JOURNAL OF CLINICAL MICROBIOLOGY, OCt. 1983, p. 779-785 Vol. 18, No. 40095-1137/83/100779-07$02.00/0Copyright C 1983, American Society for Microbiology

Granada Medium for Detection and Identification of Group BStreptococci

MANUEL DE LA ROSA,* RICARDO VILLAREAL, DOLORES VEGA, CONSUELO MIRANDA, ANDANTONIO MARTINEZBROCAL

Servicio de Bacteriologia, Ciudad Sanitaria Virgen de las Nieves, Granada, Spain

Received 16 March 1983/Accepted 30 June 1983

A new starch serum medium, Granada medium, for isolation and identificationof group B streptococci (GBS) anaerobically as red colonies is described. Themedium contains 3.8% Proteose Peptone no. 3 (Difco), 15% soluble starch 1252(Merck), 10%o coagulated horse serum, 15 ,ug of trimethoprim per ml, and 0.06 Mphosphate buffer (pH 7.8; medium pH 7.4). This medium inhibited fecal flora andat the same time supported growth of GBS. A new pigment-enhancing effect offolate inhibitors on GBS is reported and used in the formulation of the medium.The good selective and differential properties of the Granada medium favorquicker and easier detection ofGBS in heavily contaminated specimens. Since themedium is convenient to use and requires only 18 h of incubation to detect andidentify GBS, it should be useful in any clinical microbiology laboratory andwould assist in the early detection of GBS in clinical specimens.

Many methods for the detection of Strepto-coccus agalactiae (Lancefield group B strepto-cocci [GBS]) in genital or fecal specimens ob-tained from mothers before or during deliveryhave been described. The techniques reportedinclude two incubations (2), counterimmuno-electrophoresis (11, 16), fluorescence microsco-py (6), coagglutination (32), latex agglutination(26), and complex selective media (2, 12, 28, 34).Direct identification methods have also beenreported (6a, 30).Some culture media for GBS identification are

based on production of a red pigment underanaerobiosis on starch-containing media (10, 17,18, 22, 24), and, although the possibility ofrecognizing GBS directly in clinical specimenson such media has been put forward (17, 27, 33,34), they have mainly been used to identify beta-hemolytic colonies suspected of being GBS.The development of Granada medium started

from the original formulation of starch serumagar medium described by Islam (17). To makeIslam medium opaque and to avoid possibleinhibitors from horse serum, we modified it byadding the serum before the medium had cooled(90 to 95°C).With this modified medium, it was observed

that GBS colonies were more pigmented andeasier to distinguish against the opaque yellow-ish medium and that an increase in concentra-tions of starch or serum led to an increase inpigmentation.On the other hand, the use of concentrations

of soluble starch above 5% interfered with gela-

tion and caused precipitates difficult to disperse.Preliminary work showed that partial gelation ofstarch could be avoided if the medium wascontinuously stirred while being heated, that itwas not necessary to autoclave these mediabecause there were no contamination problems,and that media containing more than 10% solu-ble starch (Lintner type) became white andsolidified without adding agar, gelling at roomtemperature within 1 to 2 h.We, hypothesized that it was possible, by

applying the theory of achievement of optimumconditions (4, 5), to design from the starchserum agar medium an optimum medium onwhich GBS would grow quickly as distinctlypigmented colonies, even in heavily contaminat-ed specimens (such as vaginal or rectal sam-ples). This medium would allow rapid and accu-rate detection and identification of GBS andrapid diagnosis of colonized gravid women atrisk; in some cases it would permit the selectiveuse of intrapartum antibiotics (35), and in othercases it would alert pediatricians to the presenceof GBS at the earliest possible time.

MATERIALS AND METHODS

Organisms. One hundred consecutive isolates ofGBS were obtained from routine clinical specimensfrom different patients in the Microbiology Laboratoryof the Social Security Hospital, Granada, Spain. Sev-enty strains were from vaginal or cervical specimens,six were from urine, two were from blood cultures,nine were from wound infections, two were fromcatheters, three were from umbilici, four were from

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loquia, one was from a urethra, and three were fromcolonized newborns. Primary isolation was made onblood agar plates (Columbia agar base [Oxoid Ltd.,Basingstoke, England] plus 5% horse blood). Suspect-ed colonies were identified by accepted procedures(9). Coagglutination (Phadebact Strep B test; Pharma-cia Diagnostic, Uppsala, Sweden) was used for sero-logical identification. GBS strains were serotyped inthe Centro Nacional de Microbiologia, Majadahonda,Madrid, Spain, and the distribution of serologicaltypes was as follows: Ia, 20%; Ib, 18%; Ic, 3%; II, 9%;III, 40%. Ten percent of strains were nontypable.Strains were stored frozen (- 80°C) in brain heartinfusion broth (BHIB) (Difco Laboratories, Detroit,Mich.) plus 10% glycerol. To obtain cells for experi-ments, samples from the frozen cultures were subcul-tured onto blood agar plates and incubated overnightat 36°C. After incubation, several colonies were grownin 3 ml of BHIB for 18 h at 36°C.

