Effects of transport temperature and medium on recovery of Bordetella pertussis from nasopharyngeal swabs.

We compared relative recoveries of Bordetella pertussis from simulated nasopharyngeal (NP) specimens incubated in three separate transport media at different temperatures. Transport media included one-half-strength Regan-Lowe (RL.5), Regan-Lowe with one-half-strength agar (RL.5A), and buffered charcoal-yeast extract agar supplemented with alpha-ketoglutarate, lincomycin, and anisomycin (BCYE alpha LA). For each transport medium, recovery of B. pertussis was least efficient after storage at 25 degrees C. The highest recovery of B. pertussis from a mixed culture was achieved with RL.5 at 4 degrees C. Overall, RL.5 and RL.5A were comparable as transport media whether held at 4 or 25 degrees C, but fewer organisms were recovered from BCYE alpha LA. In addition, Regan-Lowe (RL), Bordet-Gengou, and cyclodextrin media were compared as primary isolation media for recovering B. pertussis from simulated NP swabs held at 4 and 35 degrees C in RL.5 medium. The highest recovery of B. pertussis was obtained on RL primary isolation medium. Bordet-Gengou medium recovered only 80% and cyclodextrin medium recovered less than 60% of the numbers recovered on RL medium. Based on these results, refrigeration (4 degrees C) of NP swabs shipped in RL.5 transport medium and using RL as the primary isolation medium are recommended for recovering B. pertussis from swab specimens.     

Novel, rapid optical immunoassay technique for detection of group A streptococci from pharyngeal specimens: comparison with standard culture methods.

A novel immunoassay system based on the changes in the reflection of light, termed an optical immunoassay (OIA), was utilized to directly detect group A streptococcal (GAS) carbohydrate antigen from clinical specimens. In two studies, a total of 1,275 throat swabs were tested for the presence of this antigen with the Strep A OIA rapid detection system and the results were compared with those of standard culture methods. In both studies, the Strep A OIA yielded more positive results than plating of the throat swab onto a selective agar, Trypticase soy agar containing sheep blood, or an enriched broth. In one study, the sensitivity and specificity of Strep A OIA compared with those of the broth-enriched culture were 97.4 and 95.6%, respectively. In a second study a sensitivity of 98.9% and a specificity of 98.6% were achieved. It was also shown that the carbohydrate antigen could be detected in the absence of viable GAS organisms. The Strep A OIA is an easily interpretable method and was shown to be more sensitive than routine culture methods for detecting GAS infections directly from throat swabs.     

The use of selective media for the isolation of Pseudomonas pseudomallei in clinical practice

Ashdown's selective-differential agar medium, with or without preenrichment in selective broth, was evaluated for the isolation of Pseudomonas pseudomallei from 1972 clinical specimens obtained from 643 subjects in Northeast Thailand; 226 patients proved to have meliodosis. The use of Ashdown's medium significantly increased the frequency of recovery of P. pseudomallei from sites or specimens with an extensive normal flora (throat, rectum, wounds and sputum) as compared to the recovery on blood and MacConkey agars (p less than 0.01). The isolation frequency from throat, rectal and wound swabs was further increased by the use of the broth pre-enrichment. The colonial morphology of P. pseudomallei on Ashdown's medium was sufficiently characteristic to allow presumptive identification. With the use of these selective media it was possible to culture P. pseudomallei from throat swabs taken from 87% of the patients from whom the organism could also be isolated from corresponding tracheal aspirates or sputum specimens. P. pseudomallei was isolated from rectal swabs taken from 51 patients, the first time that faecal excretion of the organism has been demonstrated in man. The diagnosis of melioidosis would not have been confirmed bacteriologically in eight patients (3.5%) without the use of the selective media. It is suggested that, in areas endemic for melioidosis, all sputum specimens should be cultured on selective media, such as Ashdown's. For the investigation of clinically suspected cases of melioidosis, and for follow-up during treatment of the disease, the use of broth pre-enrichment is recommended for specimens obtained from sites with an extensive normal flora.     

