Document Sample
					The use of fluorescent-antibody and cultural-precipitin grouping procedures
for identifying Group A streptococci from throat swabs was evaluated
with paired throat swabs. The sensitivity of the fluorescent-antibody
technic was equivalent to or greater than that of cultural-precipitin
technics. The importance of a number of conditions and factors
for successful use of fluorescent-antibody tests on a routine
basis is emphasized.

Max D. Moody, Ph.D.; Alan C. Siegel, M.D.; Bertie Pittman, M.S.; and Carrie C. Winter, M.S.

C-NVENTIONAL cultural and serologic            ent investigation include slight modifica-
     methods for detecting Group A             tions and are given in detail (see Ad-
streptococci and identifying the strepto-      dendum) inasmuch as they now have
coccal group require at least 48 hours.        been evaluated in several other labora-
An earlier laboratory diagnosis may con-       tories and are being considered as "rec-
tribute to the proper management of            ommended procedures."
Group A streptococcal infections and,
thus, help prevent rheumatic fever and         Patients Examined
other sequelae. A fluorescent-antibody
method which may make this possible               The study was done in Chicago in
was described in 1958.1 In the present         1958 and 1959 during seasons of high
investigation, the feasibility of using this   streptococcal incidence. Patients ex-
method to detect streptococci from throat      amined in 1958 were at the following
swabs was evaluated. The results of com-       locations:
paring conventional culture-precipitin            1. Northwestern University Medical School
grouping tests with fluorescent-antibody       Rheumatic Fever Clinic: Adults and children
                                               reporting for routine follow-up examination or
tests on throat swab specimens suggest         with intercurrent respiratory illnesses.
that the fluorescent-antibody method is          2. Children's Memorial Hospital Upper Re-
as specific and perhaps more sensitive         spiratory Disease Clinic: Children either with
than conventional methods.                     sore throats or those examined periodically
                                               following Group A streptococcal infections.
                                                 3. Michael Reese Hospital Upper Respira-
Materials and Methods                          tory Diseases Clinic: Children with sore
   General methods utilized for the prep-        4. Great Lakes Naval Training Center,
aration of reagents and for performing         Great Lakes, Ill.: Young male naval recruits
                                               reporting to sick call with sore throats.
fluorescent-antibody tests to identify
streptococci were given previously.'             In 1959 only patients at the Children's
However, the methods used in the pres-         Memorial Hospital were examined.

JULY, 1963                                                                              1 083
            Table 1-Effect of Broth Enrichment on Identification of
              Group A Streptococci by Fluorescent-Antibody and Culture-
              Precipitin Grouping Tests

             Kind                                   Number         Per cent Postive
            of Swab      Methods Compared          Examined       Direct      Broth

                      Direct vs. two-hour broth        63           3          46
                      Direct pour streak
                        vs. two-hour
                        broth pour streak              63          18          33

                      Direct vs. two-hour broth       163          11          37
                      Direct pour streak
                        vs. two-hour
                        broth pour streak             164          24          41

Collection of Throat Swabs and Identi-            tures of beta hemolytic streptococci and
fication of Group A Streptococci by               precipitin tests performed with anti-
Culture-Precipitin and Fluorescent-               serum for Groups A, B, C, D, F, and G
Antibody Grouping Technics                        streptococci.
                                                     Fluorescent-antibody tests were per-
   Physicians were instructed to roll             formed with the remaining swab by us-
duplicate cotton or dacron swabs simul-           ing (1) Direct Smears and (2) Two-
taneously over identical pharyngeal               Hour Broth Smears (see Addendum).
areas. One swab was selected randomly             Positive tests for Group A streptococci
for culturing and identifying Group A             were recorded only if brilliantly fluores-
streptococci in the routine bacteriology          cent cocci in several chains consisting
laboratory by culture-precipitin group-           of at least three to five cocci were ob-
ing technics. Blood agar base (Difco)             served in the smear stained with Group
plates containing 5 per cent sheep blood          A streptococcus conjugate and not in the
were streaked directly and incubated 24           control smear. Smears made immedi-
to 48 hours at 370 C (Direct Streak               ately after collection of the throat swab
Plate). In addition, throat swabs from            often contained fluorescent aggregates of
92 of the 210 patients examined in 1958           mucus and bacteria but were not called
and all of the patients in 1959 were cul-         positive unless actual fluorescent cocci
tured as follows: Poured-streaked plates          could be detected as described above.
were inoculated from broth rinsings of            In smears made from broth cultures,
the swab immediately after collection             positive specimens contained numerous
 (Direct Pour-Streak Plate) and from              fluorescent streptococcal chains which
broth rinsings in which the swab had              were differentiated readily from the
incubated two hours at 370 C (Two-                massive numbers of bacterial contami-
Hour Pour Streak Plate). In all cases             nants. In general, not more than two or
extracts were prepared from pure cul-             three minutes were required to examine

