Comparative evaluation of supplemented peptone broth with sodium polyanetholesulfonate and trypticase soy broth with sodium amylosulfate for detection of septicemia.

We compared the yield and speed of detection of clinically important microorganisms from 10,156 paired 5-ml samples of blood cultured in supplemented peptone broth (SPB) with 0.03% sodium polyanetholesulfonate (SPS) or Trypticase soy broth (TSB) with 0.5% sodium amylosulfate (SAS). The atmosphere of incubation (open venting units) and ratio of blood to broth (1:10) were the same for both samples. Only cultures with adequate blood samples (greater than or equal to 80% of stated volume) were compared statistically. Overall, SPB/SPS outperformed TSB/SAS. Bacteroidaceae and Eubacterium were found more often (P less than 0.05) and viridans streptococci were found sooner (P less than 10(-4)) in SPB/SPS than in TSB/SAS. Most importantly, staphylococci were found both more often (P less than 0.03) and sooner (P less than 10(-7)) in SPB/SPS than in TSB/SAS. In a separate experiment, SAS slowed the growth of a clinical strain of Staphylococcus aureus in TSB. Unless important advantages can be confirmed for SAS in controlled clinical trials, SAS cannot be recommended for routine use as an anticoagulant in blood culture media.     

Controlled evaluation of the effect of atmosphere of incubation on detection of bacteremia and fungemia in supplemented peptone broth.

To evaluate the role of atmosphere of incubation in the detection of clinically important bacteremia and fungemia in adults, we compared the yield of microorganisms from 10,541 paired 5-ml samples of blood incubated aerobically and anaerobically. The medium, supplemented peptone broth (SPB) with 0.03% sodium polyanetholesulfonate, and the ratio of blood to broth (1:10) were the same for all cultures. Only cultures with adequate blood samples (greater than or equal to 80% of stated volume) were compared statistically. More fungi (P less than 10(-7) ) grew in continuously vented bottles of SPB. Aerobic incubation also favored (P less than 0.01) isolation of Neisseria gonorrhoeae and Eubacterium; more than 80% of these bacterial organisms were detected only in vented bottles. Anaerobic incubation (plugged venting units) did not significantly favor the isolation of any genus of microorganisms, although an estimated 11% more Bacteroidaceae grew in the unvented bottle of SPB. By comparison of our data with published results for other media, we conclude that the need for both aerobic and anaerobic incubation of blood cultures is dependent upon the medium used and the microorganisms likely to be encountered. Vented incubation of blood cultured in SPB is crucial for detection of fungi and some bacteria. Routine use of an unvented bottle of SPB may not be worthwhile for patients in whom Bacteroidaceae cause bacteremia infrequently. However, when Bacteroidaceae are suspected as the cause of sepsis, use of an unvented bottle of SPB is prudent.     

Controlled evaluation of 5 versus 10 milliliters of blood cultured in aerobic BacT/Alert blood culture bottles.

Bottles developed for use in the BacT/Alert automated blood culture system (Organon Teknika Corp., Durham, N.C.) can accept up to 10 ml of blood without falling below a 1:5 ratio of blood to broth. We compared the yield and speed of detection of microorganisms in 13,128 adequately filled, paired, aerobic bottles inoculated with 5 versus 10 ml of blood at three university hospitals. A total of 798 microorganisms causing sepsis grew in one or both bottles. The overall recovery of microorganisms from 10-ml samples exceeded that from 5-ml samples (P < 0.001); the increased yield attributed to the additional 5 ml in the samples was 7.2%. The increased yield from 10-ml inocula was most marked for Escherichia coli (P < 0.01) and other members of the family Enterobacteriaceae (P < 0.001). Ten-milliliter samples did not yield more gram-positive bacteria, nonfermentative gram-negative rods, or yeasts. When both bottles were positive, the bottles inoculated with 10 ml of blood showed growth sooner (P < 0.001). Earlier detection with 10-ml inocula was especially notable for coagulase-negative staphylococci (P < 0.001), streptococci (P < 0.001), E. coli (P < 0.025), and other members of the family Enterobacteriaceae (P < 0.025). We conclude that an increase in the volume of blood inoculated into BacT/Alert aerobic blood culture bottles from 5 to 10 ml will increase the overall yield and the speed of detection of clinically important blood pathogens.     

Volume of blood submitted for culture from neonates.