Comparison of three-compartment Petri dishes and individual plates for routine culture of vaginal swabs.

Recovery of aerobes and facultative anaerobes from 200 consecutive randomly selected high vaginal swabs was evaluated using three-compartment Petri dishes containing Sabouraud, dextrose agar, GC selective agar, and chocolate agar. The method was compared with the traditional method using individual Petri dishes. The two methods produced comparable results both in terms and quantities of organisms recovered from the specimens. As three-compartment Petri dishes use less agar, save time in culturing specimens, yet still maintain the same standard of culture, they provide a more economical alternative to the traditional method for routine culture of vaginal swabs.     

Comparison of three methods for detection of group A streptococci in throat swabs.

Group A streptococci are generally detected in throat swabs by (i) rapid antigen tests, (ii) conventional culture, or (iii) combinations of both. Direct fluorescent-antibody testing of a 2-h enrichment broth (FA/EN) was an accepted method for same-day results before the advent of rapid antigen tests. We compared FA/EN in Todd-Hewitt Broth (THB) with conventional culture and a rapid antigen test, TestPack Strep A (TPS). Nine hundred seventy specimens were evaluated in this study. Cultures were performed for 48 h on sheep blood agar (SBA) incubated aerobically and on a selective agar for group A streptococci (SSA) incubated in 5 to 10% CO2. Following a 2-h incubation, the fluorescent-antibody test was performed. A subculture of the centrifuged sediment from the THB enrichment was also done. In comparison with a positive culture on SBA or SSA or subculture of the THB pellet, the sensitivities and specificities of the different methods were as follows: SBA, 92 and 100%; SSA, 92 and 100%; TPS, 68 and 99%; FA/EN, 88 and 98%. The FA/EN method offers the potential for definitive finalized reports on the same day as specimen collection with greater sensitivity than TPS. This study included sequential plating and rapid antigen testing of a single swab. In a separate set of experiments to validate this study design, it was shown that recovery of streptococci from swabs plated sequentially on five plates did not vary with the order of plating and the actual proportion of organisms recovered from a swab on a single plate was only 1%.     

Comparison of Escherichia coli O157:H7 antigen detection in stool and broth cultures to that in sorbitol-MacConkey agar stool cultures

We evaluated the Meridian IC-STAT direct fecal and broth culture antigen detection methods with samples from children infected with Escherichia coli O157:H7 and correlated the antigen detection results with the culture results. Stools of 16 children who had recently had stool cultures positive for this pathogen (population A) and 102 children with diarrhea of unknown cause (population B) were tested with the IC-STAT device (direct testing). Fecal broth cultures were also tested with this device (broth testing). The results were correlated to a standard of the combined yield from direct culture of stools on sorbitol-MacConkey (SMAC) agar and culture of broth on SMAC agar. Eleven (69%) of the population A stool specimens yielded E. coli O157:H7 when plated directly on SMAC agar. Two more specimens yielded this pathogen when the broth culture was similarly plated. Of these 13 stool specimens, 8 and 13 were positive by direct and broth testing (respective sensitivities, 62 and 100%). Compared to the sensitivity of a simultaneously performed SMAC agar culture, the sensitivity of direct testing was 73%. Three (3%) of the population B stool specimens contained E. coli O157:H7 on SMAC agar culture; one and three of these stool specimens were positive by direct and broth testing, respectively. The direct and broth IC-STAT tests were 100% specific with samples from children from population B. Direct IC-STAT testing of stools is rapid, easily performed, and specific but is insufficiently sensitive to exclude the possibility of infection with E. coli O157:H7. Performing the IC-STAT test with a broth culture increases its sensitivity. However, attempts to recover E. coli O157:H7 by culture should not be abandoned but, rather, should be increased when the IC-STAT test result is positive.     