Simulated specimens. To evaluate the detecting abili-ty of the media, heavily contaminated simulated speci-mens were prepared from human stools. Stool speci-mens were diluted to 1/4 in 0.05 M sodium phosphatebuffer (pH 7.2) and blended in a Vortex mixer, and 1-ml samples from each were maintained frozen (-80°C)until needed. Fecal specimens were previouslystreaked onto plates of Granada medium (see below)and incubated anaerobically for 48 h (36°C); stoolspositive for GBS were disregarded. In each replicateof the experiments, 0.01 ml of the 1/200 dilution inBHIB of the 18-h cultures of GBS were added to thediluted stools (1-ml samples) and blended vigorously ina Vortex mixer to distribute the organisms (ca. 104CFU/ml).

Plating methods. For experiments with pure culturesofGBS, overnight cultures in BHIB were diluted 1/200in BHIB, and 0.005 ml was spread onto the plates (ca.5 x 103 CFU).With simulated specimens, samples of 0.01 ml were

streaked on the plates, endeavoring to obtain isolatedcolonies (but without flaming the loop) (ca. 102 CFU ofGBS per plate).

Unless otherwise stated, plates were incubated for18 h at 36°C in an anaerobic chamber (model 1024;Forma Scientific, Marietta, Ohio); the gas mixtureconsisted of 85% nitrogen, 10% hydrogen, and 5%carbon dioxide.Pigment intensity (response variable). As our goal

was to design a culture medium on which GBS had amaximum yield of pigment, pigment intensity becamethe response variable.

After each experiment, plates were arranged ac-cording to the intensity of pigment (in each plate therewas a different strain and different medium). Thepigment intensity was scored on a 0 to 10 scale, and,since each experiment was done with at least 80 plates,there was no difficulty in ranking them. Several ob-servers usually agreed with the score assigned to eachplate.

Experimental design and analysis of data. Experi-ments with pure cultures of SGB were planned asrandomized complete block designs (7) and were runwith 10 strains (chosen at random from the populationofGBS stock cultures) and completely replicated threetimes. Each strain was considered as a block.Experiments with simulated specimens were also

randomized complete block designs (each simulated

specimen was considered as a block) with 16 blocksand also replicated three times.Data (pigment intensity) were analyzed after a rank

transformation, as suggested by Conover (8) by using afixed effects model of analysis of variance (ANOVA)(31). However, as this procedure gave results nearlyidentical to the usual ANOVA, the regular parametricanalysis was considered valid and was used (8).Data from one-way ANOVA were analyzed, com-

puting the last significant difference. Data from experi-ments planned to attain the optimum conditions werecomputed to fit a response surface equation by stan-dard statistical methods (31).

Experiments aimed to evaluate media by usingsimulated specimens were analyzed by the Cochranan4dFriedman tests for related samples (8).MIC determinations. Minimal inhibitory concentra-

tions (MICs) were determined to select suitable anti-microbial agents to make Granada medium selectivefor GBS. The MICs of gentamicin, tobramycin, amika-cin, trimethoprim (TMP), sulfadiazine (SDZ), nitrofur-antoin, nalidixic acid, pipemidic acid, and polymyxinB were determined with the agar dilution method onMueller-Hinton agar (Oxoid) plus 25 g of ProteosePeptone no. 3 (PP3) (Difco) per liter and Granadamedium, under aerobic (7% C02) and anaerobic condi-tions, using a Steer replicator. Nalidixic acid, SDZ,and nitrofurantoin were purchased from Sigma Chemi-cal Co., St. Louis, Mo., and other antimicrobial agentswere supplied as laboratory standards by pharmaceuti-cal companies. The inoculum size was approximately104 CFU, and results were read after 18 h of incuba-tion at 36°C. The MIC was defined as the lowestconcentration that gave growth inhibition.Pigment production and effect of inhibitors of folate

pathway. GBS strains were inoculated on plates ofColumbia agar (Oxoid), Islam medium (17), DMS (22),and Granada medium with a Steer replicating device(ca. 104 CFU) and incubated anaerobically (18 h at36°C). The strains were also stabbed into tubes ofDMS medium and incubated aerobically (18 h at 36°C).In all cases the presence or absence of reddish pigmen-tation of spots or stabs was recorded.GBS strains were also plated (swabs dipped in a