We prospectively examined 298 sets (298 aerobic, 299 anaerobic, and 73 resin cultures) of blood cultures from 161 critically ill newborns. The attending physicians were unaware of the study. The mean blood volume per patient (aerobic and anaerobic) was 1.05 (range, 0.11 to 3.04) ml. The mean blood volume per aerobic bottle was 0.53 (range, 0.01 to 1.90) ml. Among aerobic samples 2.7% were less than or equal to 0.1 ml, 16% were less than or equal to 0.3 ml, 33% were less than or equal to 0.4 ml, and 55% were less than or equal to 0.5 ml. For anaerobic cultures the mean blood volume was 0.52 (range, 0.01 to 1.79) ml. Among anaerobic samples 2.7% were less than or equal to 0.1 ml, 15% were less than or equal to 0.3 ml, 35% were less than or equal to 0.4 ml, and 58% were less than or equal to 0.5 ml. Blood volume did not correlate with gestational age, chronologic age, or weight. The mean volume of blood submitted in positive cultures was not significantly greater than that in negative cultures. The blood volume used for culture from ill newborns may be inadequate for detecting sepsis, and the adequacy of currently available culture methods needs to be assessed for the small samples submitted from critically ill newborns.     

Yield, clinical significance, and cost of a combination BACTEC plus Septi-Chek blood culture system.

A blood culture was performed by adding a vented Septi-Chek bottle (Roche Diagnostics, Div. Hoffmann-LaRoche Inc., Nutley, N.J.) to a standard BACTEC system (Johnston Laboratories, Inc., Towson, Md.) blood culture. The yield of bacteremic patients, the clinical significance of organisms detected, and the cost of the combination system were compared with those of the standard BACTEC system alone. Each culture included 20 ml of blood divided among a BACTEC 6B aerobic bottle (5 ml), a BACTEC 7D anaerobic bottle (5 ml), and a Septi-Chek bottle equipped with a slide subculture attachment (10 ml). Significant isolates grew in 9.6% of the 2,269 cultures evaluated. The combination BACTEC plus Septi-Chek system detected 25% more bacteremic patients than the BACTEC system alone, 129 patients versus 103. The 26 bacteremic patients detected by only the added Septi-Chek bottle included 7 whose organism was isolated from blood alone and 19 whose organism was in mixed or pure culture from a second source. Detection of the organism resulted in alteration of antimicrobial therapy in 17 of these 26 patients. The combination system, which cultured a 20-ml blood volume, cost $11,000 more during the study period than the BACTEC system alone, which cultured a 10-ml volume. Reimbursement under the diagnosis-related group system was increased by $23,000 as a result of documentation of sepsis in these 26 patients. Blood volume and, possibly, the use of multiple blood culture systems are important factors when selecting a blood culture procedure for routine use.     

Evaluation of the Cobas-Bact system for direct and rapid identification and antimicrobial susceptibility testing of gram-negative rods from positive blood culture broths.

A direct antimicrobial susceptibility test and a direct identification of positive blood culture broths for gram-negative rods confirmed with Gram stain by using a new instrument, Cobas-Bact, were compared with the conventional Kirby-Bauer agar diffusion disk method and with the in-house set of identification or API 20E, respectively. The bacterial pellet of centrifuged positive blood culture broth was used to inoculate a Cobas-Bact susceptibility and identification rotor. Bacteria from 206 cases of monomicrobial septicemia due to members of the family Enterobacteriaceae were tested. In 198 episodes (96%), direct identification and antimicrobial susceptibility testing results were obtained for the same bacterial pathogen within 5 h of detection. Of 204 direct identifications obtained, 177 (86.6%) were "high-confidence" correct identifications (percentage of likelihood [P] greater than or equal to 80%) and 25 (12.5%) "low-confidence" correct identifications (P less than 80%), whereas only 2 misidentifications occurred (1 Escherichia coli and 1 Proteus mirabilis). Direct susceptibility testing was performed in 199 episodes (96%), providing 1,885 antibiotic-microorganism combinations. Full agreement reached 86.3%, and essential agreement reached 92.8%. Minor discrepancies were found in 120 (6.5%) of the tests, major discrepancies were found in 127 (6.8%) tests, and very major discrepancies were found in only 7 (0.4%) tests. Subsequent MIC determinations in cases of major or very major discrepancies reduced the number of major discrepancies involving cephalosporins from 60 to 16, whereas all those involving aminoglycosides remained. Overall, this direct and rapid Cobas-Bact identification and susceptibility testing procedure offered accurate information with 5 to 6 h after the laboratory detection of bacteremia and septicemia due to members of the Enterobacteriacease.     

