Routine aerobic terminal subculturing of blood cultures in a cancer hospital.

Routine terminal aerobic subcultures of macroscopically negative blood culture bottles were evaluated during a 15-month period when 30,000 blood cultures were processed. Each blood culture set consisted of a vented and an unvented 50-ml broth bottle. Forty-eight pathogens and 47 contaminants were isolated only from terminal subcultures. Twenty-two of the significant isolates were yeasts (usually recovered from vented bottles), and 10 were Pseudomonas aeruginosa (usually recovered from unvented bottles). Blood cultures that were positive by terminal subculture provided clinically relevant information in many cases, whether other blood cultures were positive or not. Microbiology laboratories, particularly those in hospitals where yeasts and P. aeruginosa are commonly isolated from blood specimens, should evaluate carefully the need for terminal subcultures of blood culture bottles before abandoning their use.     

Effect of agitation of BACTEC 13A blood cultures on recovery of Mycobacterium avium complex.

The effect of agitation of BACTEC 13A bottles (Becton Dickinson) on the recovery of Mycobacterium avium complex (MAC) from blood was compared with that of static incubation. A total of 265 blood specimens was inoculated in duplicate into BACTEC 13A bottles. One specimen was statically incubated at 35 degrees C, and the other was incubated with agitation on a Gyrotory shaker at 35 degrees C for the first 2 weeks and thereafter without shaking for up to 12 weeks. Of the 265 specimens, 77 (29.1%) were positive in either one or both of the paired bottles. The average detection times for the shaken and nonshaken bottles were 12.7 and 15.9 days, respectively. A total of 10.4% of the specimens in the shaken bottles became positive 1 week before those in the nonshaken bottles, and 16.9% of the shaken cultures were positive more than 2 weeks before their counterparts. A further 46.8% of the agitated specimens became positive while the corresponding nonagitated cultures remained negative. When both specimens became positive at the same time, 88% of the shaken cultures had higher growth indices than their nonshaken counterparts. A further 11 paired blood cultures were taken from patients known to be infected with MAC to assess the effect of agitation of bottles on the utility of making twice-weekly readings during the first 2 weeks of incubation. Ten of the 11 sets of specimens in the shaken bottles were positive 1 or more weeks before those in the corresponding nonshaken bottles. In the remaining set, both specimens became positive on the same day; however, the growth index of the agitated culture was higher.(ABSTRACT TRUNCATED AT 250 WORDS)     

Controlled comparison of BacT/ALERT FAN aerobic medium and BATEC fungal blood culture medium for detection of fungemia

Yeasts are an increasingly common cause of nosocomial bloodstream infections. Methods for their detection are many; controlled comparisons are few. The vented FAN aerobic blood culture medium has been shown to be superior to the standard BacT/ALERT aerobic medium for the detection of fungemia as well as bacteremia. The BACTEC selective fungal medium (FM) (BD Biosciences, Sparks, Md.) allowed detection of more episodes of fungemia than did a resin-containing medium with equal volumes of blood cultured. Therefore, we compared vented FAN to FM for the ability to recover fungi from the blood of patients who were at increased risk of having fungemia. From 5,109 cultures processed for which both FAN and FM bottles were adequately filled, fungi were recovered from 87 cultures. Of these, 47 were detected with both bottles, 12 were detected with FAN only, and 28 were detected with FM only (P < 0.05). FAN was the first bottle positive for 36 of the 47 cultures for which both bottles yielded the same fungus, whereas the FM bottle was the first bottle positive for 11 cultures (P < 0.001). A total of 54 episodes of fungemia were identified, with 40 detected by both media, 4 detected only by FAN, and 10 detected only by FM (P value, not significant). We conclude that the vented FAN aerobic bottle is comparable to the FM bottle for detection of episodes of yeast infection but has the added benefit of detecting bacteria.     

Value of routine aerobic subculturing of unvented blood culture bottles.

The value of performing routine aerobic subcultures of both vented and unvented blood culture bottles has not been evaluated critically. We studied 4,954 pairs of blood culture bottles consisting of one vented biphasic tryptic soy broth bottle (Roche Diagnostics) and one unvented Thiol broth bottle (Difco Laboratories). A total of 736 isolates were detected, of which 124 (17%) were in the Thiol broth bottle only. Some 15 isolates were detected only by subculturing the Thiol broth, and 13 of these isolates either were contaminants or were detected in previous positive cultures. Similar results were obtained when the unvented Thiol broth bottle was paired with a vented Difco tryptic soy broth bottle. Analysis of these pairs revealed a total of 360 isolates detected in 2,669 pairs of bottles, of which 83 isolates (23%) were in the Thiol broth bottle only. There were 11 isolates seen only in subcultures of the Thiol broth bottle, and 8 of these were probable contaminants. Thus, routine subculturing of unvented Thiol broth bottles had limited value. These results may differ with the use of other culture media or subculturing procedures. We recommend that each laboratory evaluate critically its experience with aerobic subcultures from unvented bottles.     

