COMMITTEE REPORT. for the full 10 days recommended for bona fide streptococcal infections.' Compared with. many other laboratory diagnostic aids, a

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1 A Method for Culturing Beta Hemolytic Streptococci from the Throat Downloaded from by on August 20, 2018 EXCEPT in the case of scarlet fever, no completely satisfactory clinical criteria are available for distinguishing patients with streptococcal respiratory infections from those with respiratory illnesses due to other agents. Many instances of pharyngitis and tonsillitiswith or without exudate-are nonbacterial in etiology; some have been linked with the new group of adenoviruses. These nonstreptococcal infections do not lead to rheumatic fever, and patients with such infections are not benefited by antibiotic therapy. Because the diagnosis of a streptococcal infection of the upper respiratory tract on clinical groun,ds is necessarily presumptive, a culture of the throat provides valuable laboratory confirmation or rejection of the clinical impression. Throat cultures from patients with acute streptococcal infections are positive for beta hemolytic streptococci in over 95 per cent of cases. A throat culture is a relatively simple laboratory procedure to perform, and a preliminary report is available within 18 hours. In certain doubtful cases, it is possible for the physician to use his judgment in withholding antibiotic therapy until the results of the throat culture are available. A throat culture is also a useful guide for determining whether From the American Heart Association, New York, New York. Prepared at the request of the Executive Committee on Rheumatic Fever and Congenital Heart Disease. By LEWIS W. WANNAMAKER, M.D treatment with penicillin need be continued for the full 10 days recommended for bona fide streptococcal infections.' Compared with many other laboratory diagnostic aids, a throat culture is nominal in cost. The cost may be more than redeemed by the savings from withholding or discontinuing useless antibiotic therapy. As with other laboratory tests, a throat culture may be misleading unless properly taken, processed, and interpreted. For example, a common error is to confuse the nonpathogenic green (alpha) streptococci with those that produce upper respiratory infections. It is therefore essential that the practicing physician have some knowledge of the entire procedure) both to insure that the culture is correctly taken and handled and to evaluate the reports received from the laboratory. Many technics have been employed for the isolation of beta hemolytic streptococci, and a more detailed discussion of the problem may be found elsewhere.2-4 The object here is to present briefly a simple method which has proved successful in identifying these organisms in cultures of the throat. Preparation of Media A suitable medium for identifying beta hemolytic streptococci should be prepared before the throat culture is taken in order to avoid delay in inoculation. Beta hemolytic streptococci are most easily identified on sheep blood agar plates.

2 Sheep blood * is collected aseptically by venipuncture and defibrinated by slow rotation for approximately 10 minutes in a flask containing glass beads. Blood may be stored at refrigerator temperatures for 1 or 2 weeks but should be checked for contamination and hemolysis before use. Preservatives should be avoided, as these may inhibit growth on the final culture medium. A commercially available blood agar base t is prepared and sterilized according to directions. If, after sterilizing, the agar base is allowed to harden, it must be thoroughly reheated in a boiling water bath to remove lumps. The heated agar is cooled to C. A constant-temperature bath is convenient for achieving and maintaining this temperature but, with experience, the proper temperature can be judged by touch. Defibrinated sheep blood to a volume of 5 per cent t is added and mixed by gentle rotation. The blood agar mixture is poured into sterile glass or plastic Petri dishes in amounts of about 15 to 20 cc. The layer of blood agar should be about 6 mm. thick. It is difficult to differentiate the kinds of hemolysis on plates that are either too thin or too thick. A 10-cm. filter-paper disk may be placed in the top of the Petri dish to absorb excess condensed moisture. Sterile blood agar plates may be stored at room temperature for several days or in the refrigerator for 1 to 2 weeks, but with prolonged storage care must be taken to preserve sufficient moisture by sealing with parafilm strips. Plates showing evidence of contamina- *Sheet blood is preferable to rabbit, horse, or human blood. Beta streptococci are readily differentiated from alpha hemolytic (green) streptococci on sheep blood plates. Sheep blood also contains a factor which inhibits the growth of Hemophilus hemolyticus, the colonies of which may be mistaken for those of beta hemolytic streptococci. tbaltimore Biological Laboratory, Inc., Baltimore 18, Maryland; Colab Laboratories, Chicago Heights, Illinois (Oxoid medium); Difco Laboratories, Detroit, Michigan. tif the