J Clin Pathol: first published as 10.1136/jcp.8.1.58 on 1 February 1955. Downloaded from

J. clin. Path. (1955), 8, 58.

OPTOCHIN IN TIE IDENTIFICATION OF STR. PNEUMONIAE BY ERIC F. BOWERS AND LEONARD R. JEFFRIES From University College Hospital, London (RECEIVED FOR PUBLICATION MAY 17, 1954)

In 1911 Morgenroth and Levy showed that mice Tests which gave incomplete clearing in the tauro- could be protected against cholate tube were repeated by both authors and with " " (ethylhydrocupreine hydro- checked by an independent observer. chloride). The clinical use of this agent was soon abandoned as it was found to be toxic and, in doses Optochin Sensitivity which could be tolerated, did not influence the The Ditch Method.-This method was given in a course of lobar pneumonia (Moore and Chesney, personal communication from Dr. Landsman. 1917; White, 1938). Subsequent in vitro studies A ditch about 1 cm. wide was cut across the centre showed that of the streptococci, Str. pneumoniae of a blood and filled with 1 in 50,000 was the most sensitive (Moore, 1915), and that as optochin" in nutrient agar. The test organisms low a concentration as 1 in 50,000 incorporated in and a known sensitive strain were streaked up to blood agar would inhibit growth (M0rch, 1943). the ditch. copyright. Bile solubility has been generally regarded as The Disc Method.-Discs of filter paper 8 mm. in the most reliable test for the differentiation of diameter were sterilized by dry heat at 160' C. for pneumococci from other haemolytic streptococci one and a half hours. Each disc received 0.02 ml. (Wilson and Miles, 1946). The test, however, is of a 1 in 4,000 solution of " optochin," and was time-consuming and sometimes gives equivocal then dried at 37' C. This concentration of " opto- results. chin" gave inhibition zones with Str. pneumoniae slightly smaller than the ditch method. The organisms http://jcp.bmj.com/ This paper describes an investigation to deter- for test and a known sensitive strain were streaked mine whether " optochin " sensitivity can be used radially on a blood agar plate, a disc was placed in for the identification of Str. pneumoniae in place the centre, and readings were made after overnight of the bile solubility test. A large number of incubation at 37° C. alpha haemolytic streptococci were examined to Similar results were obtained with strips of filter ascertain the correlation between these tests. paper 8 x 1 cm. which had been dipped into the Mouse virulence was used as the final criterion solution, drained of excess fluid, and dried as above. of the clinical significance of some of the strains. Defibrinated horse blood agar containing 0.005% on September 30, 2021 by guest. Protected para-amino-benzoic acid was used for all experiments. Lysed blood agar without this addition and oxalated Materials and Methods blood agar were shown to give similar results. Origin of Strains.-Cultures from single colonies of Ethylhydrocupreine hydrochloride (" optochin," alpha haemolytic Gram-positive cocci were obtained Howards, Ltd., Ilford) is freely soluble in water. The from blood agar cultures of sputa, throat swabs, and base is insoluble. Solutions of 1 in 500 and 1 in pus submitted to this department for routine investi- 4,000 have proved stable in the refrigerator and at gation. A number of strains of Str. pneumoniae room temperature. The 1 in 4,000 solution was were received from other laboratories. unaffected by autoclaving at 15 lb. for 30 minutes. Bile Solubility Test.-About 1 ml. of a saline sus- Sterilization of the discs at 160' C. cannot be com- pension of growth from half a blood agar plate was bined with drying of the solution on them, as a distributed in two Dreyer tubes to one of which was considerable loss of potency results. Discs made added 2 drops of a 10% solution of sodium tauro- from 1 in 4,000 solution dried at 37' C. and stored in ;holate of proved activity and to the other 2 drops screw-capped bottles were stable for nine months in of saline. The test was regarded as positive if there the refrigerator and at room temperature in daylight. was clearing in the taurocholate tube after incubation Mouse Virulence Test.-Mice were inoculated intra- in the 37' C. water bath for one and a half hours. peritoneally with 0.5 ml. of an overnight serum broth Suspensions were consistently of pH 7.2. culture. Deaths within seven davs were recorded. J Clin Pathol: first published as 10.1136/jcp.8.1.58 on 1 February 1955. Downloaded from