Controlled evaluation of hypertonic sucrose medium at a 1:5 ratio of blood to broth for detection of bacteremia and fungemia in supplemented peptone broth.

The value of hypertonic media in the detection of bacteremia and fungemia is controversial, since prior clinical trials have yielded conflicting results with different media. Earlier, we showed that the addition of 10% sucrose to supplemented peptone broth at a 1:10 ratio of blood to broth yielded better recovery of Staphylococcus epidermidis, the Enterobacteriaceae, Pseudomonas aeruginosa, and yeasts. To evaluate the effect of 10% sucrose on blood cultured at a 1:5 ratio, we compared the yield and speed of detection of clinically important microorganisms from adult patients in 5,839 blood samples cultured in supplemented peptone broth with 0.03% sodium polyanetholesulfonate with and without 10% sucrose. The atmosphere of incubation (open venting units), 1:5 ratio of blood to broth, and methods of processing were the same for both bottles. Recovery of facultative gram-positive (P less than 0.02) and gram-negative (P less than 0.02) bacteria was improved, but the recovery of anaerobic gram-negative bacteria was both reduced (P less than 0.01) and delayed (P less than 0.02) by sucrose. The total yield of microorganisms including fungi, however, was increased with sucrose. The effect of sucrose on blood cultures appears to depend on the ratio of blood to broth as well as on the medium used and strains of microorganisms encountered.     

Optimization of detection of cytomegalovirus viremia in transplantation recipients by shell vial assay.

Cytomegalovirus (CMV) viremia is a widely used laboratory marker of CMV disease following transplantation and is additionally used to trigger preemptive antiviral therapy. Despite this, the optimal method for diagnosing CMV viremia in transplantation recipients remains unknown. To determine the sampling frequency and blood volume required for the optimal diagnosis of viremia by shell vial assay, a prospective study of 46 viremic transplantation recipients was conducted. Blood specimens (2.5 and 5 ml) were collected twice, 3 h apart, at a median of 1.4 days (range, 1 to 3 days) after the triggering shell vial-positive blood had been collected. Considering a single 2.5-ml specimen, an average of only 40% of previously viremic patients had documented CMV in their blood: this increased to 50% when a second 2.5-ml sample of blood was collected 3 h later. The yields of two 2.5-ml versus two 5-ml samples were 50 versus 61%, respectively. Viremia as detected by shell vial assay is intermittent, and increasing the frequency and volume of blood sampling increases its diagnosis. These results have implications in diagnosis of CMV infection and its preemptive therapy.     

Controlled evaluation of hypertonic sucrose medium for detection of bacteremia and fungemia in supplemented peptone broth.

Because the value of hypertonic media in detection of bacteremia and fungemia is controversial, we evaluated supplemented peptone broth (SPB) with 0.03% sodium polyanetholsulfonate with and without 10% sucrose in 5,439 paired blood cultures from adult patients. The aerobic atmosphere, 1:10 ratio of blood to broth, and methods for processing blood cultures were identical. Only cultures with adequate blood samples (greater than or equal to 4 ml) were compared statistically. More clinically important bacteria were recovered from SPB with sucrose (P less than or equal to 0.001), including Staphylococcus epidermidis, Enterobacteriaceae, and Bacteroidaceae. However, only one of nine isolates of Neisseria gonorrhoeae grew in SPB with sucrose. Staphylococci (P less than 0.001), Enterobacteriaceae (P less than 0.01), Pseudomonas aeruginosa (P less than 0.01), and yeasts (P less than 0.05) were detected 1 or more days earlier in SPB with sucrose. The effect of sucrose on blood cultures appears to be medium dependent, based on comparisons of our results with those of published reports.     

Clinical comparison of isolator and BACTEC 660 resin media for blood culture.

