! \ 1. 66 SALNE TEST FOR STREPTOCOCCAL HJEMOLYSN BY Maj9r SMON SEVTT, M.D.Dub., M.Se., D.P.H.; M.R.e.p. Late Royal Army Medical Corps: SPe.cialist in Patho~ogy. (Received April 9, 1947.] N medical bacteriology, streptococci were classifi~d primarily into a, a prime, f3 and non-hremolytic types according to the changes produced by their growth on solid blood-agar (Schotmuller, 1903; Smith and Brown, 1915, etc.).,marmorek (1895) showed t.ha~ fluid cultures of certain streptococci lysed added red cells, and BrilUn (1912) correlated the ruemolysis on blood-agar. medium with the production of a, filtrable hremolysin in serum brot.h. The. studies. of streptococcal hremolysin by McLeod (1912) and.. othets, the observations of de Kruif and reland (1920) that the hrem9lytic activity of a serum broth culture reache~ a maximum in eight hours and then diminished, and the discovery of the oxygen and heat-labile hremolysins by Todd. (1934). led to a test for the detection of soluble streptolysin. n ihis method 05 to 10 d:. of an eight to fourteen-hour whole culture of the streptococcus in 20 per cent serum broth, or of the supernatant after-centrifuging, is added to an equal volume of 5 to 10 per cent washed horse blood cells,, the mixtur~ incuba.ted for one te two hours and the presence or absence ofhremolysis. noted. This method has the disadvantage t~at owing. to the vari- ability of streptolysin production in the early period of growth and its lability, later on, false negative results might be obtained if the culture is less th~n eight,. or morethan fourteen hours old.. Moreover, the interval between commencing and reading the tes! varies froin ten to sixteen hours and this usually ~nta~ls the inoculation of the serum broth or the addition of the washed cells after nqrmal laboratory hours. n the new method, henceforth called the sajine hremolysin test; jtreptococcal c9onies are picked off.from. the overnight primary blood-agar plate and emulsi fied in about "1 c.c, of sterile saline in a small test tube. Within limits the heavier the suspt;nsion, the quicker hremolysis occurs; if too few cocci are added, false hega~ives occur. As will be shown lat@r at least ten colonies of the streptococcus shouldfbe emulsified. One drop of defib~inated or oxalated horse blood is added to the suspension, the tubes shaken a~d incubated at 37 C. A control tube of saline plus blood is desirable but not, essential. The tube should be - examined and, shaken every half to one hour for at Jeast four or five hours. Most positive results will be obtained well within this perioq but negative results should not be recorded for seyenor eight hours or preferably overnight., This test was discovered nhrough an accidental experiment set up by a serjeant-technician, in which hremolysis, within two hours of incubation, was observed in a saline suspension of a hrem?lytic streptococc~s; to which had1jeen
. Simon S~vitt added a drop or, two of,defibrinated horse blood. As this observation promised a siinple and more rapid method of detecting streptolysin, thirty strains of streptococci producingf3 zones of hremolysis on horse blood-agar. were tested. over a period. The technique was as follows: -. After isaation in subculture, the organism was grown on a horse blood-agar plate to verify its purity and /3 hremolysis: to a!lloderately heavy suspension of this growth in to 2 C.c. of sterile is()tonic saline was added one drop of sterile, defibrinated horse blood. After shaking to mix the contents, the tube was incubated a 37 C. and examilled every half to one hour for eight hours, and if necessary the following morning too. A control tube containing only saline and blood was also set up. n these and all subsequent experiments results were recorded as follows : viz. + + + complete hremolysis, + + strong but incomplete lysis, + definite lysis, tr. trace of lysis,and 0 no lysis., Of the thirty strains, Oily four gave negative results, three on one oce:asion only, giving complete or incomplete lysis in one or more repeat experiments. The fourth strain failed to produce lysis in three experiments... This was the Group D strain ref~rred to below; two of the oth~rs were Group A, one Group C., All four,strains produced soluble hremolysin against horse cells when grown in 20 per cent serum broth. Forty-two tests were perform~d on the remaining. twenty-six strains: thirty-two showed complete lysis, eight strong but inco~plete lysis, and only two the lesser lysis recorded as +. n Table the number of experiments in. which at least + + hremolysis occurred, and the minimal periods fequrred for this degree of lysis to develop, are recorded. Of the forty tests, thirty reached this stage iri less than three hours, and in only five wag it delayed lorger than seven hours. TABLE.