1 084                                                                VOL. 53. NO. 7, A.J.P.H.
                                                                               GROUP A STREPTOCOCCI

 and record results for an average smear.                   broth was significantly advantageous for
 All broth rinsings were saved in the re-                   culturing. The data would seem to indi-
 frigerator for retesting of specimens on                   cate that either cotton or dacron swabs
 which discrepant results from cultural                     offered advantages in certain cases, but,
 and fluorescent-antibody tests were ob-                    for reasons to be discussed later, these
 tained.                                                    apparent advantages may be explained
                                                            by certain uncontrolled factors.
 Effect of Broth Enrichment upon
 Identification of Group A                                  Fluorescent-Antibody Versus Cultural-
 Streptococci                                               Precipitin Tests on Paired Throat Swabs
   Decidedly more Group A streptococ-                          Of 496 paired swabs tested immedi-
cus-positive specimens were detected                        ately after collection, a total of 129
from two-hour broth cultures than di-                       positive reactions for Group A strep-
rectly from the swab, regardless of the                     tococci were obtained by cultural-pre-
kind of swab used or test performed                         cipitin and fluorescent-antibody methods.
 (Table 1). Direct smears for fluorescent-                   Fourteen of these pairs were positive by
antibody tests were considered unsatis-                     -both methods (Table 2). Thirty-three
factory, in general, because so few of the                  were positive by fluorescent-antibody
specimens were positive. It was also                        tests only, and 82 by cultural-precipitin
noted that positive fluorescent-antibody                    tests only. More positive fluorescent-anti-
reactions on direct smears were not con-                    body than conventional tests were
firmed consistently in broth smears.                        demonstrated among specimens from
Broth smears regularly contained fluo-                      two sources (Northwestern and Great
rescent chains which were easily located,                   Lakes).
and the test was interpreted readily. Al-                      Of 313 paired swabs which were in-
though more positive specimens were                         cubated in broth two to three hours and
detected in direct tests by culture than                    tested, 83 were positive by both fluores-
by fluorescent-antibody tests, plating of                   cent-antibody and cultural-precipitin

      Table 2-Identification of Group A Streptococci from Swabs Immediately
        after Collection

                                                        Results Categories of Specimenst
               Source of Patient                     FA +       FA -       FA +       FA -
             and Cultural Method*                   Conv + Conv - Conv- Conv +
      Michael Reese            (1958) CS-DS             0              20           2           3
      Northwestern             (1958) CS-DS             0              33           5           0
      Great Lakes              (1958) CS-DS             0              55          13           9
      Children's Memorial      (1958) CS-DS             3              48           2          13
      Children's Memorial      (1959) CS-DS             0              45           2          16
      Children's Memorial      (1959) CS-PS             0              49           2          12
      Children's Memorial      (1959) DS-PS            11             117           7          29
                                        Totals         14           367           33           82
             CS-DS: Cotton swab-direct streak plate conventional method.
             CS-PS: Cotton swab-pour streak plate conventional method.
             DS-PS: Dacron swab-pour streak plate conventional method.
           t Number of paired throat swvabs from which results of fluorescent-antibody (FA) and conven-
      tional (Conv) tests fell into the categories indicated.