The 10-ml Isolator system (E.I. du Pont de Nemours & Co., Inc., Wilmington, Del.) was compared with the BACTEC 16A-17A nonradiometric resin system (Johnston Laboratories, Inc., Towson, Md.) for isolation of organisms from 6,839 paired blood cultures. Equal volumes of blood (6 to 10 ml for each Isolator and 3 to 5 ml for each BACTEC bottle) were cultured in parallel in the two systems, and 600 isolates that were judged to be clinically significant by chart review were recovered during the study. The BACTEC resin system detected 510 (85%) and the Isolator system detected 435 (72%) of the clinically significant isolates (P less than 0.001). Of 45 polymicrobial blood cultures, the BACTEC system detected 32 (71%) and the Isolator system detected 21 (47%) (P less than 0.05). Of 253 gram-negative bacilli isolated during the study, 30% were detected only in the BACTEC system and 16% were detected only in the Isolator system (P less than 0.001), and of 56 nonfermentative or fastidious gram-negative bacilli detected, 46% were recovered only in the BACTEC system, while 14% were detected only in the Isolator system (P less than 0.001). Of 86 streptococci isolated during the study, 30% were detected only in the BACTEC system, and 4% were detected only in the Isolator system (P less than 0.001). Recoveries of anaerobic bacteria, staphylococci, and yeasts were equivalent in the two systems. Organisms judged to be contaminants were detected in approximately 1% of the cultures in each system. The results suggest that use of resin media renders the BACTEC nonradiometric system equivalent or superior to the Isolator system for detection of clinically significant organisms in blood cultures.     

Determination of formaldehyde in blood plasma by high-performance liquid chromatography with fluorescence detection

An HPLC method was developed for the determination of formaldehyde in human blood plasma. The method was based on the determination of the fluorescent product of the chemical reaction between formaldehyde and ampicillin. A 0.2-ml aliquot of blood plasma was reacted directly with ampicillin under acidic and heating conditions. The reaction product was extracted from the matrix with diethyl ether and analyzed by reversed-phase HPLC with fluorescence detection. Recoveries of spiked formaldehyde at the low ppm (microg/ml) level were between 93% and 102% with relative standard deviations less than 8%. The limits of detection and quantitation of formaldehyde in blood plasma samples were 0.46 microg/ml and 0.87 microg/ml, respectively.     

A semiquantitative culture method for identifying intravenous-catheter-related infection.

We evaluated a semiquantitative culture technic for identifying infection due to intravenous catheters: rolling the catheter segment across blood agar. This method was compared to broth culture. Of 250 catheters studied, 225 (90%) had low-density colonization on semiquantitative culture (less than 15 colonies on the plate) although 49 (19.6%) of these grew some organisms in broth or on the plate. None of these catheters led to septicemia. Twenty-five catheters (10%) grew greater than or equal to 15 colonies by the semiquantitative technic; most gave confluent growth. Septicemia originated from four of these catheters (P = 0.008). Of 37 catheters exposed to bacteremias from distant foci of infection, four yielded matching growth in broth, whereas none were concordant with the blood isolate on semiquantitative culture. Local inflammation was associated with high-density colonization semiquantitative culture (P less than 0.001). The semiquantitative technic distinguishes infection (greater than or equal to 15 colonies) from contamination and is more specific in diagnosis of catheter-related septicemia than culture of the catheter in broth.     

Significance of coagulase negative staphylococci in neonates with late onset septicemia

Present study was undertaken for establishing significance of coagulase negative staphylococci isolated from cases of late onset neonatal septicemia. 660 neonates admitted to NICU with clinical suspicion of late onset septicemia, over a period of nine months, were included in study. After skin preparation 1.5-ml blood for culture was collected from two different sites by venipuncture and each was inoculated into a blood culture bottle. All CONS thus isolated were further analysed. Laboratory criteria for significant CONS bacteremia was defined as recovery of CONS with in 48 hours of specimen collection from both sites of a blood culture set that displayed uniform antibiotic susceptibility and biochemical reactions. Due to technical difficulties two samples for blood culture were obtained only from 338 cases, CONS were recovered from 52 (22.7%) cases; only 13 (25%) were considered significant. Only single blood sample was available from remaining 322 subjects and CONS were recovered from 36/322 (24.3%). CONS isolation rate was similar in both subject groups. Using double specimen protocol we found majority of CONS recovered from neonates, to be probable contaminants. Recovery of CONS from blood of a septicemic neonate needs to be viewed with caution since not all of them are true bacteremic agents.     