1/200 dilution in BHIB of overnight broth cultureswere streaked evenly over the surface) onto two platesof Granada medium without TMP and a plate ofGranada medium. Two filter paper disks, one contain-ing 50 ,ug of TMP and the other containing 300 ,ug ofSDZ, were applied to the inoculated surface of theplates of the medium without TMP. Two disks, onecontaining 25 ,ug of thymine (Sigma) and the othercontaining 25 p.g of thymidine (Sigma), were applied tothe plates of Granada medium. One plate of eachmedium was incubated aerobically (18 h at 36°C in 7%C02), and the other plate (medium without TMP) wasincubated anaerobically (18 h at 36°C). The formationof a zone of enhanced pigmentation around the TMPor SDZ disk was recorded as a positive pigment-enhancing effect.

Optimization and evaluation of Granada medium.Experiments to optimize the medium started from thefollowing formulation: NaCl, 3 g; soluble starch 1252(Merck, Darmstadt, Federal Republic of Germany),135 g; PP3, 25 g; horse serum, 70 ml. Sodium phos-phate buffer (0.06 M; pH 7.4) was added to the NaCl,starch, and PP3 to make up 930 ml; they were dis-

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solved by boiling for 30 min in a water bath, and then70 ml of horse serum was added while stirring continu-ously.The optimization of the medium was achieved by

using experiments with pure cultures of GBS:(i) Determining the optimum starch concentration,

pH, and time of heating with the one-factor-at-a-timemethod (7). Concentrations of starch from 125 to 265g/liter (in steps of 35 g/liter) were tested. The optimumpH was investigated by testing media prepared byusing 0.06 M sodium phosphate buffer with pH rangingfrom 6.6 to 8 (in steps of 0.2). The effect of the durationof heating was evaluated by testing the media preparedhaving varied the heating time from 20 min to 3 h (20and 40 min and 1, 2, and 3 h).

(ii) Determining (linear regression- analysis) the pathof steepest ascent (5) related to changes in concentra-tion of PP3, horse serum, and TMP to increase theyield of pigment in the most efficient manner.

(iii) Fitting a quadratic response surface to theexperimental data (pigment intensity) from a factorialexperiment (1) and calculating the optimum combina-tion of factors (concentrations of PP3, horse serum,and TMP) that maximized the response variable (pig-ment intensity).The theoretically optimized medium, using simulat-

ed specimens, was evaluated against: (i) the mediumwithout TMP, (ii) media containing 10, 20, and 40 ,ug ofpipemidic acid per ml, (iii) media containing 5, 10, 15,20, and 30 ,zg of TMP per ml, and (iv) Islam medium.After incubation, plates were scored from 0 (no GBScolonies visible) to 3+ (more than 50 red colonies).The following formulation of Granada medium was

adopted after the experiments with simulated speci-mens: Proteose Peptone no. 3 (Difco), 38 g; solublestarch (1252 Merck), 150 g; sodium chloride, 3 g; TMP(as lactate), 15 mg; horse serum, 100 ml; and 0.06 Msodium phosphate buffer (pH 7.8), up to 1,000 ml.

Preparation directions. Dissolve PP3 and NaCl in200 ml of buffer by heating gently in a flask. Then addthe following in this order: 400 ml of cold buffer, starch(stirring continuously), TMP, and buffer up to 900 ml.Place the flask in a boiling bath, and heat with frequentagitation for 20 min. Mixing during heating is essentialto avoid the formation of starch precipitates.Remove the flask from the boiling bath and, stirring

continuously, immediately add 100 ml of sterile horseserum. Allow to cool to 60 to 65°C, with frequentagitation, and pour plates (thin plates are prone todesiccation and crack easily). Allow to stand at roomtemperature for 2 to 3 h and store in sealed plastic bagsat 4 to 100C.As the gelling capacity and pigment-enhancing abili-

ty of starch may vary from lot to lot, it is imperative totest the performance of each batch of Granada mediumthat is prepared with an appropriate GBS strain. Poorresults can be obtained with some lots of starch.