JULY. 1963                                                                                                1 085
        Table 3-Identification of Group A Streptococci after Incubation of
          Swabs in Broth    x

                                                         Results Categories of Specimenst
                 Source of Patient                    FA +       FA -       FA +       FA -
               and Cultural Method*                  Conv + Conv- Conv- Conv +
        Michael Reese            (1958) CS-PS             1            18           5            1
        Northwestern             (1958) CS-PS             0            12           1            0
        Great Lakes              (1958) CS-PS             1             6           4            1
        Children's Memorial      (1958) CS-PS            14            13           5            4
        Children's Memorial      (1959) CS-PS            18            31          11            3
        Children's Memorial      (1959) DS-PS            49            86          11           18
                                          Totals         83          166           37           27
             * CS-PS: Cotton swab-pour streak plate conventional method.
               DS-PS: Dacron swab-pour streak plate conventional method.
             t Number of paired throat swabs from which results of fluorescent-antibody (FA) and conven-
        tional (Conv) tests fell into the categories indicated.

methods, and 166 were negative by both                    initiated immediately after collecting
(Table 3). That is, in 249 (80 per cent)                  the throat swab. Fifty-two pairs of swabs
of 313 specimens examined, results from                   were positive by both methods, and 287
both methods were in agreement. Thirty-                   negative by both. In all groups of speci-
seven pairs of swabs were positive by                     mens there were more positive fluores-
fluorescent-antibody tests and negative                   cent-antibody than cultural-precipitin
by cultural-precipitin tests. Conversely,                 tests.
in 27 other pairs cultural-precipitin tests
were positive and fluorescent-antibody                    Specificity of Fluorescent-Antibody and
tests negative. Of the 147 positive tests                 Cultural-Precipitin Tests
demonstrated by both methods, 120 (81
per cent) were positive by fluorescent-                     Divergent results obtained with cer-
antibody and 110 (74 per cent) by cul-                    tain paired swabs tested by fluorescent-
tural-precipitin tests.                                   antibody and cultural-precipitin tests,
                                                          described in the foregoing, indicated a
Immediate Cultural-Precipitin Versus                      need for further examination of the
Delayed Fluorescent-Antibody Tests                        specimens involved. Remaining saline
                                                          suspensions from all swab washings were
   Data obtained in this study make it                    tightly stoppered and stored at 00 C to
possible to compare results of what may                   50 C after sampling for the initial test-
be considered commonly used cultural-                     ing of specimens collected in 1959. After
precipitin grouping technics (immediate                   results of all initial tests were obtained
culturing of throat swab followed by                      and recorded, 31 specimens which gave
grouping of cultures of beta hemolytic                    positive fluorescent-antibody but nega-
streptococci) with grouping from young                    tive cultural-precipitin tests were re-ex-
 (two-hour) broth cultures by fluores-                    amined intensively by cultural-precipitin
cent-antibody tests. Of 482 paired swabs                  methods. Group A streptococci were iso-
examined, 172 (88 per cent) of the                        lated from 14 of the 31 specimens (47
broth cultures gave positive fluorescent-                 per cent). Cultural failure in the remain-
antibody reactions, and 75 (38 per                        ing 17 specimens probably resulted dur-
cent) were positive in conventional tests                 ing the storage period in saline for ap-

1 086                                                                           VOL. 53, NO. 7. A.J.P.H.
                                                            GROUP A STREPTOCOCCI

