TYPES OF BACTERIA ON BLOOD AND CHOCOLATEAGAR AND THE IMMEDIATE CAUSE OF THESE

TYPES

EINAR LEIFSONDepartment of Pathology and Bacteriology, Johns Hopkins University, Baltimore,

Maryland'

Received for publication March 30, 1932

The action of certain bacteria on blood agar is so striking andconstant a phenomenon that it has come to be of considerableimportance in diagnosis. This is especially true in the case ofthe cocci. The effect on blood agar of bacteria other than thecocci has been described from time to time but no systematicstudy of a large number of types seems to have been made. Paul-son and Brown (1931) have recently described colonies of colonbacilli of the alpha, beta, and gamma types.The present paper deals with the effect on blood agar of some

260 strains of Gram-negative bacilli and 14 selected strains ofvarious types of streptococci and pneumococci. The probablecause of the green coloration produced by some of these bacteriahas been determined, and the various factors which influence theproduction of the green coloration have been investigated. Astudy has also been made of the effect on chocolate agar of bothbacilli and cocci. It must be emphasized at this time that thetypes of bacterial colonies in blood agar about to be described areall deep colonies. Many deep colonies of the gamma (non-hemo-lytic) type may be considerably more hemolytic as surfacecolonies.The appearance of different types of colonies of streptococci in

blood agar has been described by a number of investigatorsand especially by Brown (1919) in whose paper wrln be found a

I The writer wishes to express his appreciation to Dr. J. Howard Brown formany helpful suggestions, and kindly criticism.

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review of the literature up to that time. The three distinct typesof deep colonies of streptococci in blood agar are now well known,as are also the designations alpha, beta, and gamma given bySmith and Brown (1915). A fourth type, delta, was described byBryant (1925), but the difference between this type as describedby Bryant, and the alpha type seems to have been too slight togain general recognition. Tunnicliff (1930) has found that somebeta streptococci will give a green coloration on chocolate agarwhile others will not. From the literature referred to and thedata which will be presented in this paper, we may now recognizetwo distinct kinds of bacteria which produce the alpha type ofcolony. The beta streptococci may also be divided into twotypes. A brief description of these types follows:

1. The gamma type produces no visible change of the cor-puscles of a blood agar plate. None of the strains studied pro-duced any visible effect on chocolate agar. This does not excludethe possibility that such strains exist, however. There are alsoa number of bacteria, both cocci and bacilli, which produce deepcolonies of the gamma type but whose surface colonies are hemo-lytic. Staphylococci of this type are often found.

2. The beta type produces clear zones of hemolysis around thedeep colonies in a blood agar plate. Occasionally bacteria arefound which produce deep colonies having beta-like zones of par-tial hemolysis. These have been designated as alpha primeby Smith and Brown (1915). They are perhaps either to be con-sidered as atypical beta types or atypical alpha. On chocolateagar some streptococci of the beta type produce a green colorationwhich according to McLeod and Gordon (1922) is due to the pro-duction of hydrogen peroxide. Others produce no visible effect.The former of these may be designated beta-H202 positive andthe latter beta-H202 negative. More exhaustive investigationis needed to show whether the production of hydrogen peroxideby some streptococci of the beta type is sufficiently constant andclear-cut to be of classificatory significance.

3. The alpha type generally produces a green coloration aroundthe deep colonies in a blood agar plate. In case of the bacilli ofthe alpha type the green coloration is very pronounced *nd the

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accompanying hemolysis rather slight. Streptococci and pneu-mococci of the alpha type often produce a barely perceptibleamount of green with considerable hemolysis. In many casesonly microscopic exami ation of the colonies will determinetheir type. For a detailed description of the microscopic appear-ance of the alpha type the reader is referred to the monograph byBrown (1919). All of the strains of alpha streptococci and pneu-

TABLE 1Types of bacterial colonie8 in blood and on chocolate agar

TYPE

Gamma.................Alpha-H2S positive......

Alpha-H202 positive.....

Beta-H202 negative.....

Beta-H202 positive......