OPTOCHIN IN THE IDENTIFICATION OF STR. PNEUMONIAE 59

From dead animals, cultures of the heart blood were 1ABLE II made, and stained smears of heart blood and peri- toneal exudate examined for the presence of Str. Limits of Zones of Inhibition of pneumoniae. Optochin Str. pneumoniae (mm.) 1/4,000 5 to 9 1/8,000 4 ,, 8 Experimental 1/16,000 2 ,,6 Complete insensitivity to " optochin" was shown by growth of organisms right up to the edge of the use an " optochin " concentration of less than 1 in disc or ditch. Sensitivity was of two grades, which 8,000 for routine diagnostic work. In practice, could be very easily distinguished. Full sensitivity we have found that a concentration of 1 in 4,000 resulted in a zone of inhibition of more than 5 mm. is satisfactory and does not give rise to any diffi- from the edge of the disc. Partially sensitive culty in distinguishing between the inhibition zones strains gave very small zones not exceeding 2 mm. of pneumococci and of the anomalous strains of TABLE I bile-insoluble streptococci. As an additional check we also record the size of the inhibition zone of a from Edge of Optochin Inhibition from Edge of Optochin control Str. pneumoniae for each disc. The sen- Disc in mm. sitivity of this control strain has not altered with Nil |I lor2 5to9 daily subculture on blood agar for one year. 1-l 1 242 17 1 Experimental Dissociation of Bile Solubility and Optochin Sensitivity Table I records the " optochin " sensitivities of In view of the natural occurrence of strains 243 bile-soluble strains and 452 bile-insoluble showing no correlation between " optochin " sen- strains. Of the former, all except one were fully sitivity and bile solubility an attempt was made sensitive to "optochin"; the exception was parti- to separate these characteristics experimentally. copyright. ally sensitive and was found to be completely aviru- Str. pneumoniae type I (N.C.T.C. 7465) was lent for the mouse. Of the bile-insoluble strains, shown to be bile soluble, "optochin" sensitive, only one was fully " optochin " sensitive, but 17 and mouse virulent. A series of dilutions of were partially so. The wholly sensitive strain and " optochin " in 5% serum broth was seeded from 13 of the 17 partially sensitive strains were tested an overnight serum broth culture of this organism. for mouse virulence and all were found to be After 24 hours' incubation the culture containing completely avirulent. the highest concentration of "optochin" and http://jcp.bmj.com/ There is thus almost complete correlation showing turbidity was used to inoculate a second between bile solubility and full " optochin " sen- series of " optochin " serum broths. This pro- sitivity, but 2.4% of the 695 strains examined cedure was repeated through a number of serial showed slight "optochin " sensitivity in spite of subcultures. being bile insoluble. As all of these which were After two subcultures the organism became tested were found to be completely avirulent for "optochin" resistant. In the standard disc test mice it is reasonable to assume, either that they it grew up to the disc, and its sensitivity, tested by on September 30, 2021 by guest. Protected were not true pneumococci, or that they repre- titration in serum broth, had decreased from 1 in sented completely rough variants unlikely to be of 500,000 to 1 in 50,000. It was bile soluble. any clinical significance. After eight subcultures "optochin " sensitivity The proportion of bile-insoluble streptococci had fallen to 1 in 5,000, though the organism still giving visible evidence of slight sensitivity to remained bile soluble. At this stage the mouse " optochin" can be reduced by reducing the con- virulence test was repeated. To exclude the possi- centration of " optochin " used. This, however, bility of a bacteriostatic effect from the presence is accompanied by a reduction in the size of the of traces of " optochin," the pneumococci were zone of inhibition given by true pneumococci. washed in peptone water and resuspended before The relation between " optochin " concentration injection. Furthermore the test was repeated after and zone size was determined by tests on 55 strains three serial subcultures in serum broth to elimi- of Str. pneumoniae by the disc method, with nate " optochin " which may have been adsorbed various concentrations of " optochin." The results on the cells. In each test the mice survived, in- are tabulated in Table II. dicating an association between loss of "opto- As inhibition zones of 2 mm. might be missed chin " sensitivity and loss of mouse virulence, with by inexperienced observers it would be unwise to dissociation from the bile solubility. J Clin Pathol: first published as 10.1136/jcp.8.1.58 on 1 February 1955. Downloaded from

60 ERIC F. BOWERS and LEONARD R. JEFFRIES

A second attempt to separate the properties of amined before making a report. Alternatively bile solubility and " optochin" sensitivity of the several single colonies may be tested by the original strain (N.C.T.C. 7465) was made by serial " optochin " disc or strip on one blood agar plate. culture in 10% type specific antiserum in serum broth. Through 14 daily subcultures the organism Summaqar remained bile soluble and " optochin " sensitive, "Optochin" sensitivity offers a satisfactory *though losing mouse virulence. means of identification of Str. pneumoniae. It is less time-consuming than the bile solubility test, Applcation Of 695 strains of alpha haemolytic streptococci tested, only two showed lack of correlation between some strains of The of bile solubility and full " optochin " sensitivity. and Str. viridans is very similar. Str. pneumoniae The " optochin " disc method facilitates detec- appear a mixed culture When two such strains in tion of Str. pneumoniae on overnight plate cultures go the proportion of either may unrecognized and of sputum or pus containing a mixed bacterial each may be difficult to assess. In a routine diag- practicable to flora. nostic laboratory it is not always One strain of pneumococcus was experimentally numerous colonies from avoid this error by testing " " while retaining its method is to rendered optochin insensitive each plate. An alternative simple bile solubility. place an " optochin " disc on the pool of inoculum of the original blood agar plate. The presence of We wish to thank Professor Wilson Smith, F.R.S., large numbers of pneumococci will be revealed by and Dr. E. J. Stokes for their guidance and encourage- are a zone of sparser growth round the disc due to ment in the preparation of this paper. We inhibition of the pneumococci. The effect- is grateful to those who have sent us strains of Sir. Mr. E. Y. Bridson. striking even when fairly heavy growth of other pneumoniae, especially occurs up organisms, including Str. viridans, right REFERENCES copyright. to the edge of the disc. Plates incubated anaero- M0rch, Erna (1943). Serological Studies on the Pneumococci. the effect even more clearly, because Munksgaard, Copenhagen; Oxford University Press, London. bically show Moore, H. F. (1915). J. exp. Med., 22, 269. many bacterial species are inhibited under these -and Chesney, A. M. (1917). Arch. intern. Med., 19, 611. Morgenroth, J., and Levy, R. (1911). Bert. klin. Wschr., 48, 1560. conditions. Nor does the addition of 10% carbon White, B. (1938). The Biology of Pnewnococcus. Commonwealth to affect the phenomenon. Fund, New York. dioxide the atmosphere Wilson, G. S., and Miles, A. A. (1946). Topley and Wilson's Prin- A confirmatory Gram-stained film should be ex- ciples of Bacteriology and Immunity, 3rd ed. Arnold, London. http://jcp.bmj.com/ on September 30, 2021 by guest. Protected