proximately four weeks. Conversely, 62       growth or staining were removed by di-
specimens which gave positive cultural-      lution in broth. The advantage gained
precipitin but negative fluorescent-anti-    by enrichment of throat swabs in broth
body reactions were re-examined by           was greater for fluorescent-antibody
staining additional smears with fluores-     than for cultural tests.
cent-antibody. In all 62 cases Group A          Dacron swabs were used in one phase
streptococci were demonstrated in a          of the study based upon the assumption
single additional smear. Control smears      that a better recovery of organisms
were negative.                               would be possible with a fiber less ab-
   In the course of the investigation, 156   sorptive than cotton.2 The results did not
cultures of beta hemolytic streptococci      bear this out consistently. The fact that
were isolated in pure culture and found      dacron swabs were much smaller than
to be Group A by precipitin tests. All       the cotton swabs used may have been a
were brilliantly stained with Group A        factor which affected the number of
streptococcus fluorescent-antibody ab-       positive tests by culture or fluorescent-
sorbed with Group C cells and not by         antibody methods. The qualities of swab
normal rabbit globulin conjugate ab-         fiber undoubtedly affect the ease with
sorbed with Group A cells. In addition,      which organisms detach from the swab.
three Group C and four Group G strains          Since two-hour broth cultures gave
were isolated and failed to stain with       considerably more positive cultural-pre-
Group A fluorescent-antibody. Most           cipitin and fluorescent-antibody tests, a
specimens contained alpha streptococci;      better comparison of the true sensitivity
17 strains were tested with fluorescent-     of the two technics was possible. In all
antibody and none reacted. Similarly,        groups of patients more positive fluores-
two strains of gamma streptococci were       cent-antibody tests than cultural-precipi-
tested and failed to stain.                  tin tests resulted. Eighty-one per cent of
                                             all positives were detected by fluorescent-
Discussion                                   antibody, while 74 per cent were posi-
                                             tive from cultural isolations. The results
   The results obtained by cultural-pre-     of both tests were in agreement in 80 per
cipitin grouping tests for identifying       cent of the specimens. It seems likely
Group A streptococci from throat swabs       that the group represented by the 20 per
were compared with those obtained by         cent disagreement reflects inherent defi-
fluorescent-antibody tests. Although cul-    ciencies of either test. Regardless of the
tural-precipitin tests cannot be con-        comparisons made in this study, there
sidered 100 per cent accurate, they were     usually were some paired swabs in which
accepted as standard procedures for the      one swab was positive and the other
purpose of this study. It will be as-        negative. There were some indications
sumed that the technical skills employed     that disagreement of results from dupli-
were at least average. One of the most       cate swabs occurred more often in speci-
important observations in the study was      mens from which only a few beta hemo-
that the holding of swabs in broth for       lytic streptococci were cultured on
two hours at 370 C resulted in consider-     plates. Yarashus and Siegel3 have
ably more positive cultural-precipitin       demonstrated that a closer correlation
and fluorescent-antibody tests for Group     between cultural and fluorescent-anti-
A streptococci than did testing immedi-      body tests occurs when many colonies of
ately after collection of the swabs. Two     Group A streptococci are found on the
reasons for these results may be that (1)    plate than when few are isolated. In
multiplication of organisms occurred, or     another sense, the greater sensitivity of
(2) mechanical or chemical inhibitors of     fluorescent-antibody tests over conven-

JULY, 1963                                                                         1 087
tional tests is based upon the fact that     a satisfactory means of ruling out
clear-cut specific fluorescence reactions    Groups C and G cross-reactions.
are possible on smears containing mas-          Wolfe and Cameron" observed occa-
sive bacterial contamination. Pure cul-      sional strains of Staphylococcus aureus
tures of beta hemolytic streptococci are     in stained smears made from Trypticase
required for reliable results in grouping    Soy Agar slant throat cultures mailed to
by precipitin tests.                         the state laboratory. Our conjugates
   The fluorescent-antibody tests ap-        were tested with approximately 90
peared to be specific for Group A            strains of S. aureus. All became well
streptococci. It was possible to demon-      stained with fairly high dilutions of both
strate positive reactions on all subcul-     Group A and normal rabbit conjugates.
tures of Group A streptococci as well as     Additional experiments indicate that
on all broth sediments containing these      conjugates prepared from globulin of a
organisms. By staining one additional        large number of nonimmunized rabbits
smear of stored broth sediment in those      consistently stain S. aureus strains.13 The
cases originally called fluorescent-anti-    problem of removing cross-reacting anti-
body negative and culturally positive, it    body without affecting the Group A re-
was possible to demonstrate fluorescent      action has not been solved. Absorptive
chains of streptococci. Confirmatory cul-    qualities of S. aureus strains are con-
turing of those saline-washed broth sedi-    siderably variable. Thus, in reading the
ments that gave positive fluorescence re-    test, brilliantly fluorescing cocci should
actions originally was delayed; Group A      be observed in the smear stained with
streptococci could not be isolated from      Group A conjugate but not in the con-
all, although several did reveal positive    trol. If S. aureus is present, fluorescent
cultures. It is most likely that viability   cocci not in chains should be observed in
was reduced during the storage period        both smears.
in saline. None of the Group A strains          Warfield, et al.,7 appear to have
stained with normal rabbit globulin con-     demonstrated Group A streptococci more
jugate absorbed with Group A cells.          successfully by direct staining of throat
   Cross-reacting strains of Groups C and    swab smears than by staining smears
G streptococci were not encountered, al-     from incubated broth. Their conjugate
though such strains have been found in       was absorbed with Group C streptococci.
other clinical studies.4'5 Usually such      It is not clear whether a control con-
cross-reactions are of lower intensity       jugate was used with each specimen to
than specific Group A reactions but may      rule out the presence of S. aureus.
pose a problem unless adequate control       Specimens which contained unchained
strains of Groups C and G are included       fluorescent Group A streptococci would
with each series of tests. Because of the    be particularly difficult to evaluate with-
close serological relationships existing     out the additional control conjugate
among strains of A, C, and G, complete       which has been absorbed with Group A
removal of cross-reactions by absorption     cells. In our experience, absorption of
is not always possible. Therefore, a dilu-   the Group A conjugate with Group C
tion of absorbed conjugate which no          streptococci does not affect the reaction
longer reacts with these unusual C and       of the conjugate with S. aureus (over 80
G strains, but one which stains represen-    strains). The absorbed conjugate pre-
tative Group A strains well, is recom-       pared by Warfield, et al.,7 did fail to
mended. Redys, et al.,4 suggested that a     stain two strains of S. aureus. Redys, et
combination of an inhibition test with       al.,4 employed Streptosel broth for en-
Group C antiserum and Group A con-           richment of throat swabs since it seemed
jugate absorbed with Group C provided        to eliminate problems with S. aureus.