Comparison of the BacT/Alert PF pediatric FAN blood culture bottle with the standard pediatric blood culture bottle, the Pedi-BacT

The performance of the BacT/Alert PF (Organon-Teknika Corp., Durham, N.C.), a new nonvented pediatric FAN blood culture bottle, was compared to that of the original pediatric bottle, the Pedi-BacT, with matched aerobic cultures obtained from two separate facilities. A total of 244 clinically significant isolates were recovered from 4,015 compliant pairs. Among the positive cultures, 170 (70%) isolates were detected in both the BacT/Alert PF and the Pedi-BacT bottles, while 47 (19%) isolates were recovered in the BacT/Alert PF bottle only and 27 (11%) isolates were recovered in the Pedi-BacT bottle only. Although isolation of specific microorganisms was comparable for the two bottles, the total number of organisms recovered by the BacT/Alert PF was greater than that by the Pedi-BacT (P = 0.0272). In addition, more organisms were recovered by the BacT/Alert PF bottle from the blood of patients receiving antimicrobial therapy (P = 0.0180). Overall time to detection was similar for the two bottles; however, a significantly decreased mean time to detection was recorded for yeast from the BacT/Alert PF bottle (22.9 h; P = 0.0001) and staphylococci from the Pedi-BacT bottle (22.5 h; P = 0.0056). One false-negative culture and five false-positive cultures occurred with the Pedi-BacT bottle, compared to one false-positive culture with the BacT/Alert PF bottle. The BacT/Alert PF bottle is a reliable blood culture bottle for pediatric blood culture specimens and may offer improved recovery of microbes from patients on antimicrobial therapy. The use of the nonvented bottle will both facilitate bottle processing and decrease expenditures for materials due to the elimination of the venting needles required for the original vented bottles.     

Clinical laboratory comparison of a slide blood culture system with a conventional broth system

The recovery of bacteria and fungi from blood cultures was compared in conventional tryptic soy broth (TSB) bottles and in TSB bottles with an agarcoated slide attachment. A total of 2,662 sets of blood cultures, including 413 that were positive (15.5%), were evaluated. Significantly more gram-positive and gram-negative bacteria were recovered in the slide culture bottles than in conventional bottles (299 versus 253 isolates). Growth of gram-positive organisms and fungi was detected in the slide culture bottles 24 to 48 h earlier than in the TSB bottles. In addition, 76% of the isolates in the slide culture system were detected on the agar slide. In comparison, only 40% of the isolates in the TSB bottles were detected initially by blind subculturing. The incidences of contamination were 2.7% (71 cultures) for the slide culture system and 1.5% (39 cultures) for the TSB bottles.     

Critical analysis of hypertonic medium and agitation in detection of bacteremia.

Over 18,000 clinical specimens collected in Vacutainer tubes with sodium polyanethol sulfonate were inoculated into modified Columbia broth (MCB) with and without 10% sucrose. The effects of venting and shaking on recovery were studied. The volume of the blood had a definite effect on the recovery rate. When inoculum size was held constant, recovery of aerobic and facultative organisms was maximal in vented and shaken bottles; the presence of sucrose had no demonstrable effect, recovery of anaerobes was maximal using an unvented bottle incubated under stationary conditions; a significantly greater recovery of facultatives and a marginally greater recovery of anaerobes was obtained with the hypertonic formulation. We conclude that a hypertonic formulation of MCB offers no advantage in the recovery of anaerobes but is of value in the recovery of facultatives and anaerobes. It is recommended that blood cultures be routinely inoculated into isotonic MCB and then vented and shaken for at least 4 hours, and hypertonic MCB incubated without venting or shaking.     

Determination of the optimum incubation period of blood culture broths for the detection of clinically significant septicemia.

The value of incubating blood culture broths for more than 7 days was analyzed. A total of 20,155 blood cultures, consisting of a vented Roche tryptic soy broth (R-TSB) bottle and an unvented Difco thiol broth bottle, was processed; 2,509 organisms were recovered in the R-TSB bottles, and 1,865 organisms in the thiol bottles. Only 32 organisms isolated in the R-TSB bottles and 10 organisms isolated in the thiol bottles were detected after incubation for more than 7 days; 15 of the 32 isolates in the R-TSB bottles and all 10 of the isolates in the thiol bottles were either recovered in other blood cultures or were not considered to be clinically significant. Thus, incubation of the R-TSB bottles and unvented thiol bottles for more than 7 days is unnecessary for the detection of most clinically significant septicemias.     