hematocrit level of the sheep blood is low, 7 to 10 per cent blood may be necessary to obtain satisfactory plates tion or hemolysis on storage should be discarded. Although readily prepared in the hospital or office laboratory, sheep blood agar plates may also be obtained commercially. Technic of Obtaining Throat Culture To insure reliable results, throat cultures should be taken before antibiotic therapy is initiated. Failure to isolate beta hemolytic streptococci may also result from faulty technic in obtaining and handling cultures before they reach the laboratory. Small cotton or Dacron swabs on 2 by 150 mm. sticks are sterilized in individual cottonplugged test tubes. An adequate culture of the throat cannot be obtained blindly. The tongue should be depressed and the throat adequately exposed and illuminated. Routinely, the swab should be rubbed over each tonsillar area and the posterior pharynx. Any area exhibiting exudate should also be touched. Care should be taken to avoid contaminating the swab by touching the tongue and lips. The swab is returned to the test tube, labeled, and inoculated onto a blood agar plate as soon as possible. No more than 1 or 2 hours should elapse before plates are inoculated. If a longer delay is inevitable, drying may be prevented by placing the swab in a test tube containing 0.5 ml. of broth. The broth-tube method has the disadvantage of permitting overgrowth by other organisms and of interfering with an estimation of the number of beta hemolytic streptococci present. To facilitate prompt inoculation of plates, some physicians keep a stock of blood agar plates in their office and carry a few in their examining bags. For this purpose plates made of disposable plastics and sealed with scotch tape are particularly desirable. A convenient disposable sterile wire loop for immediate streaking of the blood agar plate can be incorporated in the test tube or in a sealed paper envelope along with the sterile throat swab. A partially unbent paper clip sterilized in a match flame may also serve as a handy substitute for a streaking loop.

3 1056 Other procedures for handling throat swabs, including mail-in procedures, are being used and evaluated in certain communities. Technic of Streaking Plate The objectives in streaking a blood agar plate are to avoid drying of the specimen by delay, to insure adequate distribution so that well-isolated colonies will be present for examination, and to provide for observation of subsurface as well as surface hemolysis. Upon arrival in the laboratory, the swab should be immediately processed by rubbing and twisting onto the edge of a moist sheep blood agar plate. This initial inoculation with the swab should cover only about one sixth of the plate. (The area of inoculation with the swab should not be too large, since growth on this area is usually too thick for proper identification.) A sterile wire loop * is streaked through the primary inoculum onto about one half of the plate in 10 to 20 to-and-fro strokes. Without re-entering the site of primary inoculation, the loop is streaked through this area of secondary inoculation onto the remainder of the plate. Finally, several stabs are made into the agar (for observation of subsurface hemolysist). Reheating of the loop is not necessary at any stage of the streaking or stabbing of the plate. Inoculated plates are incubated overnight at 37 C. Satisfactory small incubators can be inexpensively constructed or purchased commercially. Technic of Reading Plate Streptococci causing acute respiratory infections (beta hemolytic streptococci) are recognized by the clear zone of hemolysis surrounding the colonies. In general, streptococci surrounded by no hemolysis (gamma *Loops may be sterilized by heating in a flame just prior to using or may be packaged sterilely. tas an alternative method of observing subsurface (or anaerobic) hemolysis, some laboratories employ pour plates (inoculum incorporated into the blood agar) or anaerobic incubation of surface-inoculated plates. streptococci) or by green or partial hemolysis (alpha streptococci) are nonpathogenic inhabitants of the throat. Cultures from normal throats usually show numerous streptococci (or sometimes pneumococci) which produce such partial or green hemolysis, and care must be taken not to confuse these with the beta hemolytic streptococci, which cause infections of the throat. This important differentiation may be difficult to make if blood agar plates are too thin or too thick, if growth is too heavy, or if plates are incubated more than 24 hours (e.g., over a week end). Occasionally, streptococci-causing respiratory infections produce partial or green hemolysis on the surface and clear hemolysis in the subsurface (or stab) growth. Other organisms that may produce clear hemolysis on sheep blood agar are hemolytic staphylococci and gram-negative cocci. To the experienced eye, the appearance of the colony may be helpful, but a Gram stain should be done in all doubtful instances. Chaining of streptococci is more likely to be observed on smears made from broth cultures. On smears made from blood agar plates, it is sometimes difficult to distinguish staphylococci from streptococci. The differentiation may be made by the catalase test.