Comparison of NNA Agar Culture and Selective Broth Culture for Detection of Group B Streptococcal Colonization in Women

In 1996, the Centers for Disease Control and Prevention recommended the use of a selective broth culture for the improved detection of genital tract or anorectal carriage of group B streptococci (GBS) in pregnant women. In order to verify this recommendation in our laboratory, we compared the sensitivity of Todd-Hewitt medium with gentamicin and nalidixic acid (SBM) with our current method of direct plating on blood agar medium containing neomycin and nalidixic acid (NNA). Five hundred consecutive cervicovaginal and anorectal specimens submitted for GBS culture were included in the study. Swabs were plated onto NNA and the swabs were immersed in SBM, followed by overnight incubation at 35°C. On the following day, the NNA plates were examined for colonies typical of GBS and the organisms were identified by the CAMP test or by latex agglutination. SBM cultures were subcultured onto blood agar and CNA agar plates, and the plates were reincubated for 24 h. Negative specimens from either medium were incubated for an additional 24 h and were examined again before finalization of the results. GBS were recovered from 78 specimens by both methods; from SBM only for 17 specimens (sensitivity, 86%) and from NNA only for 16 specimens (sensitivity, 85%). A moderate to heavy growth of Enterococcus faecalis was observed on plates containing NNA-positive, SBM-negative specimens. Competitive growth studies suggested that E. faecalis suppressed the growth potential of GBS in SBM. Our study suggests that direct plating on NNA, as a single method, is equivalent in sensitivity to SBM for the recovery of GBS, and the results are often available 24 h sooner. However, it appears that both direct plating and selective broth amplification techniques are required for the maximum level of identification of colonization with GBS in pregnant women.     

Evaluation of a vanA-Specific PCR Assay for Detection of Vancomycin-Resistant Enterococcus faecium during a Hospital Outbreak

We investigated the use of PCR as an alternative to culture of fecal samples for detection of vanA-containing Enterococcus faecium during a recent hospital outbreak. Rectal swabs collected consecutively from 223 patients were analyzed by culture with and without enrichment broth and by vanA-specific PCR of enrichment broth samples. Fifty-five specimens were positive for vanA-containing E. faecium by at least one method. The sensitivities of the vanA-specific PCR assay and agar culture with and without enrichment broth were 94.5, 98, and 89%, respectively. All three methods were 100% specific. Final results were obtained much more rapidly by PCR (within 24 to 30 h of specimen submission) than by the culture methods (4 to 5 days). Thus, PCR is an accurate and rapid alternative to culture for detection of vancomycin-resistant enterococci during hospital outbreaks.     

Rapid detection of vanA and vanB genes directly from clinical specimens and enrichment broths by real-time multiplex PCR assay

A real-time PCR assay previously developed for use on the Roche LightCycler platform was investigated as an alternative to culture for the direct detection of vancomycin-resistant enterococci (VRE) in clinical specimens. PCR primers and fluorescence resonance energy transfer hybridization probes specific for the vanA and vanB genes were combined in a multiplex real-time PCR assay performed directly with fecal material obtained by rectal swabbing and with enrichment broth samples. DNA was prepared from the rectal swabs and enrichment broths with a commercially available DNA preparation column designed specifically for use with fecal specimens. One hundred eighty duplicate rectal swabs were obtained from 42 patients who were previously found to be positive for VRE and who were being monitored for carriage of VRE. Direct and enrichment broth cultures were performed with one swab, while PCR was performed with the other swab as well as any corresponding presumptive positive enrichment broth. In total, 100 specimens from 30 patients remained positive for VRE by at least one method. The multiplex real-time PCR was positive for 88 enrichment broths of rectal swabs from 27 patients but for only 45 rectal swabs from 15 patients. Direct culture was positive for VRE for only 43 specimens from 11 patients, while enrichment broth culture was positive for VRE for 75 specimens from 22 patients. Inhibition studies for the multiplex real-time PCR assay, performed by spiking the DNA extracts from 50 negative rectal swabs and the corresponding enrichment broths with between 1 and 10 CFU of a VanB Enterococcus faecium strain, detected inhibition rates of 55.1 and 10%, respectively. PCR performed directly with enrichment broths was found to be significantly more sensitive than enrichment broth culture (P < 0.025). Negative samples were identified significantly earlier by PCR than by culture alone.     