Three days of incubation may be sufficient for routine blood cultures with BacT/Alert FAN blood culture bottles

BacT/Alert FAN blood culture bottles have been shown to enhance the recovery of bacteria and yeast from blood compared with standard BacT/Alert bottles. It is well established that standard BacT/Alert blood culture bottles require no more than 5 days of incubation for the detection of routine bacteria and yeast. It is less clear, however, whether FAN bottles also routinely require 5 days of incubation. To address this question, we recently reviewed the results of 17,887 blood culture sets collected in FAN blood culture bottles at Geisinger Medical Center. Of these cultures, 1,780 were positive for bacteria or yeast, yielding a total of 1,242 clinically significant isolates. The numbers of isolates recovered on days 1, 2, 3, 4, and 5 were as follows: (values in parentheses are percentages of total significant isolates): 877 (71%), 269 (22%), 65 (5%), 18 (1%) and, 13 (1%), respectively. In total, 97.5% of all clinically significant isolates were detected in the first 3 days of incubation. Of the 31 significant isolates detected on day 4 or 5 of incubation, 17 were detected in concurrent blood cultures within the first 3 days of incubation. Chart reviews were conducted for the 13 patients with the remaining 14 isolates detected on day 4 or 5 to determine whether therapy was changed due to this blood culture result. Therapy was changed for only 1 patient. These results suggest that it may not be necessary to routinely incubate FAN blood culture bottles for more than 3 days.     

Effects of Blood on Blood Culture Medium

The morphological and biochemical changes that occur after inoculation of sterile blood into a blood culture medium (tryptic soy broth) with sodium polyanetholesulfonate and CO(2) were investigated. Cellular changes, pH, PCO(2), and PO(2) were monitored and evaluated. Erythrocytes became crenated and developed precipitated hemoglobin inclusions within 4 h. The lymphocytes appeared morphologically intact at 24 h, and by 48 h a few cells had undergone transformation. Many neutrophils were vacuolated at 24 h. Neutrophils capable of phagocytizing Staphylococcus aureus were observed after 18 h of incubation. Identifiable eosinophiles were present on day 6 of the study. A decrease in PO(2) in the unvented bottles from 44.4 to 8 mm of Hg occurred by 24 h. PO(2) remained low for 6 days, after which a slight increase occurred. An increase in PO(2) in the vented bottle from 51 to 58 mm of Hg occurred by 24 h of incubation. In both the vented and unvented bottles the PCO(2) increased. This increase was markedly more rapid in the unvented bottle. From a pH of 7.06 a decrease occurred for the first 24 h after inoculation, with the pH stabilizing at 6.8 in the vented bottles and at 6.6 in the unvented bottles. The biochemical changes that occurred in the vented culture bottles stabilized more rapidly than those of the unvented bottles. Changes caused by the addition of sterile blood to a blood culture medium resulted in conditions which departed considerably from accepted optima for the isolation of clinically important microorganisms. The phagocytosis of organisms that occurred may also have reduced the yield.     

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.     

Variability in CO2, O2, and pH levels in blood culture bottles from five different manufacturers

The CO2, O2, and pH levels of commercially available blood culture bottles with tryptic soy broth medium from five different manufacturers were compared. Ranges of 1.3 to 6.9% for CO2, 1.1 to 6.0% for O2, and pH 6.94 to 7.26 were found. Different venting procedures revealed that blood culture bottles from which the rubber diaphragm was removed equilibrated the most rapidly (24 h) to the atmosphere (10, 5, and 2.5% CO2) they were incubated in. In contrast, blood culture bottles vented with cotton-plugged needles required 48 h to achieve similar CO2 levels in the medium. The ability of these venting procedures to support bacterial growth was confirmed by measuring the growth of a CO2-dependent Escherichia coli isolate in such vented bottles; blood culture bottles that showed rapid atmospheric (5 and 10% CO2) equilibration had the fastest growth curves. Our results suggest that the differences in the recovery of certain microorganism from blood culture bottles may be due in part to the large variability seen in CO2 and O2 concentrations and the use of various venting procedures.     

Effect of hydrogen peroxide on growth of Candida, Cryptococcus, and other yeasts in simulated blood culture bottles.