* On horse, rabbit, or human blood agar, Hemophilus hemolyticus may closely resemble hemolytic streptococci, but staining reveals gram-negative rods rather than gram-positive cocci in chains. A complete laboratory report should indicate the relative number of colonies of beta hemolytic streptococci present as well as the kind of hemolysis. Further Laboratory Confirmation Final determination of the kind of hemolysis sometimes cannot be made on the original tadd a small amount of growth from blood agar plate to 0.5 cc. of 3 per cent hydrogen peroxide solution. If bubbles of gas appear, the organism is not a streptococcus. Care must be taken not to transfer any of the red blood cells of the medium, because this may give a false-positive test. A more reliable test can be obtained on cultures grown on nutrient or serum agar without blood.

4 *Group A antiserum is now commercially available (Baltimore Biological Laboratories; Difco Laboratories). For methods, see reference to Swift.4 tcommercially available (Difco Laboratories) plate. In such instances, the organism should be isolated in pure culture for further study of the colony formation and of the surface and subsurface (stab) hemolysis. The procedure outlined does not differentiate between the various groups of beta hemolytic streptococci. Since most strains causing upper respiratory infections in man and all those resulting in rheumatic fever belong to group A., identification of the serologic group can be of importance in determining whether the full 10-day treatment with penicillin is required. Therefore, it is recommended that whenever possible it should be established whether the organism isolated is group A. The conventional method of determining the serologic group is by a precipitin test, using a hot acid extract of the bacterial cells and specific grouping antisera.* Several alternate methods are now available, including the use of a Streptomyces albus enzyme.t One of the simplest methods of differentiating group A streptococci is by their bacitracin sensitivity.5 Colonies of beta hemolytic streptococci are subcultured onto blood agar plates, and a bacitracin-containing paper strip or disc is placed over the area of inoculations so as to influence both heavily and lightly inoculated zones. After 18 to 24 hours' incubation, plates are examined for bacitracin inhibition. If any colonies grow up to the edge of the disc, the strain is recorded as resistant, i.e., non-group A. If inhibition of hemolysis but not of growth is observed, it must be ascertained that the subculture is not a mixture of a sensitive group A strain and a resistant non-beta hemolytic strain. In cases of acute infection, growth of beta hemolytic streptococci may be sufficiently heavy to make a dependable interpretation of inhibition or lack of inhibition around a bacitracin disc placed on the primary blood agar plate. The bacitracin method correlates well with serologic grouping when special differentiation discs containing 1 to 2 units of bacitracin* are used, but an appreciable number of false-positive results will be obtained if commercial discs designed for routine antibiotic sensitivity screening, which may contain larger quantities of bacitracin, are employed. The use of old discs may produce false-negative results. For these reasons it is advisable to include control strains of known bacitracin sensitivity. It should be emphasized that the bacitracin test only applies to beta hemolytic streptococci. Fluorescent antibodyt 6 may be used to identify group A streptococci on smears made from beta hemolytic colonies picked from blood agar plates or on smears made from original throat swabs incubated in broth for 4 hours. Although results are somewhat delayed, the former method appears to be preferable for general purposes, since valuable quantitative information is obtained from the conventional blood agar plates and since it avoids tedious screening of many negative cultures with the fluorescent microscope. By additional serologic technics, group A strains can be subdivided into some 50 different typest but classification according to specific types is ordinarily beyond the interests or needs of the routine diagnostic laboratory. Clinical Interpretation of Laboratory Reports WVith the simple precautions outlined above, it is rare that beta hemolytic streptococci are missed on throat cultures obtained from active untreated streptococcal infections. Negative cultures are often obtained on patients in whom treatment has been initiated. Only a few hours of antibiotic therapy may result in suppression of the organism so that isolation becomes difficult or impossible. In active, untreated streptococcal infections, colonies of beta hemolytic streptococci are often present in large numbers on the *Commercially available (Baltimore Biological Laboratories; Difco Laboratories). tcommercially available (Difco Laboratories; Sylvana Chemical Co., Orange, New Jersey). ttyping antisera are not commercially available.