Modifications of Granada medium. Several modifica-tions of the medium were investigated by means ofexperiments using GBS pure cultures. (i) Replacementof Merck starch 1252 with other Litner-type starches:Sigma S-2630, Merck 1253, Probus (Barcelona, Spain),Panreac (Barcelona, Spain), and eight products ob-tained in our laboratory by partial hydrolysis (HCI, 1N, 36°C, 1 to 8 days) followed by elimination of solublesugars of washed (0.05 M NaOH) and methanol-water(80:20 [vol/vol]) defatted (13) corn starch (Maizena).

(ii) Replacement of PP3 with other protein hydroly-sates: Casitone (Difco), tryptose (Oxoid), tryptone(Difco), or Casamino Acids (Difco). Two concentra-tions of each (25 and 35 g/liter) and a medium contain-ing no protein hydrolysate were tested. (iii) Replace-ment of horse serum with 5 g of beef extract (Difco),Lab lenco (Oxoid), or bovine albumin (fraction IX;Sigma) per liter. A formulation from which horseserum was eliminated was also tested. (iv) Addition of1% yeast extract (Difco), 5% Fildes extract (Oxoid),2% Vitox (Oxoid), 1% RNA (Sigma), 1% DNA (Sig-ma), 0.05 and 0.1% sodium pyruvate (Sigma), or 0.1%sodium acetate (Merck). (v) Replacement of PP3 by 35g of tryptose, tryptone, or Casamino Acids per literplus 1% yeast extract.

RESULTSOptimum pigmentation of GBS was obtained

with the medium at pH 7.4, obtained by incorpo-rating a sodium phosphate buffer at pH 7.8.When the pH of the buffer was below 7 (pH ofthe medium below 6.8), the formation of pigmentdiminished significantly.Maximum pigment yield was obtained by us-

ing a boiling time of 20 min, noting that themedium significantly lost its ability to supportpigment production when boiled for 40 min orlonger.The calculated optimum concentrations of

medium constituents were starch, 150 g/liter;PP3, 38 g/liter; horse serum, 100 ml/liter; andTMP, 25 ,ug/ml.The GBS strains tested were resistant (when

tested in Granada medium or in Mueller-Hintonbroth supplemented with PP3) to polymyxin B,nalidixic acid, pipemidic acid, TMP, SDZ, gen-tamicin, tobramycin, and amikacin and fairlysusceptible to nitrofurantoin (Table 1). Occa-sional isolates showed only moderate resistanceto the aminoglycosides. It was also found thatnalidixic and pipemidic acids, even when pres-ent at low concentrations in Granada medium(10 .LgIml), lead to a slower growth of GBS.Experiments with simulated specimens (ca.

102 CFU of GBS) showed that the mediuminhibited the fecal flora (when incubated anaero-bically), and hence there was no problem indetecting red GBS colonies.An unexpected result from the determination

of the MICs of TMP and SDZ was that, anaero-bically, these inhibitors of folate strongly en-hanced GBS pigment. This effect could be dem-onstrated in all the pigmented strains tested withconcentrations of TMP and SDZ above 2 jxg/ml.In the experiments planned to confirm this, allpigmented strains showed, after 18 h of anaero-bic incubation, a distinct zone of enhanced pig-mentation around the TMP and SDZ disks (Ta-ble 2), although the zone was more intensearound the TMP disk. This effect of TMP andSDZ could not be observed aerobically with any

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TABLE 1. Summary of antimicrobial susceptibility of 100 isolates of S. agalactiaea

Medium and atmosphere Gentamicin Tobramycin Amikacin Nalidixic acidMIC5 MIC50 MIC5 MIC50 MIC5 MIC50 MIC5 MICm

Mueller-Hinton + 2.5%pp3bAerobiosis (7% CO2) 32 32 16 32 64 64 >256 >256Anaerobiosis 32 32 16 32 64 128 >256 >256

GranadaAerobiosis (7% CO2) 64 >128 64 >128 256 >256 >256 >256Anaerobiosis 64 >128 64 >128 256 >256 >256 >256

a MIC5, Concentration required to inhibit 5% of strains; MIC50 concentration required to inhibit 50% ofstrains. Data are in micrograms per milliliter.

b PP3, Proteose Peptone no. 3 (Difco).

GBS strain and aerobically or anaerobically withthe nonpigmented strains.

Inhibition of pigment around the thymine orthe thymidine disk was not observed in any caseon Granada medium plates.