The addition of hydrogen peroxide to blood contained in liquid culture medium increased the dissolved-O2 partial pressure in direct proportion to the volume injected. The effect of hydrogen peroxide on the growth of subcultured clinical isolates of Candida albicans, Cryptococcus neoformans, Torulopsis glabrata, and other yeasts and on the growth of blood culture isolates of representative pathogenic bacteria was compared with its effect on their growth in vented and unvented stationary bottles. C. albicans and C. neoformans grew significantly better in bottles to which hydrogen peroxide had been added than in vented or unvented bottles. The advantage of hydrogen peroxide over venting was most marked with several slowly growing strains. Similar results were obtained in shaker cultures with strains of C. albicans which were inoculated directly from positive blood cultures. The effect of hydrogen peroxide tended to diminish during serial passage. T. glabrata grew less well when hydrogen peroxide was added, perhaps because of the absence of oxidase. The growth of Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis was not significantly inhibited or augmented by the addition of hydrogen peroxide. The growth of Escherichia coli was inhibited slightly. The value of the addition of hydrogen peroxide to blood cultures to improve the isolation of yeasts needs to be established by a clinical trial which would compare this method with established methods.     

Comparison of the Roche Septi-Chek blood culture bottle with a brain heart infusion biphasic medium bottle and with a tryptic soy broth bottle.

In a comparison of 1,368 positive blood cultures, a vented Roche Septi-Chek (V-RSC) blood culture bottle was superior to an unvented tryptic soy broth-containing bottle (Difco) for the recovery of all aerobic and facultatively anaerobic microorganisms. Anaerobic bacteria were recovered more frequently and earlier in the unvented tryptic soy broth-containing bottle. A separate comparison of 529 positive blood cultures was conducted to examine the performance of the V-RSC bottle with that of a vented brain heart infusion biphasic medium. The V-RSC bottle recovered significantly more isolates of Enterobacteriaceae and of anaerobic bacteria than did the vented brain heart infusion biphasic medium. The V-RSC bottle is a reliable blood culture system for all aerobic and facultatively anaerobic microorganisms. Because of its suboptimal recovery of anaerobic bacteria, it is recommended that the V-RSC bottle be used in combination with an unvented vacuum blood culture bottle.     

Effect of volume of blood cultured on detection of bacteremia.

The rates of recovery of bacteria from vented vacuum blood culture bottles containing 50 and 100 ml of a soybean-casein digest broth were compared. Overall, more isolates were recovered from the larger bottle; moreover, gram-negative bacilli and especially Pseudomonas aeruginosa were recovered significantly more frequently (P less than 0.01) from the 100-ml bottle.     

Recovery of yeast from vented blood culture bottles.

Rates of isolation of yeasts from blood cultures were significantly enhanced by venting vacuum blood culture bottles in studies of both stimulated and patients' blood cultures; however, the time interval to detection of positivity of yeasts in the clinical studies was significantly (P less than 0.01) shorter in a vented bottle with biphasic brain heart infusion medium than in a vented bottle with soybean-casein digest broth. The mean time intervals to detection of positivity were 2.6 days in the former and 5.2 days in the latter.     

Evaluation of a hypertonic sucrose medium for the detection of fungi in blood cultures.

A comparison was made between biphasic brain heart infusion medium without and with 15% sucrose in vented blood culture bottles. A higher recovery rate (P less than 0.01) of fungi was noted in the medium without sucrose.     

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.     

Evaluation of a biphasic medium for blood cultures.

A study comparing the recovery of microorganisms from a transiently vented biphasic brain heart infusion medium bottle and a vacuum bottle containing tryptic soy broth demonstrated that growth was initially detected on the slant of the biphasic bottle or on a routine subculture of either broth in nearly 50% of instances. All organisms were isolated equally well in both bottles, with the exception of Staphylococcus aureus, which was isolated significantly more frequently from the biphasic bottle, and anaerobic bacteria, which were isolated significantly more frequently from the tryptic soy broth bottle.     

Evaluation of a lysis-centrifugation system for recovery of yeasts and filamentous fungi from blood

A lysis-centrifugation system (Isolator; E. I. du Pont de Nemours & Co., Inc., Wilmington, Del.) was compared with a biphasic brain heart infusion (BHI) medium in a prospective study of 5,125 fungal blood cultures. The Isolator recovered 90.3% of the positive cultures, compared with 63.4% recovered by the biphasic BHI medium. Overall, the detection of fungemia was increased 36.6% by the Isolator. Mean recovery times for yeasts were 2.12 and 4.90 days for the Isolator and BHI bottles, respectively. Cultures of Histoplasma capsulatum required 8.0 and 24.14 days for recovery by the Isolator and BHI bottles, respectively. The Isolator provided a more rapid and sensitive means of detecting organisms associated with fungemia.