1088                                                          VOL. 53, NO. 7. A.J.P.H.
                                                                 GROUP A STREPTOCOCCI

However, extreme caution in the prepa-            cent-antibody and 5,408 (87 per cent)
ration of the broth was indicated in              by culture-precipitin tests. All compari-
order that growth of Group A strepto-             sons were made following incubation of
cocci would not be inhibited. Since               the swabs in broth for approximately
hemolytic S. aureus is demonstrated in            two hours. Despite the variable condi-
throat swab cultures and because of cer-          tions existing in different laboratories,
tain disadvantages of using inhibitory            the correlation and sensitivity is high. In
media, we feel at the present time the            some cases it becomes necessary to mod-
fluorescent-antibody test should be so            ify the over-all procedure in order to
controlled that S. aureus can be differ-          accomplish the laboratory workload
entiated definitely from Group A strep-           more efficiently.
tococci in every specimen tested. A                  For example, substantial testing indi-
point not to be overlooked is the possi-          cates that Group A streptococci can be
bility that some fluorescent-antibody re-         identified as to group by testing smears
actions could arise from nonbeta hemo-            of colonies, or more satisfactorily, smears
lytic Group A streptococci which would            of young broth subcultures of colonies,
not be detected ordinarily in the search          with fluorescent-antibody.'2 Thus, the
for beta hemolytic colonies. Such organ-          presence of Group A streptococci can be
isms have been encountered in nature,             confirmed within 24 hours after the
but their frequency is not known.                 swab is collected. Modifications such as
   The procedures described here (see             this should be evaluated by the individ-
Addendum) were recommended as reli-               ual laboratories involved.
able for routine use in state public health
laboratories approximately four years
ago with the following qualifications:            Summary
   1. That bacteriologists who plan to use the
technic obtain thorough training in the per-         The use of fluorescent-antibody and
formance of both culture-precipitin and fluo-     cultural-precipitin grouping procedures
rescent-antibody grouping technics.               for identifying Group A streptococci
   2. That well-standardized reagents and re-     from throat swabs was evaluated with
liable equipment be used according to tested
procedures.                                       paired throat swabs. Significantly more
   3. That the use of culture-precipitin group-   positive specimens were obtained by
ing methods be continued at least until com-      both methods when swabs were incu-
petency in the use of the fluorescent-antibody    bated in broth for two hours, rather
tests becomes well established, and that after    than being tested immediately after col-
this period representative isolants be grouped
by precipitin tests as a control on new or        lection. The sensitivity of the fluores-
unusual situations.                               cent-antibody technic was equivalent to
                                                  or greater than that of cultural-precipitin
   Bacteriologists from 63 state, terri-          technics. Furthermore, reliable reactions
torial, and major metropolitan public             were possible with highly contaminated
health laboratories have received such            specimens and with those which failed to
training, equipment, and reagents.                yield positive cultures. Under the con-
   Results received from 51 of the labor-         ditions described, the fluorescent-anti-
atories which tested 27,966 paired                body test was specific for Group A
throat swabs by culture-precipitin and            streptococci. The importance of using
fluorescent-antibody methods were in              trained personnel, standardized reagents,
agreement in approximately 95 per cent            properly controlled tests, and reliable
of the specimens. Of 6,223 pairs that             equipment was suggested as necessary
were positive by either method, 5,574             for successful use of fluorescent-antibody
(89 per cent) were positive by fluores-           tests on a routine basis.