BLOOD AGAR

No visible effectLarge green zone with

little if any hemolysis.Appearance due to theproduction of H2S, andsubsequent oxidation byair of the sulfhemoglobinformed

Green zones with partialhemolysis. Appearancedue to the formation ofhematin, and H202 whichreact to form the greencoloration

Large zones of completehemolysis. No green

Large zones of completehemolysis. No green

CHOCOLATE AGAR

No visible effectNo visible-effect

Green coloration. Due tointeraction of hema-tin and H202

No visible effect

Green coloration. Dueto interaction of hema-tin and H20OX

mococci studied produced a green coloration on chocolate agar.None of the alpha bacilli studied produced any green colorationon chocolate agar. Subsequent discussion will show that the twotypes of alpha bacteria, as determined by chocolate agar, differfundamentally physiologically as well as morphologically in allcases which we have studied. The alpha bacilli produce a greencoloration of blood agar by virtue of the hydrogen sulphide whichthey produce. The alpha cocci change hemoglobin to hematinand also produce hydrogen peroxide which reacts with hematin togive a green coloration. Rather than introduce a new name it

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was decided to refer to the one group of alpha bacteria as alpha-H2S positive, and to the other as alpha-H202 positive. The termalpha as here used is purely descriptive of the appearance of thedeep colonies in blood agar. In a report of this work given beforethe last meeting of the Society of American Bacteriologists theterm delta was used for what we now call alpha-H2S positive.

THE CAUSE OF THE GREEN COLORATION

The alpha-H202 positive typeThe bacteria which produce this type of colony are often re-

ferred to as methemoglobin producers. This designation hascome about perhaps from the work of Cole (1914) and Blake(1916) who showed that some green-producing streptococci andpneumococci generally produce methemoglobin when blood isadded to a broth culture. Lyall (1914) found that some green-producing streptococci produce little if any methemoglobin whileothers produce considerable. Our own observations confirmthose of Lyall. The green-producing bacilli which we have desig-nated as alpha-H2S positive do not seem to produce demonstrableamounts of methemoglobin. From these observations it is clearthat there is no general correlation between the production ofmethemoglobin and a green coloration of blood agar. The sug-gestion is made that the term "methemoglobin producers" as re-ferring to green producing cocci should be discontinued. McLeodand Gordon (1922) found a perfect correlation between the pro-duction of hydrogen peroxide and a green coloration of heatedblood agar. These observations have been abundantly confirmed,especially by the exhaustive work of Avery and Neirl (1924), Neilland Avery (1924, 1925), and Neill (1925). Although such oxidiz-ing agents as sodium perborate will also produce a green colora-tion of heated blood agar, hydrogen peroxide is most likely re-sponsible for the green coloration produced by bacteria in heatedblood agar. The inference seems to have been made by some thathydrogen peroxide is responsible for the green coloration producedby bacteria in blood agar as well as in chocolate agar. Hydrogenperoxide, however, does not produce a green coloration of bloodagar and cannot, therefore, alone be the cause of the green colora-

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tion. Hagan (1925) suggested that the simultaneous productionof acid and of hydrogen peroxide might be the cause of the greencoloration. The amount of acid however, in combination withhydrogen peroxide, which is necessary to produce a green colora-tion is far in excess of that generally produced by these bacteriain blood agar. This explanation is therefore not very probable.Ruediger (1906) found that certain hemolytic streptococci wouldproduce a green coloration in blood agar if a fermentable carbo-hydrate (glucose) was added. He concluded that the greencoloration was due to the action of some acid on the erythrocytesof the blood. No such acid is known, however, and this explana-tion is not very probable.When blood is treated with acid, sodium nitrite, or heated to

70°C. or above, the hemoglobin is broken down and hematin isformed. Hematin will react with hydrogen peroxide to give agreen coloration but hemoglobin will not. Blood which has beentreated with potassium ferrocyanide does not react with hydro-gen peroxide to give a green coloration. This may be taken toshow that hydrogen peroxide does not react with methemoglobinto give a green coloration. Any bacteria, therefore, which pro-duce hydrogen peroxide will give a green coloration on blood agarwhich has been heated or treated with chemicals in such a waythat hematin is produced, but will not necessarily give a greencoloration on whole blood agar or on methemoglobin agar. Theexact mechanism of the production of hematin from hemoglobinis still somewhat obscure. For a detailed discussion of this sub-ject the reader is referred to the work of Avery and Neill. Ac-cording to these authors the hemoglobin is first changed tomethemoglobin by means of oxidizing enzymes. The methemo-globin is further changed by enzymes to form hematin. Fromthe work of Lyall (1914) and also from our own observations itseems that in some cases methemoglobin is not formed to anappreciable extent. This does not prove that methemoglobin isnot always an intermediary product in the change of hemoglobinto hematin, but rather that in some cases it is too transitory to bedetected. The change of hemoglobin to methemoglobin and hem-atin takes place only under aerobic conditions and consequently