Rates of GBS isolation, using simulated speci-mens, did not show (Cochran test) a significantdifference between the theoretically optimizedGranada medium and media with different con-centrations ofTMP or pipemidic acid. However,when the scores of the plates (0 to 3+) werecompared (Friedman test), the medium contain-ing 15 ,g ofTMP per ml performed significantlybetter; therefore, this TMP concentration wasincluded in the final formulation of the medium.

In experiments with simulated specimens, Is-lam medium was not able to detect GBS in anycase because of the confluent growth that hin-dered detection.

Experiments with modifications of Granadamedium showed the following. (i) The type ofstarch is important, and poor results (pigmentintensity values lower than 5) were obtainedwith all the other starches tested, except withthe soluble starch prepared by hydrolyzing de-fatted corn starch for 4 days. (ii) PP3 cannot beeliminated or substituted by any of the proteinhydrolysates tested, as media prepared withthem and the medium without any protein hy-drolysate were not able to support the growth ofGBS as pigmented colonies. (iii) Pigment pro-duction was significantly diminished if horseserum was eliminated or substituted, except bybeef extract, but this medium had a strongbrown coloration, and GBS colonies were moredifficult to observe. (iv) Sodium acetate andyeast extract did not influence pigment produc-tion. Sodium pyruvate and RNA significantlylowered the pigment yield. Pigment was notobserved in media containing Vitox, Fildes ex-tract, or DNA. (v) Media in which PP3 wassubstituted by any of the mentioned proteinhydrolysates plus 1% yeast extract did not sup-

port pigment production of GBS.A summary of the results of pigment produc-

tion, pigment induction and other identificationtests on the 100 isolates of GBS tested is shownin Table 2. Each of the GBS presumptive tests,CAMP, hippurate hydrolysis, and pigment pro-duction, gave about the same proportion ofpositive results. All the strains that producedpigment were also hemolytic.

DISCUSSIONAlthough the identification of GBS can be

accomplished by different tests, most of themare time consuming, labor intensive, or unreli-able (33). The use of selective media has beensuggested to enhance the recovery of GBS fromvaginal cultures (2, 26, 34), but inhibitory agentsmay actually impede the growth of some GBSstrains (12), and their usefulness has been ques-tioned (20).

Recently, the ability of GBS to produce (an-aerobically) a red pigment has received consid-erable attention as a distinctive and characteris-tic identification test (10, 14, 15, 17, 18, 22-24,33). This pigment has characteristics of a carot-enoid localized in a membranous fraction of thecell (23), and, although similar pigments areproduced by other gram-positive cocci (23),false identification of GBS by pigment detectionhas not been reported (33). In addition, it hasbeen suggested (R. A. Nemergut and K. Merrit,Abstr. Annu. Meet. Am. Soc. Microbiol. 1982,B147, p. 42. and 1983, B32, p. 28) that pigment isimportant in the pathogenicity of GBS; there-fore, this characteristic might help in the evalua-tion of its role in disease.

Furthermore, a plating medium relying onpigment to detect GBS has the advantage that itcan be read by nontrained personnel, and, with adisposable anaerobic system, it may be used inany laboratory or maternity unit.Columbia agar has been proposed as a plating

medium to detect pigment production by GBS,

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TABLE 1-Continued

Pipemidic acid Polymyxin B Nitrofurantoin Sulfadiazin TMP

MIC5 MIC50 MIC5 MIC50 MIC5 MIC50 MICs MIC50 MIC5 MIC50

>256 >256 >512 >512 8 16 512 >2,048 128 256>256 >256 >512 >512 4 8 512 >2,048 32 64

>256 >256 >512 >512 8 16 512 >2,048 >1,024 >1,024128 >256 >512 >512 4 4 512 >2,048 >1,024 >1,024

but irregular results, ranging from 97% of posi-tives (19) to 0% (22) (12% in this series), havebeen reported.

In 1977 Islam (17) proposed a starch serumagar as a pigment-enhancing medium for detec-tion of GBS pigment. During 1981 we used thismedium routinely for the identification of GBS.Although it performed perfectly when used withpure cultures, GBS colonies were easily over-grown when contaminated specimens, such asthose of vaginal or rectal origin, were directlyplated, and therefore they could not be distin-guished. Islam medium was chosen as the start-ing point for the development of Granada medi-um because it supports adequate growth of GBSanaerobically as pigmented colonies (17, 33).To develop the new medium, the methodology

on the attainment of optimum conditions (4, 5)has been applied. Although this approach iscommonly used in other fields (1, 4, 7), to thebest of our knowledge, it has not been applied tothe design of a culture medium, perhaps becauseof the problems of variability of bacteria andmedium components and the difficulties ofchoosing and analyzing a suitable response vari-able, which usually has to be measured in anominal or ordinal scale. However, these prob-lems could be partially overcome if the mediumhad a definite goal, if the response variable couldbe measured at least in an ordinal scale, if asuitable experimental design were applied, and ifenough replication were used in the experi-ments.We hypothesized that this theoretical ap-

proach could be applied to the design of anoptimum culture medium for detection of GBSas pigmented colonies.

