Microbial populations involved in cycling of dimethyl sulfide and methanethiol in freshwater sediments

Although several microorganisms that produce and degrade methanethiol (MT) and dimethyl sulfide (DMS) have been isolated from various habitats, little is known about the numbers of these microorganisms in situ. This study reports on the identification and quantification of microorganisms involved in the cycling of MT and DMS in freshwater sediments. Sediment incubation studies revealed that the formation of MT and DMS is well balanced with their degradation. MT formation depends on the concentrations of both sulfide and methyl group-donating compounds. A most-probable number (MPN) dilution series with syringate as the growth substrate showed that methylation of sulfide with methyl groups derived from syringate is a commonly occurring process in situ. MT appeared to be primarily degraded by obligately methylotrophic methanogens, which were found in the highest positive dilutions on DMS and mixed substrates (methanol, trimethylamine [TMA], and DMS). Amplified ribosomal DNA restriction analysis (ARDRA) and 16S rRNA gene sequence analysis of the total DNA isolated from the sediments and of the DNA isolated from the highest positive dilutions of the MPN series (mixed substrates) revealed that the methanogens that are responsible for the degradation of MT, DMS, methanol, and TMA in situ are all phylogenetically closely related to Methanomethylovorans hollandica. This was confirmed by sequence analysis of the product obtained from a nested PCR developed for the selective amplification of the 16S rRNA gene from M. hollandica. The data from sediment incubation experiments, MPN series, and molecular-genetics detection correlated well and provide convincing evidence for the suggested mechanisms for MT and DMS cycling and the common presence of the DMS-degrading methanogen M. hollandica in freshwater sediments.     

Microbial Populations Involved in Cycling of Dimethyl Sulfide and Methanethiol in Freshwater Sediments

Although several microorganisms that produce and degrade methanethiol (MT) and dimethyl sulfide (DMS) have been isolated from various habitats, little is known about the numbers of these microorganisms in situ. This study reports on the identification and quantification of microorganisms involved in the cycling of MT and DMS in freshwater sediments. Sediment incubation studies revealed that the formation of MT and DMS is well balanced with their degradation. MT formation depends on the concentrations of both sulfide and methyl group-donating compounds. A most-probable number (MPN) dilution series with syringate as the growth substrate showed that methylation of sulfide with methyl groups derived from syringate is a commonly occurring process in situ. MT appeared to be primarily degraded by obligately methylotrophic methanogens, which were found in the highest positive dilutions on DMS and mixed substrates (methanol, trimethylamine [TMA], and DMS). Amplified ribosomal DNA restriction analysis (ARDRA) and 16S rRNA gene sequence analysis of the total DNA isolated from the sediments and of the DNA isolated from the highest positive dilutions of the MPN series (mixed substrates) revealed that the methanogens that are responsible for the degradation of MT, DMS, methanol, and TMA in situ are all phylogenetically closely related to Methanomethylovorans hollandica. This was confirmed by sequence analysis of the product obtained from a nested PCR developed for the selective amplification of the 16S rRNA gene from M. hollandica. The data from sediment incubation experiments, MPN series, and molecular-genetics detection correlated well and provide convincing evidence for the suggested mechanisms for MT and DMS cycling and the common presence of the DMS-degrading methanogen M. hollandica in freshwater sediments.     

Enumeration of micro-organisms in processed soy products with an automated most probable number method compared with standard plate method

Aim: The automated TEMPO system (bioMerieux) is based on the most probable number (MPN) method for the enumeration of micro‐organisms in foods. In this study, we evaluated the performance of the TEMPO system as a diagnostic tool in comparison with the standard method in processed soy products. Methods and Results: A verification study was conducted using artificially contaminated soy product samples such as soy protein isolate, water‐soluble soy polysaccharides, soy milk and processed soy food. Five types of micro‐organisms were analysed using the automated MPN method (total aerobic bacteria, total coliforms, Enterobacteriaceae, yeast and mould and Staphylococcus aureus) vs the standard plate method. The results from each of the methods were highly correlated (r > 0·95). Naturally contaminated processed soy products on the market were also studied. There were no discrepancies observed between the respective methods. Conclusions: TEMPO methods were equivalent to the corresponding standard plate methods with very good rates of agreement. Significance and Impact of the Study: The automated MPN method is more practical and reliable for in‐house microbiological testing in processed soy products.     