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no green coloration is produced by the alpha cocci under anaero-bic conditions.The cause of the hemolytic zone around the colonies of alpha

cocci is somewhat obscure. In some ways it would seem to bedue to an agent distinct from that which causes the green colora-tion. Six strains of alpha streptococci and pneumococci, forexample, produced a distinct hemolysis but no green on a synthe-tic blood agar the composition of which is given later (see fig. 4,plate 1). On the other hand, 5 of the 6 cocci when grown understrictly anaerobic conditions produced no hemolysis and no green,while one produced both hemolysis and a green coloration. Thosealpha strains which are gamma under strictly anaerobic conditionsbecome somewhat hemolytic when the oxygen tension is slightlyincreased, but no green coloration is apparent until the oxygentension is increased still further. The beta type of hemolysisdoes not seem to be influenced by the oxygen tension. Fromthese data it seems that the production of hemolysis and of a greencoloration by alpha cocci are not independent phenomena, butthat the two are caused by the same agent. Hemolysis may bea manifestation of a slight injury to the erythrocytes and may bebrought about in a variety of ways. For example, if part of ablood plate is heated on a steam bath a wide zone of hemolysisappears between the zones of chocolate agar and of unchangedblood. Hemolysis may be brought about also by the action ofacids, hydrogen peroxide, etc. From these data it seems mostlikely that hemolysis produced by the alpha type of cocci is dueto very much the same substances which produce the greencoloration, and is associated with a slight injury of theerythrocytes.To summarize the course of the formation of the alpha-H202

positive type of colony: The oxyhemoglobin is first reduced tohemoglobin. In some way, as yet poorly understood, the hemo-globin is changed either first to methemoglobin and then to hema-tin and globin, or the hematin may be formed directly from thehemoglobin. The hematin reacts with the hydrogen peroxideformed by the bacteria to produce a green pigment. Both ofthese reactions take place only in the presence of air. Due, per-

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haps, to hydrogen peroxide or some other substance produced bythe bacterial metabolism, some of the erythrocytes are slightlyinjured and a zone of partial hemolysis is formed around thecolony.

The alpha-H2S positiVe typeThe alpha-H2S positive type has been distinguished from the

alpha-H202 positive type by its failure to produce a green colora-tion on chocolate agar, and by its large zone of green colorationon blood agar, with little or no hemolysis. All green-producingbacilli studied belong to the alpha-H2S positive type. The alphatype of bacilli produces no hydrogen peroxide, and neither met-hemoglobin nor hematin from hemoglobin. From the study ofsome 260 strains of Gram-negative bacilli a perfect correlation wasfound between the formation of a green coloration on blood agarand the production of hydrogen sulphide. Investigation of theeffect of hydrogen sulphide on blood showed that it reacts withhemoglobin to form purple sulfhemoglobin. Sulfhemoglobin hasa strong absorption band at about 620,u,. This absorption bandmay be distinguished from that of methemoglobin by the additionof ammonium hydroxide which causes the methemoglobin bandto disappear but leaves the sulfhemoglobin band intact. Carbonmonoxide will cause a shift of the sulfhemoglobin band to about610,$M but has no effect on the methemoglobin band. Sulfhemo-globin is easily oxidized either by air or by oxidizing agents suchas sodium perborate to form a green compound. Sulfhemoglobinis rather difficult to demonstrate in blood cultures of alpha bacilliunless substances such as cysteine or thiosulphates are present.Blood plates exposed aerobically to hydrogen sulphide turn greenimmediately and no sulfhemoglobin can be detected in the plates.Neither can any sulfhemoglobin be detected in the green zonesaround the colonies of alpha bacilli. The green compound doesnot seem to have a characteristic spectrum. One typical strainof an alpha colon bacillus grown on blood agar plates under anaer-obic conditions for forty-eight hours produced no green colora-tion. Exposure of these plates to air in the refrigerator did notlead to the production of a green coloration, but exposure at room