It should be mentioned that the result of ourinvestigation on the effect of pH on pigmentproduction supports the findings of Fallon (10)and Noble et al. (27) that the medium pH shouldnot be below 7.3.Our results have also confirmed the previous

recommendation of including starch as a compo-nent of media (10, 17, 22) for detecting GBSpigment. They have also shown the influence ofconcentration, type, degree of hydrolysis, andresidual fatty acid content of starches on theirpigment-enhancing properties. The effect ofsuch a high concentration of starch in the Grana-da medium might be explained by a nonspecificabsorption effect (21) related to the absorptiveaffinity of amylose (25, 29).Although we have not systematically investi-

gated the component or components of PP3 andserum that enhance pigment production, ourdata agree with the observations of Islam (17)and Merrit and Jacobs (22) about the importanceof PP3 and show striking differences with thepigment-enhancing ability of the protein hydro-lysates and meat extracts.

It is interesting that, despite the high MIC ofTMP (in Granada medium), there was an inhibi-tory effect on the growth of GBS when lowinocula were used (as in simulated specimens)that prevents its utilization as a pigment-en-hancer in concentrations above 15 ,ug/ml.These results coincide with the findings of

Bopp et al. (3) on the inactivity of TMP againstother TMP-susceptible gram-positive cocci inmedia containing nutritionally rich ingredientswhich are likely to contain high levels of thymi-dine.

Furthermore, our observations on pigmentinhibition on media containing DNA, Fildesextract, and Vitox and our findings on the pig-ment-enhancing effect, anaerobically, of folateinhibitors (an effect that we have not been ableto antagonize with either thymine or thymidine)suggest a metabolic pathway of pigment synthe-sis that can be triggered or repressed. Furtherwork would be necessary for clarification of thispoint.Our results also coincide with previous re-

ports (17, 22, 27) on the association betweenhemolysis and pigment production in GBS. To

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TABLE 2. Reactions exhibited by 100 clinicalisolates of S. agalactiae

No. (%) of positive strainsTest Pigmented' Nonpigmentedb

(n = 96) (n = 4)

Pigment onIslam medium plates 96 (100) 0 (0)Granada medium plates 96 (100) 0 (0)Columbia agar plates 11 (12) 0 (0)DMS plates 0 (0) 0 (0)DMS stabs 86 (91) 0 (0)

Pigment-enhancing effect ofTMP 96 (100) 0 (0)SDZ 96 (100) 0 (0)

Hemolysis (5% horse blood) 96 (100) 0 (0)

Hippurate hydrolysis 96 (100) 4 (100)

CAMP reaction 96 (100) 4 (100)

Bile-esculin reaction 0 (0) 0 (0)

Growth in 6.5 NaCl 36 (38) 1 (25)

Susceptibility toSXTC 0 (0) 0 (0)Bacitracin 0 (0) 0 (0)

Coagglutination 96 (100) 4 (100)a Strains pigmented on any of the media.b All nonpigmented strains were serotype III.' SXT, TMP-sulfamethoxazole.

explain this association, a close genetic linkagehas been suggested between the genes that de-termine hemolysin and pigment production (27).The results of the present study and the data

from our laboratory, using pigment productionfor identification of GBS in more than 5,000clinical specimens, first with Islam medium andlater with Granada medium, indicate that thischaracteristic (pigment production anaerobical-ly, in a starch-containing medium) is specific forGBS.Our results have also shown that Granada

medium is suitable for detection of GBS inclinical specimens because colonies are readilydistinguishable, even in specimens that exhibitconfluent growth when plated on non-inhibitorymedia.

ACKNOWLEDGMENTS

We thank J. Casals and A. Fenoll, Centro de MicrobiologiaVirologia e Inmunologia, Majadahonda, Madrid, Spain, whokindly performed the serotyping, and E. Bridson, I. Sanz, andM. A. Gillies for review of the manuscript.

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