Importance of blood volume cultured in the detection of bacteremia

The influence of the volume of blood cultured on the rate of detection of bacteremia was evaluated in a routine 12-tube blood culture system using 1693 samples from 1502 patients. Blood samples were drawn simultaneously into two transport tubes. The blood volume cultured was the only varying parameter. Generally, 17 % more cultures with clinically significant microorganisms (bothEnterobacteriaceae and gram-positive cocci) were found when blood from two instead of one tube was used (in most cases comparing 13–16 ml of blood with 6.5–8 ml). Of the most prevalent species, the maximum average extra yield was observed forStaphylococcus aureus (26 %) followed byEscherichia coli (16 %) andStreptococcus pneumoniae (12 %). In adults most cases of bacteremia are low-grade. The grade of bacteremia in our patient population was on average as low as 0.25 CFU/ml blood. Therefore, all patients suspected of having bacteremia should have the benefit of a sufficient volume of blood cultured. Since the volume of blood cultured seems to be the single most important factor in the detection of bacteremia, it is imperative that the volume is the same in comparative studies of different blood culture systems.     

Cost-Effectiveness of 30- Compared to 20-Milliliter Blood Cultures: a Retrospective Study

The importance of blood culture (BC) volume for detection of bloodstream infections (BSIs) is documented. Recently, improved diagnostic sensitivity was demonstrated for 30- versus 20-ml BCs in adults (Cockerill FR, Wilson JW, Vetter EA, Goodman KM, Torgerson CA, Harmsen WS, Schleck CD, IIstrup DM, Washington JA, Wilson WR. Clin Infect Dis 38:1724–1730, 2004, http://dx.doi.org/10.1128/JCM.01314-11). Hospitals receive higher reimbursement for patients with documented septicemia. We determined the cost-effectiveness of 30-ml versus 20-ml BCs using results from our institution and previously published data. Positive BC results from 292 bacteremic episodes were reviewed. The costs of the reagents, equipment, phlebotomist, and technologist time were determined. The medical records department provided Medicare reimbursement (MR) data for patients with selected ICD-9 codes. These data provided an estimate of the annualized increase in MR versus costs associated with conversion to 30-ml BCs. MR for 464 annual primary BSIs was $24,808/episode. An expected 7.2% increase in BSIs detected using 30-ml BCs would add 34 additional cases annually and increase MR by $843,472. Comparative MR data for cases where septicemia complicated another diagnosis were available for 4 International Classification of Diseases, Ninth Revision (ICD-9) codes: laparoscopic cholecystectomy, biliary tract disorders, pneumonia, and cellulitis. The mean incremental MR was $9,667 per episode, which projected to a $483,350 revenue increase annually. The annual cost associated with conversion to 30-ml BCs was estimated to be $157,798. Thus, the potential net increase in hospital revenue would be $1,169,031 for 30-ml versus 20-ml BCs. Our results suggest that conversion to 30-ml BCs may not only improve patient care by detecting more BSIs but also increase hospital revenue substantially.     

Performance of nucleic acid amplification following extraction of 5 milliliters of whole blood for diagnosis of Mycobacterium tuberculosis bacteremia

To investigate the performance of a nucleic acid amplification test (NAAT) for the diagnosis of Mycobacterium tuberculosis bacteremia, 5-ml aliquots of blood were inoculated into bioMérieux mycobacterial (MB) bottles and incubated, and 5-ml aliquots of blood were extracted and tested by real-time PCR. Of 25 samples from patients with M. tuberculosis bacteremia, 9 (36.0%) were positive and 1 (1.5%) of 66 control samples was positive by NAAT. The NAAT shows promise, but modifications should focus on improving sensitivity.     

Accuracy of calibrated-loop transfer.

The accuracy of the 0.001-ml calibrated platinum loop was tested by two methods: visible absorption spectroscopy and weight determinations. By both methods it was demonstrated that the volume of the loop delivery is related to the angle at which the loop is withdrawn from the solution being sampled and the diameter of the container, provided that the volume in the container is adequate to cover the loop. Vertical sampling from small-diameter containers (less than or equal to 7-mm inside diameter) delivered approximately 50% of 0.001 ml, and sampling at a 45 degree angle from larger containers (greater than or equal to 22-mm inside diameter) gave a delivery volume of greater than 150% of 0.001 ml, resulting in a threefold difference in the amount delivered and an error rate of +/- 50%.     