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temperature did. Compounds such as cysteine and sodium thio-sulphate greatly stimulate the production of a green coloration byalpha bacilli.Hydrogen sulphide does not produce a green coloration of choc-

olate agar, nor does it react with hematin to give a green colora-tion. Neither do alpha bacilli, in our experience, ever producea green coloration on chocolate agar.The effect of hydrogen sulphide on blood has been known for

some time. Harnach (1899) studied the effect of hydrogen sul-phide on blood and noted the development of a green colorationin the presence of air. V. d. Bergh (1905) found that the bloodof some of his enteritis patients showed a typical sulfhemoglobinspectrum. Addition of blood to bouillon cultures of the specificorganisms gave, after twenty fours of incubation, a typical sulf-hemoglobin spectrum. He concluded that the sulfhemoglobinin the blood of his patients was caused by hydrogen sulphideformed by the organsms in the intestine.The data presented above seem to the writer to be adequate

proof that the alpha type of colony, produced by the bacilli in-vestigated, results from the interaction of hydrogen sulphide andhemoglobin to form sulfhemoglobin. The sulfhemoglobin thusformed is quickly oxidized to form a green compound. Any hemol-ysis formed around this type of colony is probably due to somemetabolic product of the bacteria, perhaps hydrogen sulphide.

EFFECT OF THE PHYSICAL AND CHEMICAL ENVIRONMENT ON THE

PRODUCTION OF THE GREEN COLORATION

The effect of the physical and chemical environment on a bio-logical reaction does not seem to be fully appreciated. It is wellknown that chemical reactions are modified and at times com-pletely changed by changing the environment. This is even moretrue in the case of biological reactions. Not only must weconsider the effect of the environment upon the chemical reactantsand resultants, but also the effect of the environment upon thelving matter producing the chemicals. The environment isespecially important in cases where it forms, or may form, one

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of the reactive agents. Such is the case with bacterial oxidationsor reductions in which the atmospheric oxygen may play an im-portant r6le. The formation of a green coloration by alpha bac-teria in blood agar is partly an oxidation reaction and dependsfor its comipletion upon atmospheric oxygen.As stated above, the alpha-H2S positive type of colony results

from the formation of sulfhemoglobin and the subsequent oxida-tion of the sulfhemoglobin to a green compound. Any changeof environmental conditions affecting the production of hydrogensulphide would also affect the production of the green coloration.In a medium consisting of 0.2 per cent ammonium sulphate, 0.1per cent disodium phosphate, 0.5 per cent sodium chloride, 0.5per cent glucose, and 2 per cent agar the alpha bacilli examinedproduced no hydrogen sulphide as determined by lead acetate.On blood agar plates made with this medium without the glucosethe same alpha bacilli produced no green coloration and thecolonies had the gamma appearance (see fig. 2, plate 1). In thissame blood medium a number of strains of alpha streptococciand pneumococci also failed to produce a green coloration, al-though some hemolysis was produced (see fig. 4, plate 1). Onchocolate agar made with this medium a beta-H202 positive strep-tococcus and some strains of alpha streptococci produced nogreen coloration, indicating that they were unable to produce anyhydrogen peroxide on the medium. Addition of peptone to thesynthetic medium generally restored the ability of the alphabacilli to produce hydrogen sulphide, and of the alpha cocci toproduce hydrogen peroxide. Different bacteria differ greatlyin their ability to produce hydrogen sulphide or hydrogen peroxideon different media. The same can be said for the productionof a green coloration. Salmonella schottmislleri, for example,will produce a good green coloration in extract blood agar, whileEberthella typhosa ordinarily will not do so. If infusion agaris used E. typhosa produces a green coloration. Many strains ofE. typhosa which fail to produce a blackening of extract leadacetate agar may be found to blacken infusion lead acetate agar.If cysteine or sodium thiosulphate is added to infusion blood agar

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the alpha type of colony is formed by many bacilli which havethe gamma type of colony on the standard infusion blood agar. Insome cases even a change of peptone concentration from 1 to 2per cent may be sufficient to change the appearance of an organ-ism from that of a non-green-producer to a green-producer, thehigher peptone concentration producing more green. The na-ture of the blood used, the concentration of salts, and the hydro-gen ion concentration may materially effect the type of colonyproduced. Tun icliff (1930) showed that some hemolytic strep-tococci will produce a green coloration on chocolate agar incu-bated at 320C. but not at 370C. The kind of agar used, and themanner in which the chocolate agar is produced also greatly influ-ence this reaction. If the action of beta bacteria on chocolateagar is to be of any differential value, a standard technique for thepreparation of chocolate agar must be adopted.