~To SHOW THE NUMBER OF fists DEvELO,niG AT LEAST + +.. D~GREE OF HlElJOL YSS N VAROUS PERODS.. 1. 1 No. of tests.. 11. 5 Appearance of + + lysis in hours. 11 2. 3 4 5 6 4 4 6 2 1 2 Eighteen of the thirty strains were grouped by Lancefields (1933) method;, thirteen were Group A, one Group D, and two Gro,up G~ The C and G strains and five of the Group A strains were o,btained from the Lister nstitute; twelve of the others were isolated from human throat infectiois, the Group D,strain which was a typical enterococcus, from ~ case of pyelocystitis; and the remainder from various purulerit infections. :..,... Twenty-one tests for production of soluble hremolysin were carried out. The organism was grown ifi20 per cent horse serum broth for ten to twelve hours, the culture centrifuged and washed horse cells added to the supernatant fluid. Results were read after two hours incubation at 37 C. >7 5 Three tests were negative, but when repeated wer,e strongly positive: all others were positi~e..,. The results of such small numbers cannot be too}videly interpreted, but it seems that the saline hremolysis test which gave 87 per cent of positives if + + hremolysis is taken as the minimal end"point, or 91 per cent if + degree of
. \ \ 68 Saline Test for Streptococcal Ha:molysin, hremolysis is accepted, is at least as efficient as the longer m~thod which gave 85 per cent of positive results. CONTROL EXPERMENTS:, A series of experiments were performed on organisms other than tj hremolytic stre.ptococci to discover the specificity and accuracy of the new test. First, ~ourteen strains PJ viridans~ imd two of non-hremolytic streptococci w~re, tested for hremolysin by the saline test; Fifteen of these gave completely negative results even when left overnight and read the following day: but in one test, hremolysis commenced in one and a half hours and was + + in four hours~ This was, the sfrain which showed a zone of (3 hremolysin in,!;ddition to the: green discoloration around the colonies, and it was considered possible that, if cultured anaerobically, this may be classified as atrue (3 hremolytic streptococcus (see Fry, 1933).,Unfortunately th~ organism died out in subculture and this theory, could not be tested. Eighteen organisms, ~ther th:instreptococci~ were also tested. These were, eight strains of!;oagulese-positive staphylococci, five of which showed zones of f hremolysis around their colonies on horse blood-agar, two hremolytic coliform strains, three diphtheroids (one (3 hremolytic) and one strain each of C. diphtheriagr(jjis, B. dysenteria:, Sonne and Flexner, Ps. fluorescens and B. paraty~, phosum A. All tests gave uniformly negative results in.eighteen to twenty-fo\lr llours, although certain Ol:ganisms, particularly the staphylococci, produced a dirty purplish discoloration of the non-hremolysed blood. OPTMUM "BLOOD-SALiNE PROPORTON~., The success of the saline hremolysin test is probably due to one of two mechanisms. Prefermed "lysin ma~ be extracted from the saline suspension of intact cocci into the serum or plasma,of the added blood: alternatively formation of new hremolysin may occur by growth of the!}tieptococcal suspe?sion in the blood-salineinedium. Whichever is true there may bean optimal serum concentration for,the test. To examine this possibility, tubes containing descending dilutions of horse serum in,saline were set up in final volumes of 1 c.c. Experiments on four strains kp,own to produce hrer).olysin, were performed. A heavy suspension from an overnight culture oil blood-agar was made in each. tube and washed,, horse cells added. n some experiments, one "standard", drop of packed cells was added to each tube, in others OSe.c. of alo per cent suspension of cells in saline was used. A typical1,"esult is shown in Table n. TABLE ri.-tt> SHOW THE RELATONSHip BETWEEN CONCENTRATON OF HORSE- SERUM AND THE RATE AND EXTENT OF HlEMOLYSS, Time in Final concentrations of horse-serum in normal saline Uninoculated, 1 1 16 11"2 0 saline control hours t! i 0 0 0 0: TR. o O. 1 0 0 O + ++ 0 0 2 0 0 +, ++ +++ TR~ 0,3 TR. + +++ +++ +++ + 0 6 + +-+: +++ +++ +++ + 0 i One of these produced a zone or fj hremolysis in addition to green discoloration,.