5 1058 blood agar plate. The beta hemolytic streptococcus is usually the predominant organism. Quantitation is therefore an important aspect of culture reports. It should be borne in mind that the isolation of beta hemolytic streptococci does not necessarily indicate active streptococcal infection. A significant number of normal individuals harbor beta hemolytic streptococci which may be incorrectly accepted as the etiologic agent when isolated during the course of a nonstreptococcal respiratory infection. In such chronic carriers, only a few colonies of beta hemolytic streptococci are usually found and, these may be non-group A (i.e., not associated with rheumatic fever). In interpreting the laboratory report, attention should be paid to the number of colonies of beta hemolytic streptococci present and the serologic group should be obtained whenever possible. In all instances, the laboratory results should be considered in conjunction with clinical and epidemiologic evidence in order to arrive at the most reasonable diagnosis. Additional evidence of active streptococcal infection may be obtained by streptococcal antibody studies; e.g., antistreptolysin 0 or ASO titers, on patients' sera.7 These are not commonly obtained except in epidemiologic studies or in patients with suspected complications of streptococcal infection (acute rheumatic fever or acute nephritis).* *A brochure on "Jones Criteria (Revised) for Guidance in the Diagnosis of Rheumatic Fever" is available from your Heart Association or the American Heart Association. References 1. COMMITTEE ON PREVENTION OF RHEUMATIC FE- VER AND BACTERIAL ENDOCARDITIS: Prevention of Rheumatic Fever, 4th revision, American Heart Association, 44 East 23 Street, New York, N. Y , WILSON, A. T., ET AL.: Streptococcus infections. In Diagnostic Procedures and Reagents, American Public Health Association, 1790 Broadway, New York, N. Y In press. 3. WILLIAMs, R. E. O.: Laboratory diagnosis of streptococcal infections. Bull. Wld. Health Org. 19: 153, SWIFT, H. F.: The Streptococci. In Bacterial and Mycotic Infections of Man, 1st Edition, edited by R. J. Dubos, C. B. Lippincott Company, Philadelphia, 1948, p MAXTED, W. R.: The use of bacitracin for identifying Group A hemolytic streptococci. J. Clin. Path. 6: 224, CHERRY, W. B., GOLDMAN, M., AND CARSKI, T. R., WITH THE COLLABORATION OF MOODY, M. D.: Fluorescent Antibody Techniques in the Diagnosis of Communicable Diseases. Public Health Service Publication No U. S. Department of Health, Education and Welfare, Public Health Service, Bureau of State Services, Communicable Disease Center, Atlanta, Georgia, a) MCCARTY, M.: The antistreptolysin 0 titer in rheumatic fever-methods and significance Bull. on Rheumatic Diseases, 7:S-23, No. 7, 1957 (supplement). b) WANNAMAKER, L. W., AND AYUB E. M:. Antibody titers in acute rheumatic fever. Circulation 21: 598, 1960.