SUMMARY

A study of the effect of various bacteria on blood agar and onchocolate agar has resulted in the differentiation of five distincttypes. The cause of the green coloration of blood agar has beendetermined with a fair degree of probability. The effect of vari-ous media and environmental conditions on the formation of thedifferent types of colonies is discussed.

REFERENCES

AVERY, 0. T., AND NEILL, J. M. 1924 Jour. Exper. Med., 39, 347.AVIERY, 0. T., AND NEILL, J. M. 1924 Jour. Exper. Med., 39, 543.AVERY, 0. T., AND NEILL, J. M. 1924 Jour. Exper. Med., 39, 745.BERGH, A. A. HIJMANS V.D. 1905 Deut. Arch. fur klin. Med., 83, 86.BLAKE, FRANCIS G. 1916 Jour. Exper. Med., 24, 315.BROWN, J. HOWARD. 1919 Monograph No. 9, Rockefeller Inst. for Med. Research.BRYANT, CARRIE KIRK 1925 Jour. Bact., 10, 53.CLARKE, T. WOOD, AND HURTLEY, W. H. 1907 Jour. Physiol., 36, 62.COLE, RuFuS. 1914 Jour. Exper. Med., 20, 363.HAGAN, WILLIAM H. 1925 Jour. Infect. Dis., 37, 1.HARNACH, ERICK 1899 Zeit. physiol. Chem., 26, 558.LYALL, H. W. 1914 Jour. Med. Research, 30, 487.MCLEOD, J. W., AND GORDON, JOHN 1922 Biochem. Jour., 16, 499.NEILL, J. M., AND AVERY, 0. T. 1924 Jour. Exper. Med., 39, 757.NEILL, J. M., AND AVERY, 0. T. 1925 Jour. Exper. Med., 41, 285.

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NEILL, J. M. 1925 Jour. Exper. Med., 41, 299.PAULSON, M., AND BROWN, J. HOWARD 1931 In press.RuIEDIGER, G. F. 1906 Jour. Infect. Dis., 3, 663.SMITH, T., AND BROWN, J. HOWARD 1915 Jour. Med. Res., 31, 455.TUNNICLIFF, R. 1930 Jour. Amer. Med. Assoc., 94, 1213.WALLIS, R. L. MACKENZIE 1913-1914 Quart. Jour. Med., 7, 73.

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PLATE IFIG. 1. Deep colonies of an alpha-colon bacillus in pork infusion blood agar

after incubation at 37°C. for forty-eight hours. The green coloration is notapparent in the photograph but it is very strong. The hemolysis seems moreprominent in the photograph than it appears to the naked eye.

FIG. 2. Same organisms as in figure 1 in synthetic blood agar. The coloniesshow no green coloration and practically no hemolysis.

FIG. 3. Deep colony of a pneumococcus (alpha type) in pork infusion bloodagar after incubation at 37°0. for forty-eight hours. To the naked eye the colonyappears very hemolytic with a touch of green coloration.

FIG. 4. Same organism as in figure 3 in synthetic blood agar. The colonyshows no green coloration but decided hemolysis.

FIG. 5. Same organism as in figure 1 in pork infusion blood agar to which wasadded 1 per cent sodium thiosulphate. The green coloration is much increaseddue to the addition of the thiosulphate.

JOURNAL OF BACTERIQLOGY, VOL. XXIV

(Einar Leifscn: Bacteria on Blood and Chocolate Agar)

PLATE 1,

EINAR LEIFSON

PLATE 2FIG. 1. Surface culture on chocolate agar of the same organism as in figurc 1,

plate 1. There is no green coloration.FIG. 2. Surface culture on chocolate agar of a pneumococcus (alpha type) show-

ing a strong green coloration around the colonies.FIG. 3. Surface culture on chocolate agar of a strain of Str. epidemicus (beta-

H202 positive type) showing a green coloration around the colonies.FIG. 4. Surface culture on chocolate agar of a streptococcus of the beta-H202

negative type showing no green coloration around the colonies.The chocolate agar plates were all incubated at 37°C. for forty-eight hours.For the photographs in plates 1 and 2 the writer is indepted to Dr. J. Howard

Brown.

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Einar Leifson: Bacteria on Blood and Chocolate Agar)

PLATE I11