JULY. 1963                                                                              1 089
   ACKNOWLEDGMENTS-The authors wish to                      Antibody Technique versus Cultural Methods as a
acknowledge the support for this evaluation                 Routine Procedure for Finding Beta-Hemolytic
provided by the PHS Heart Disease Control                   Streptococci in Throat Cultures. Pub. Health Lab.
                                                            17:76-82, 1959.
Program; the cooperation and advice of Dr.               7. Warfield, M. A.; Page, R. H.; Zuelzer, W. W.;
Gene Stollerman, Northwestern University;                   and Stulberg, C. S. Immunofluorescence in Diag-
and Mr. Paul Frank, Great Lakes Naval Medi-                 nostic Bacteriology. II. Identification of Group A
                                                              Streptococci in Throat Smears. A.M.A. Am. J.
cal Research Unit No. 4, for arranging for                   Dis. Child. 101 :160-163, 1961.
and collecting throat swabs from patients.                8. Lancefield, R. C. Micro-Precipitin-Technique for
                                                             Classifying Streptococci. Proc. Soc. Exper. Biol. &
                                                             Med. 38:473-478, 1938.
                                                          9. Gornall, A. G.; Bardawil, C. J.; and David, M. M.
 REFERENCES                                                  Determination of Serum Proteins by Means of the
                                                             Biuret Method. J. Biol. Chem. 177:751-766, 1949.
 1. Moody, M. D.; Ellis, E. C.; and Updyke, E. L.       10. Riggs, J. L.; Seiwald, R. J.; Burckhalter, J.;
    Staining Bacterial Smears with Fluorescent Anti-         Downs, C. M.; and Metcalf, T. G. Isothiocyanate
    body. IV. Grouping Streptococci with Fluorescent         Compounds as Fluorescent Labelling Agents for
    Antibody. J. Bact. 75:553-560, 1958.                     Immune Serum. Am. J. Path. 34:1081-1097, 1958.
 2. Hollinger, N. F., and Lindberg, L. G. Delayed       11. Marshall, J. D.; Eveland, W. C.; and Smith, C. W.
    Recovery of Streptococci from Throat Swabs.              Superiority of Fluorescein Isothiocyanate (Riggs)
    A.J.P.H. 48:1162-1169, 1958.                             for Fluorescent Antibody Technique with a Modifica-
 3. Yarashus, D., and Siegel, A. Unpublished results,        tion of Its Application. Proc. Soc. Exper. Biol. &
    1961.                                                    Med. 98:898-900, 1958.
 4. Redys, J. J.; Ross, M. R.; and Borman, E. K.        12. Moody, M. D. Unpublished material, 1961.
    Inhibition of Common-Antigen Fluorescence in        13. Pittman, B., and Moody, M. D. "Staining Staphy-
    Grouping Streptococci by the Fluorescent Antibody        lococcus aureus with Fluorescein-Labeled Globulin
    Method. J. Bact. 80:823-829, 1960.                       from Nonimmunized and Streptococcus-Immunized
 5. Updyke, E. L. Unpublished material, 1961.                Animals." Bacteriological Proc. Detroit, Mich.:
 6. Wolfe, M. D., and Cameron, G. M. Fluorescent             American Society for Microbiology, 1960, p. 140.

            Dr. Moody is chief, Staphylococcus-Streptococcus Unit, Bacteriology Section,
          Laboratory Branch, Communicable Disease Center, Atlanta, Ga.; Dr. Siegel is
          in charge of the Streptococcus Laboratory, Children's Memorial Hospital,
          Chicago, Ill.; Miss Pittman and Mrs. Winter are medical bacteriologists,
          formerly associated with Dr. Moody.
            This study was conducted by the Communicable Disease Center, Public
          Health Service, U. S. Department of Health, Education, and Welfare, Atlanta,


