A haemo-lytic streptococci (streptolysin S). Brit. J. Exptl. Med., 25, 242-254.

ISAACS, A. 1947 The production of viridans variants of haemolytic streptococci. J.Path. Bact., 69, 487-489.

MORGAN, H. J., AND NEILL, J. M. 1924 Methemoglobin formation by sterile culturefiltrates of pneumococcus. J. Exptl. Med., 40, 269-279.

MORTON, H. E., AND STINEBRING, W. 1950 Some factors which influence the typicalalpha hemolysis of streptococci on blood agar. Bact. Proc., 1950, 85.

NEILL, J. M. 1925a Studies on oxidation-reduction of hemoglobin and methemoglobin.I. Changes induced by pneumococci and sterile animal tissues. J. Exptl. Med., 41,299-313.

NEILL, J. M. 1925b Studies on oxidation-reduction of hemoglobin and methemoglobin.II. The oxidation of hemoglobin and reduction of methemoglobin by anaerobic bacilliand by sterile plant tissue. J. Exptl. Med., 41, 535-549.

NEILL, J. M. 1925c Studies on oxidation-reduction of hemoglobin and methemoglobin.III. The formation of methemoglobin during oxidation of autoxidisable substances.J. Exptl. Med., 41, 551-559.

NEILL, J. M. 1925d Studies on oxidation-reduction of hemoglobin and methemoglobin.IV. The inhibition of "spontaneous" methemoglobin formation. J. Exptl. Med., 41,561-570.

NEILL, J. M., AND AVERY, 0. T. 1924 Studies on oxidation and reduction by pneumo-coccus. V. The destruction of oxyhemoglobin by sterile extracts of pneumococcus.J. Exptl. Med., 39, 757-775.

ROTHER, W. 1925 Ueber die Vergriunung des Blutagars durch Streptokokken. Deut.med. Wochschr., 51, 1031.

RUEDIGER, G. 1906 The cause of green coloration of bacterial colonies in blood agarplates. J. Infectious Diseases, 3, 663-665.

TODD, E. W. 1928 The conversion of hemolytic streptococci to non-hemolytic forms.J. Exptl. Med., 48, 493-511.

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