Evaluation of the Granada Agar Plate for Detection of Vaginal and Rectal Group B Streptococci in Pregnant Women

Granada medium was evaluated for the detection of group B streptococci (GBS) in vaginal and rectal swabs compared with selective Columbia blood agar and selective Lim broth. From May 1996 to March 1998, 702 pregnant women (35 to 37 weeks of gestation) participated in this three-phase study; 103 (14.7%) of these women carried GBS. In the first phase of the experiment (n = 273 women), vaginorectal specimens were collected on the same swab; the sensitivities of Granada tube, selective Columbia blood agar, and Lim broth were 31.4, 94.3, and 74.3%, respectively. In the second and third phases (n = 429 women), vaginal and rectal specimens were collected separately; the sensitivities of Granada plate, selective Columbia blood agar, and Lim broth (subcultured at 4 h on selective Columbia agar in the second phase and at 18 to 24 h in Granada plate in the third phase) were 91.1, 83.9, and 75%, respectively, in the second phase and 88.5, 90.4, and 63.5%, respectively, in the third phase. There were no statistically significant differences in GBS recovery between the Granada agar plate and selective Columbia blood agar, but the Granada plate provided a clear advantage; the characteristic red-orange colonies produced overnight by GBS can be identified by the naked eye and is so specific that further identification is unnecessary. The use of the Granada tube and Lim broth did not result in increased isolation of GBS. In conclusion, the Granada agar plate is highly sensitive for detecting GBS in vaginal and rectal swabs from pregnant women and can provide results in 18 to 24 h.     

New York City medium for enhanced recovery of Mycoplasma pneumoniae from clinical specimens.

Modified New York City (MNYC) medium and PPLO medium without methylene blue (PPLO agar) were compared for their ability to support the growth of Mycoplasma pneumoniae from clinical specimens. Pharyngeal specimens were collected from 1,070 college students who visited the Syracuse University Student Health Center. Of these patients, 623 were symptomatic for respiratory infection, and the remaining 447 were asymptomatic for respiratory illness. Throat swabs were inoculated into PPLO broths, and these broths were subcultured onto MNYC medium and PPLO agar after 3 and 14 days of incubation. A total of 222 (20.7%) clinical isolates of M. pneumoniae were recovered on these solid media, with the majority of the isolates (196) recovered from symptomatic patients. All isolates grew on MNYC medium, whereas five isolates failed to grow on PPLO agar. All isolates of M. pneumoniae recovered from symptomatic patients were detected on MNYC medium within 1 to 5 days of incubation, whereas 5 to 7 days of incubation were required before mycoplasmal growth was detected on PPLO agar. Over 86% of these mycoplasma isolates were detected on MNYC medium within 3 days of incubation and before the detection of any mycoplasmal growth on PPLO agar. A similar pattern of recovery times was observed for mycoplasmas isolated from asymptomatic patients. The results of this study have shown that MNYC medium is better than PPLO agar in supporting the rapid growth of M. pneumoniae from clinical specimens after 72-h blind subculture in PPLO glucose broth.     

Detection of vancomycin-resistant enterococci in fecal samples by PCR.