Simon Sevitt 69 n general the results shqwed that neith~r no~mal saline ~lone, nor concen.trationsof serum greater than 25 per cent, favoured rapid or extensive hremo. lysis, and that under the conditions of the experiments, the. optimum serum concentration was in the region of 2 to 10 percent. Happily the addition of one or two drops of blood to or 2 C.c. of saline gives a serum concentration of this order, so that the ratio of blood to saline originally and stibseqtiently used in the tests, is the optimum. one for the experiment.. SENSlTviTY OF THE TEST. The real valu.e of the hew method, besides its theoretical nterest: is the rapidity and ease with which it tests for hremolysin: n addition it is unnec~ssary to subculture the streptococcus from. the primary blood-agar plate. n practice it would not be always of value if a heivy suspension of the test streptococcus was essential, since the colonies are small, and if only a few were available, a false negative result may be obtainfd. Experiments were set up to.discov~r the minimal suspensioj?s of streptococci in saline necessary to ensure rapid hremolysis.. Owing to the granularity of streptococcal suspensions and the related, diffi~ culty of calculating density of cocci per c.c., the strength of the suspensions,,,ere recorded in colonies. per c.c.. This of course Js not very accurate, hut has a practical advantage. The sensitivity of the test was inv.estigated by removing colonies of average size from an eighteen to twenty-four hour blood-agar plate culture arid suspending increasing numbers of them in saline volumes of c.c. Numbers of colonies varying from one to sixty-four ~ere used, and as usual qne, drop of defibrinated horse blood was add~d to each tube. These were shaken, incubated at 37 C. and read every half to one hour, when they,were again shaken. Ten strains known to produce hremolysi~ were selected for these.ex-. periments. Typical results ire recorded in Table ll. TABLE.-ToSHow THE RELATONSHP BETWEEN THE STRENGTH OF THE SUSPENSON AND THE RATE AND EXTENT OF HlEMOLYSS. Time in No. of colonies in 1 C.c. of sali,ne Strain hours 0 3 to 4 6 to 8 12 to 16 24 to 32 Strep.! 0 0 0 0 ++ +++. Poole. 1 0 0 0,+ +++ +++ 2 0 0 TR. ++ +++ +++ 4. 0 Q +++ +++ +++ +++ 6 0 TR. +-+-+ +++ +++ +-+-+ Strep. v! 0 0 ~ 0 0 0 0 Fadey 1 0 0 0 0 0 0 2 0 0 0 0 TR. ++ 4 O. 0 TR. + ++ +++ (j 0 0 +++ +++ +++ +++ Strep.! 0,0 0 0 0 0 Spires. 1 0 0 0 0 0 0 2 0 0 Q 0 0 + 4 o ~ 0 0 TR. +. ++ 6 0 0 TR. + ++ ++
70 Saline Test for Streptococcal Hcemolysin t was found that the greater the mmberof colqnies used, the more rapid the hremolysis. n some cases as few as three or four colonies produced strong or complete lysis in three or four hours, but more often six to twelve colonies were necessary to pro~uce a simjlar change in the same peri.od.. n general it may be said that at least ten colonies should be emulsified in the lc.c. of saline,". and; if possible, h~ilvy emulsions should be made to ensure a quicker and more accurate- result.. DSCUSSON. Compared with testing for soluble hremol sin production in serum broth culture, the advantages of the saline hreinolysin test are first, its simplicity. and relative rapidity, and secondly-that it is unnecessary to subculture the streptococcus; as the test can be pet:formed from colonies on the overnight primary blood-agar plate. Moreover, as it se(;!ms that other. organisms, even those producing (3 haemolysis on solid blood agar, give a negative test, the accidental picking off of colonies other than streptococci has no influence on the final result. However this admixture. should be avoided, as far as possible. f possible a heavy saline suspension of streptococci should be made to avoid false negatives... The origin of the hremolysin involved in the test, and its relationship to the o and S lysins described by Todd (1934) are under inv~stigation. SUMMARY... ~ nex method fort~e detection of streptococcalhremolysih is describe~j.in: which an emulsion of streptococci from the primary blood-agar plate is made.. in about 1 C.c. of normal saline and one drop of defibrinated or oxalated horse blood is added... ". The test is as reliable as McLeods method but is simpler and more rapidly carried out. ACKNOWLEDGMENT.. wish to thank Colonel J. D. Gripper, and Li~ut.-Colonel E. H. Hall, R.A.M.C., A.D.P. Southern Command, for permission to forward this article for publication:. \ REFERENCES. BRAUN, H. (1912). Zbl. Bakt., 62, 383, as tep9rted by Topley and Wilson. FRY; R. M. (193~). ]. Path. Bact., 37,337.. KRUF,P. H. de, and RELAND, P. M. (1920). ]. nject. Dis., 2~, 285.. LANCEFELD, R. C. (1933). J. expo Med., 57, 57l., MARMOREK, A. (1895). Ann. nst. Pasteur, 9, 593. McLEOD, J. W. (1912). ]. Path. Bact., 16,,321.. SMTH,T., and BROWN, J. H. (1915). J. Med. Res., ;U, 455. TODD, KW. (1934). 1. Path. Bact., 39, 299. TOPLEY and WLSONS "Pri.nciples of Bacteriology and i:m:ij.1itity" (1946l. EditiQ,11, revised by G. S. Wilson and A. A.. Miles. Third