Cumulative positivity rates of multiple blood cultures for Mycobacterium avium-intracellulare and Cryptococcus neoformans in patients with the acquired immunodeficiency syndrome

We examined the occurrence of low-grade Mycobacterium avium-intracellulare bacteremia and Cryptococcus neoformans fungemia in patients with the acquired immunodeficiency syndrome and the consistency of positive cultures obtained using a sensitive blood culture system (Isolator, E. I. Du Pont de Nemours, Wilmington, Del) for the recovery of these organisms. The blood culture records were reviewed, and the proportion of positive blood cultures yielding less than 1 colony-forming unit per milliliter of M avium-intracellulare or C neoformans was calculated. To determine consistency, a period of potentially detectable septicemia was defined as the period between 1 week before the first positive blood culture and the last positive blood culture, providing consecutive positive blood cultures were separated by less than 2 weeks. All positive and negative blood cultures obtained during the period of potentially detectable septicemia were considered in the data analysis. Overall, 40 (16.9%) of 236 cultures positive for M avium-intracellulare and 36 (57.1%) of 63 for C neoformans yielded less than 1 colony-forming unit per milliliter. Mycobacteremia was detected in 52 of 57 periods of potentially detectable septicemia in the first culture and in 56 of 57 in the first two (cumulative detection rates of 91.2% and 98.2%, respectively). Cryptococcemia was detected in 12 of 17 periods of potentially detectable septicemia in the first culture and in 15 of 17 in the first two (cumulative detection rates of 70.6% and 88.2%, respectively). Because of the sensitivity of the blood culture system and the consistency of M avium-intracellulare bacteremia and C neoformans fungemia in patients with the acquired immunodeficiency syndrome, it appears that two blood cultures are sufficient for the detection of most septic episodes caused by these organisms.     

Evaluation of blood culture procedures in a pediatric hospital.

To determine optimal clinical laboratory techniques for detecting pediatric bacteremia, we studied 7,768 consecutive blood cultures in a 1-year period. Blood was inoculated into one vented 50-ml bottle of brucella broth with 0.05% sodium polyanetholsulfonate and one unvented 50-ml bottle of Columbia broth with 0.05% sodium polyanetholsulfonate and 0.05% cysteine. Bottles were visually examined for growth on days 1 through 7 and blindly subcultured aerobically and anaerobically on days 1, 2, and 7. There were 724 (9.3%) positive cultures, and 484 (6.2%) were clinically significant. The most frequent isolates from bacteremic patients were Haemophilus influenzae (24%) and Streptococcus pneumoniae (17%). Growth was noted in only one bottle in 25% of clinically significant isolates. Bottles inoculated with greater than or equal to 1 ml of blood became positive earlier than bottles inoculated with less than 1 ml. After 1 day of incubation, 48% of the clinically significant cultures showed growth on visual examination, whereas 85% showed growth on subculture. Only 19% of Haemophilus isolates were detected visually on day 1, whereas 88% were recovered on subculture. By day 7, 3.5% of all isolates (including 18% of pneumococcal isolates and 1% of Haemophilus isolates) could no longer be recovered on subculture. We conclude that a two-bottle blood culture system and blind subculture within 24 h will optimize detection of pediatric bacteremia.     

Controlled evaluation of blood culture medium containing gelatin and V-factor-analog for detection of septicemia in children.

Both Neisseria meningitidis and Haemophilus influenzae are important isolates recovered in blood cultures from septicemic children. Sodium polyanetholsulfonate is present in most blood culture media and can inhibit the growth of certain bacteria, including N. meningitidis. The addition of gelatin to blood culture media neutralizes this inhibition. The growth of H. influenzae is enhanced by specific growth factors such as hemin and NAD. The addition of gelatin and V-factor-analog (a proprietary supplement for enhancing the growth of H. influenzae) might have a positive effect on the yield and on the speed of detection of septicemia in children. To evaluate this possibility, we did 4,565 paired comparisons of blood cultured in BACTEC 6B (aerobic) medium with and without the addition of both 1.2% gelatin and V-factor-analog. More aerobic and facultative bacteria grew in the 6B than in the 6B-gelatin-V-factor-analog medium (P less than 0.01). Only seven isolates of Neisseria spp. were recovered during this study period, with the 6B medium performing as well as the supplemented medium. When microorganisms grew in both bottles, they did so at the same time except for H. influenzae and Candida albicans. H. influenzae was recovered earlier from the 6B-gelatin-V-factor-analog bottle (P less than 0.01), with a mean time to detection of 8.5 h compared with 15.9 h for the 6B bottle. C. albicans was recovered earlier from the 6B bottle (P less than 0.02), with a mean time to detection of 34.9 h compared with 71.6 h for the 6B-gelatin-V-factor-analog bottle. We conclude that the 6B medium in its present formulation is superior to bB supplemented with gelatin and V-factor-analog.