Surveillance cultures for vancomycin-resistant enterococci (VRE) are time-consuming and expensive for the laboratory to perform. Therefore, we investigated the use of PCR as an alternative method of detecting and identifying VRE directly in fecal samples. PCR primers directed to vanA, vanB, vanC1, vanC2, and enterococcal ligase genes were used to detect and identify VRE in fecal material obtained by rectal or perirectal swabbing. Although PCR-inhibitory substances were present in DNA prepared directly from the swabs, the inhibitory substances could be reduced by processing the nucleic acid with two commercially available DNA preparation columns. Fecal material from 333 swabs was cultured on several selective agar media before and after broth enrichment. DNA was extracted from the fecal material and was analyzed by PCR. By using all four primer sets, only 59 (67.8%) of the samples were positive for vanA. However, after retesting the negative samples with only the vanA primer set, 77 (88.5%) of 87 specimens that were culture positive for Enterococcus faecium containing vanA were positive by PCR. One specimen was PCR positive for the vanA gene but culture negative for enterococci. The specificity of the vanA assay was 99.6%. PCR analysis of enrichment broth samples with all four primers sets after 15 to 18 h of incubation detected 74 (85.1%) of the 87 culture-positive specimens. The specificity of the vanA assay after the enrichment step was 100%. No vanB-containing enterococci were recovered by culture. Since 16 samples can be tested by PCR in 4 h (including electrophoresis), identification of VRE is possible within 8 h of specimen submission at a cost of approximately $10.12/assay. Thus, PCR may be a cost-effective alternative to culture for surveillance of VRE in some hospitals.     

Evaluation of a novel medium for screening specimens from hospitalized patients to detect methicillin-resistant Staphylococcus aureus

A novel medium, Oxacillin Resistant Screening Agar (ORSA) medium, was evaluated for the screening of specimens for methicillin-resistant Staphylococcus aureus (MRSA) in the hospital setting. Screening swabs (swabs of the nose, throat, perineum, and infected sites) were inoculated onto the new ORSA medium and into an enrichment broth (Muller-Hinton broth supplemented with NaCl and oxacillin). After 24 h of incubation, the enrichment broth was subcultured onto one ORSA plate and one lipovitellin Chapman salt agar plate. The sensitivities for the detection of MRSA were calculated for each medium alone and for the media in combination. A low sensitivity (74%) was obtained when ORSA medium was used alone as a primary culture, whereas the sensitivity was 88% when a single selective enrichment broth was used. Among the 414 blue colonies observed on ORSA plates, only 47% were found to be MRSA, 40% were coagulase-negative staphylococci, 7% were Enterococcus species, and 2% were methicillin-sensitive S. aureus. The optimal incubation time for the ORSA plates was evaluated. On primary culture, 38% of the blue MRSA colonies were visible only after 48 h of incubation (no blue colonies were not seen after 24 h of incubation), whereas 94% of the colonies were already visible at 24 h when ORSA plates were used for subcultures. In conclusion, the advantage of the novel ORSA medium is the ease of recognition of mannitol-fermenting bacteria, but further identification tests are needed to confirm the identification of S. aureus. An enrichment broth is still needed to ensure a good sensitivity for the recovery of MRSA, and an incubation time of 48 h is required for primary culture on ORSA medium.     

Use of supplemented Stainer-Scholte broth for the isolation of Bordetella pertussis from clinical material.

The use of Stainer-Scholte broth supplemented with (2,6-O-dimethyl)beta-cyclodextrin (heptakis) for the isolation of Bordetella pertussis from clinical specimens was evaluated with 3,632 nasal swabs from children and adults with suspected whooping cough or from their family contacts. The liquid enrichment medium was subcultured on charcoal agar with 10% defibrinated horse blood. Charcoal agar and soft charcoal agar served as the standard procedure to detect B. pertussis. We isolated 772 strains of B. pertussis (21%). Charcoal agar alone detected 87% of all strains (n = 668), soft charcoal agar grew 78% (n = 602), and 637 strains (83%) were isolated when Stainer-Scholte broth with heptakis was used. We detected 590 isolates with all three media. Whereas 65 strains grew only on charcoal agar, 27 strains were detected by soft charcoal agar. Supplemented Stainer-Scholte broth allowed the isolation of an additional 77 strains which did not primarily grow on charcoal media (P less than 0.05). Our data indicate that Stainer-Scholte medium supplemented with heptakis can be effectively used as an enrichment medium for detection of B. pertussis in clinical specimens.     