I. Preparation of Serums                                 C. Resuspend precipitate in cold half-satu-
A. Group A streptococcus antiserum                          rated ammonium sulfate, centrifuge, dis-
    1. Prepare antiserum in rabbits by inject-               card supernatant fluid.
       ing a formalin-killed whole cell antigen          D. Repeat step C
       of Group A streptococcus, preferably              E. Dissolve the final precipitate in distilled
       from a strain in which "M" protein can               water and adjust to the volume of the
       no longer be demonstrated.                           original serum.
    2. Prepare a pool of antiserums which                F. Remove ammonium sulfate by dialysis
       react strongly only with Group A                     against 0.85 per cent sodium chloride at
       streptococci in precipitin tests.8                   00 C to 50 C.
B. Normal rabbit serums                                  G. Determine protein content by a biuret test9
   Using nonimmunized rabbits, prepare a pool               using a standard protein solution of bovine
of serums which fail to give precipitin reac-               albumin (Armour Laboratories) for estab-
tions with streptococcal Groups A, B, C, D,                 lishing a standard curve. If the protein
F, and G.                                                   content is at least 1.0-g per cent, proceed
                                                            with labeling, or if less than l-g per cent,
IL Preparation of Globulin                                  concentrate at room temperature by sus-
A. Mix equal parts of serum and saturated                   pending the globulin in dialysis tubing
   ammonium sulfate and allow to stand over-                in front of a fan.
   night at 00 C to 50 C.                               III. Labeling Globulin with Fluorescein Iso-
B. Centrifuge and decant supernatant fluid.                   thiocyanate

1 090                                                                        VOL. 53. NO. 7. A.J.P.H.
                                                                    GROUP A STREPTOCOCCI

A. Method (modification of Riggs, et al.,         5. Blot, mount with glycerol-saline and a
    195810)                                          coverslip, and examine on the fluorescence
    1. Place globulin in Erlenmeyer flask and,       microscope.
       while stirring, add an equal volume of
       0.85 per cent NaCl.                        V. Absorption of Conjugates with Streptococci
   2. Add carbonate-bicarbonate buffer (0.05      A. Grow large quantities of Group C and
       M concentration, pH 9.0) using a              Group A streptococci in an enriched Todd-
       volume equal to 15 per cent of the            Hewitt Broth prepared as follows: Prepare
       volume of globulin-saline mixture.            two liters of enrichment solution by dis-
   3. Add acetone (Reagent grade) dropwise           solving in distilled water 120-g dextrose,
       using a volume equal to 12 per cent of        80-g sodium bicarbonate, 32-g sodium phos-
       the volume of globulin-saline mixture.        phate dibasic, and 80-g sodium chloride;
   4. Check the pH rapidly. If under pH              sterilize by filtration. Use 750 ml of sterile
       9.0, add a few drops of pH 11.0 car-          enrichment solution for each 15 liters of
       bonate buffer.                                Todd-Hewitt Broth (Difco).
   5. Suspend fluorescein isothiocyanate (0.05
       mg per mg protein) in 1.5-ml acetone       B. Add formalin (0.25 per cent final concen-
       and add slurry to buffered globulin.          tration) and refrigerate 48 hours.
   6. Stopper flask with rubber stopper and       C. Collect cells by centrifugation and wash
       place on slowly rotating shaker over-         packed cells three times with phosphate
       night at O°C to 50 C.                         buffered saline (pH 7.2).
   7. Sorb conjugate with Dowex 2- x 4            D. Mix two parts labeled globulin with one
       (Dow Chemical Company) by mixing              part of packed cells (Group A conjugate
       equal volumes of conjugate and Dowex          with Group C cells and normal rabbit
       and incubating two hours at 370 C or          conjugate with Group A cells). Allow to
       overnight at 00 C to 50 C.                    stand at 370 C for two hours or at 00 C
   8. Collect sorbed conjugate by pipetting          to 50 C overnight with occasional stirring.
       through glass wool.
   9. Dialyze at 00 C to 50 C against 0.85        E. Centrifuge and collect absorbed conjugates.
       per cent sodium chloride buffered at pH       More complete removal of cells used for
       7.2 with 0.01 M phosphate buffer for          absorption may be obtained by filtration.
       approximately 48 hours or until un-        F. If necessary, repeat the absorption if a
       reacted fluorescein ceases to appear in       greater level of specificity is desired. Re-
       the dialysate.                                peated absorptions may be expected to
B. Method 2 (modification of Marshall, et al.,       reduce homologous staining titers, however.
   195811)                                        G. Check pH. Adjust to approximately 7.2-
   Method 2 is similar to Method 1 except            7.4 with phosphate buffer.
that dry fluorescein isothiocyanate is added
directly to the globulin-saline-buffer mixture.   VI. Testing Absorbed Conjugates
Extreme care is required to bring all particles   A. Determine staining titers as indicated in
of dye into solution at the beginning of the         step IV.
reaction period.
                                                  B. Dilutions of Group A conjugate selected
IV. Testing of Conjugates Before Absorption          for routine use should stain representative
   Determine staining titers by testing several      strains of Group A streptococci at a 3-4+
strains of Groups A, C, and G streptococci           level of brilliancy. At this dilution occa-
and Staphylococcus aureus using varying dilu-        sional strains of Group C and G strepto-
tions of conjugate as follows:                       cocci may stain at moderate levels of bril-
1. Make smears from cells grown two to five          liancy, and with lower dilutions, more
    hours in Todd-Hewitt Broth and washed            brilliantly. Hence, such possible cross-
    in 0.85 per cent NaCl. (Smears may be            reactions must be known when the reagents
    prepared and frozen until ready for testing      are used. The normal rabbit and Group A
    conjugates.)                                     conjugates should be used at the same
2. Fix smears gently with heat or with               dilutions.
    ethanol (95 per cent or absolute for one
    minute).                                      VII. Preservation and Storage of Conjugates
3. Stain 15 to 30 minutes at room tempera-          Conjugates may be preserved with either
    ture in a moist atmosphere.                   Merthiolate or phenylmercuric borate (1:
4. Rinse ten minutes in buffered saline (pH       10,000), dispensed in small amounts, and
    7.2).                                         stored at 00 C to 50 C, frozen, or lyophilized