Use of GBS Media for Rapid Detection of Group B Streptococci in Vaginal and Rectal Swabs from Women in Labor

 In order to evaluate the differences in efficacy, three methods were used to detect group B streptococci (GBS) in women in labor. The recommended method for detecting GBS carriage in pregnant women is to culture vaginal and anorectal swabs in a selective broth medium and to subculture them onto blood agar. This method was compared with the use of GBS agar and GBS broth, both of which produce an orange pigment in response to GBS strains. A total of 319 women in labor were screened. Among the 638 specimens tested, 134 (21%) were positive in the selective Todd-Hewitt broth subcultured onto sheep blood agar, 133 (20.8%) were positive on the GBS agar and 126 (19.7%) were positive in the GBS broth. Altogether, 89 (27.9%) women in labor were found to be colonized with GBS; 87 (97.8%) of them were identified as carriers using the Todd-Hewitt broth, 87 (97.8%) with the GBS agar and 86 (96.6%) with the GBS broth. These results indicate that both GBS agar and GBS broth are reliable methods that can be used to screen for maternal and neonatal GBS colonization.     

Evaluation of new blood culture processing systems

The Antimicrobial Removal Device (ARD; Marion Scientific) was evaluated in vitro with simulated blood culture samples in fresh blood and clinically with samples from potentially septic patients to test its ability to remove antimicrobial agents and recover bacteria from blood culture specimens containing these drugs. In simulated specimens, the ARD was evaluated for adverse affects on microorganisms as well as compared with lysis-centrifugation (Isolator; Du Pont Co.), biphasic brain heart infusion bottles, and tryptic soy broth bottles for antimicrobial inactivation and organism recovery. There was no adverse effect of the ARD on organisms during a 4-h test period. The ARD was the only system to actually inactivate antimicrobial agents and removed greater than 99.2% of all antimicrobial agents tested from spiked and clinical specimens. Overall, with simulated blood culture specimens, the ARD recovered 90% of bacteria spiked into fresh blood containing antimicrobial agents, Isolator recovered 73%, biphasic brain heart infusion bottles recovered 31%, and tryptic soy broth bottles recovered 24%. In the clinical study, 43 of 86 clinically significant isolates were recovered only by ARD-assisted processing, 6 were recovered only by conventional processing, and 37 were recovered by both methods (the advantage of ARD processing over conventional processing in the clinical study was significant at P less than 0.001). Both clinical and simulated specimens demonstrated the ARD-associated blood culture processing to be the most efficient method for the isolation of microorganisms from specimens containing antimicrobial agents.     

Evaluation of the impact of direct plating, broth enrichment, and specimen source on recovery and diversity of methicillin-resistant Staphylococcus aureus isolates among HIV-infected outpatients