JULY. 1963                                                                                   1 091
and held at room temperature for at least two          from a small saline suspension of beta
years without serious deterioration.                   hemolytic colonies from blood agar plates.
                                                       If time permits, a young broth subculture
VIII. Equipment for Fluorescence Microscopy            of such colonies is desirable, in which case
   For examining the smears, use a good                the broth would be handled as in step C.
quality monocular microscope fitted with a        E.   Allow smears to air-dry and fix one min-
cardioid darkfield condenser and oil immer-            ute in 95 per cent or absolute ethanol.
sion lens. For illumination use an Osram               Rinse off in buffered saline, pH 7.2, and
High Pressure HBO-200 Mercury Vapor                    blot gently.
Lamp. Proper wave lengths of light are ob-
tained with a Schott BG-12 pass filter, 3-mm      F.   Stain one smear each with appropriate
thickness, and a 2-mm Schott OG-1 barrier              dilutions of absorbed Group A strepto-
filter. Satisfactory photographs may be ob-            coccus and normal rabbit conjugates for
tained with Super Anscochrome or Tri XXX               15 to 30 minutes under a petri dish lid
film.                                                  containing moist filter paper. If the fluo-
                                                       rescence-inhibition test is used, each of the
IX. Examination of Throat Swabs for Group              reagents is mixed with an equal volume of
     A Streptococci by Fluorescent-Antibody            an appropriate dilution of Group C strep-
     Tests                                             tococcus precipitin grouping antiserum and
                                                       the mixtures are used to stain smears.
A. Place throat swab into 1-ml Todd-Hewitt                Equally effective inhibition of cross-reac-
   Broth and incubate two to five hours at             tions, however, of the Group A and normal
   370 C.                                              rabbit conjugates for Group C and G strep-
B. Drain swab against side of tube, replace in         tococci and S. aureus can be accomplished
   sterile test tube, and store at 00 C to 50 C        by using mixtures of dilutions of either
   in case cultural tests are desired.                 conjugate and a dilution of serum from
                                                       most normal rabbits (unpublished material).
C. Centrifuge broth three to four minutes to
   pack sediment. Carefully decant superna-       G.   Rinse ten minutes in buffered saline, then
   tant fluid, resuspend sediment in 1-ml buf-         momentarily in distilled water. Blot gently.
   fered 0.85 per cent NaCl, centrifuge, and      H.   Add a drop of glycerol-saline and a cover-
   discard supernatant fluid.                          slip.
D. Using a capillary pipette and most of the      I.   Examine on the fluorescence microscope
   sediment, prepare duplicate smears on a             with oil immersion. Not more than three
   slide inscribed with two circular areas.            minutes should be required to read a
   Stopper tube with wax-coated cork and               smear. Note the presence of fluorescent
   refrigerate in case additional smears are           cocci, whether or not they are in chains,
   needed. Smears may also be prepared                 and the intensity of fluorescence.

1 092                                                                  VOL. 53, NO. 7. A.J.P.H.

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