We compared recovery of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) from nasal and groin swab specimens of 600 HIV-infected outpatients by selective and nonselective direct plating and broth enrichment. Swabs were collected at baseline, 6-month, and 12-month visits and cultured by direct plating to mannitol salt agar (MSA) and CHROMagar MRSA (CM) and overnight broth enrichment with subculture to MSA (broth). MRSA isolates were characterized by pulsed-field gel electrophoresis (PFGE), staphylococcal cassette chromosome mec (SCCmec) typing, and PCR for the Panton-Valentine leukocidin. At each visit, 13 to 15% of patients were colonized with MRSA and 30 to 33% were colonized with methicillin-susceptible S. aureus (MSSA). Broth, CM, and MSA detected 95%, 82%, and 76% of MRSA-positive specimens, respectively. MRSA recovery was significantly higher from broth than CM (P ≤ 0.001) or MSA (P ≤ 0.001); there was no significant difference in recovery between MSA and CM. MSSA recovery also increased significantly when using broth than when using MSA (P ≤ 0.001). Among specimens collected from the groin, broth, CM, and MSA detected 88%, 54%, and 49% of the MRSA-positive isolates, respectively. Broth enrichment had a greater impact on recovery of MRSA from the groin than from the nose compared to both CM (P ≤ 0.001) and MSA (P ≤ 0.001). Overall, 19% of MRSA-colonized patients would have been missed with nasal swab specimen culture only. USA500/Iberian and USA300 were the most common MRSA strains recovered, and USA300 was more likely than other strain types to be recovered from the groin than from the nose (P = 0.05).     

Isolation and characterisation of intestinal spirochaetes.

Faeces or rectal swabs from 1527 subjects were examined for the presence of intestinal spirochaetes by anaerobic culture on blood agar incorporating spectinomycin (400 mg/l). Twenty three specimens (1.5%) were positive, and only one of these came from a patient with diarrhoea. All positive specimens came from either Asians or known homosexuals. Comparative tests showed a close phenotypic similarity between the human isolates and non-pathogenic porcine intestinal spirochaetes. These organisms differ from Brachyspira aalborgi, a spirochaete isolated from subjects with histologically confirmed intestinal spirochaetosis.     

Comparative evaluation of the oxoid signal and Roche Septi-Chek blood culture systems.

The Oxoid Signal blood culture system (Oxoid USA, Inc., Columbia, Md.) was compared with the Roche Septi-Chek system (Roche Diagnostics, Div. Hoffmann-La Roche Inc., Nutley, N.J.), with the latter consisting of a tryptic soy broth (R-TSB) bottle with an attached agar slide unit and a Columbia broth bottle. A total of 5,034 cultures with equal volumes of blood in each bottle were processed. Overall, more organisms were recovered in the R-TSB bottle than in the Signal bottle, with significantly more aerobic organisms (Pseudomonas spp., Acinetobacter spp., and yeasts) recovered in the R-TSB bottles and anaerobes and viridans group streptococci recovered in Signal bottles. Approximately equivalent numbers of organisms were recovered in the Signal and Columbia broth bottles. The times of detection were essentially identical with the three blood culture broth systems. During the study, 30.6% of the Signal bottles had a positive indicator of growth, of which 1,103 (71.7%) were false-positive cultures. Additionally, nonviable organisms resembling streptococci were observed in 13.7% of the Signal bottles that were Gram stained and in unioculated blood culture bottles. With appropriate modifications of the preparation of the media, the latter problem can be eliminated.     

Primary plate identification of group A beta-hemolytic streptococci utilizing a two-disk technique.

A two-disk system is described which allows primary plate identification of group A beta-hemolytic streptococci. Group A beta-hemolytic streptococci could be visualized on primary throat culture plates by using trimethoprim-sulfamethoxazole to inhibit normal flora. In the heavily inoculated area of Trypticase soy agar plates containing 5% sheep blood, a 25-microgram/ml trimethoprim-sulfamethoxazole disk was placed contiguous to a 0.04-U bacitracin disk. A total of 259 throat specimens were examined with this two-disk system. The swabs from these throat specimens were incubated in Todd-Hewitt broth. The bacterial pellet from the broths was stained by fluorescent antibody as a control. Of the cultures that were determined to be positive on the plates, 75% could be read unequivocally after overnight incubation, whereas the remaining 25% required subculture. The plates recovered 91% of the cultures which were considered as true positives by the broth-fluorescent-antibody technique. This method provided a significant savings in time compared with standard plate methods and in cost of materials compared with broth-fluorescent-antibody methods. This technique is particularly valuable for producing rapid results in laboratories where fluorescence microscopy would not be cost-effective.