Methods for detecting recombinant DNA in the environment

The successful introduction of genetically modified and genetically engineered microorganisms into the environment requires a quantitative evaluation of the survival and dispersion of the microorganisms and specific gene(s) in the environment. The objective of this article is to examine the applicability, suitability, and significance of existing and new methods for detecting and monitoring the recombinant genes or organisms introduced into the environment. Conventional microbiological method(s) involving the selective and differential growth of microorganism(s) adn other quantitative approaches such as the most-probable-number (MPN) method and direct microscopic observation (e.g., acridine orange direct count analysis) have drawbacks and are not specific or universally applicable. Direct enumeration by immunofluorescence by the use of fluorescent dye seems more sensitive although still not perfect. However, the molecular methodologies such as the use of gene probes, plasmid epidemiology, antibiotic resistant marker strains, and protein electrophoresis and bacteriophage sensitivity are receiving more attention. As yet, the technology of DNA:DNA hybridization appears to be very useful, sensitive, and accurate for detecting and monitoring the microorganisms in the environment, although improvements are required. New approaches can be developed which may include biochemical signature compounds as well as gene cassettes to be used in a complementary fashion with conventional and molecular techniques for quantifying specific genotypes and genes in the environment.     

Hygienic quality of traditional processing and stability of tomato (Lycopersicon esculentum) puree in Togo

Microbiological and physicochemical qualities of a tomato (Lycopersicon esculentum) puree production line (ripe tomato, washing, cutting, pounding, bleaching, straining, bottling and pasteurization) and its preservation in Togo, West Africa, were studied using the HACCP method. Samples generated during the steps described previously were analyzed by determining sensory, chemical and microbiological characteristics. Samples were analyzed using MPN for coliform populations and plate count methodology for other bacteria. The microorganisms involved in spoilage of the opened products were moulds of genera Penicillium, Aspergillus, Fusarium, Geotrichum, Mucor and gram-positive Bacillus bacteria. The preserved tomato puree exhibited a pH value of 4.3, 90% water content, 0.98 water activity (aw) and an average ascorbic acid level of 27.3mg/100g. Results showed that the critical control point (CCP) of this tomato puree processing line is the pasteurization stage. The analysis of selected microbiological and physicochemical parameters during the preservation of bottled tomato puree indicated that this product was stable over 22 months at 29 degrees C. But the stability of the opened product stored at 29 degrees C did not exceed two months.     

A rapid culture independent methodology to quantitatively detect and identify common human bacterial pathogens associated with contaminated high purity water

Background

Water and High Purity Water (HPW) distribution systems can be contaminated with human pathogenic microorganisms. This biocontamination may pose a risk to human health as HPW is commonly used in the industrial, pharmaceutical and clinical sectors. Currently, routine microbiological testing of HPW is performed using slow and labour intensive traditional microbiological based techniques. There is a need to develop a rapid culture independent methodology to quantitatively detect and identify biocontamination associated with HPW.

Results

A novel internally controlled 5-plex real-time PCR Nucleic Acid Diagnostics assay (NAD), was designed and optimised in accordance with Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines, to rapidly detect, identify and quantify the human pathogenic bacteria Stenotrophomonas maltophilia, Burkholderia species, Pseudomonas aeruginosa and Serratia marcescens which are commonly associated with the biocontamination of water and water distribution systems. The specificity of the 5-plex assay was tested against genomic DNA isolated from a panel of 95 microorganisms with no cross reactivity observed. The analytical sensitivities of the S. maltophilia, B. cepacia, P. aeruginosa and the S. marcescens assays are 8.5, 5.7, 3.2 and 7.4 genome equivalents respectively.Subsequently, an analysis of HPW supplied by a Millipore Elix 35 water purification unit performed using standard microbiological methods revealed high levels of naturally occurring microbiological contamination. Five litre water samples from this HPW delivery system were also filtered and genomic DNA was purified directly from these filters. These DNA samples were then tested using the developed multiplex real-time PCR NAD assay and despite the high background microbiological contamination observed, both S. maltophilia and Burkholderia species were quantitatively detected and identified. At both sampling points the levels of both S. maltophilia and Burkholderia species present was above the threshold of 10 cfu/100 ml recommended by both EU and US guidelines.

Conclusions

The novel culture independent methodology described in this study allows for rapid (<5 h), quantitative detection and identification of these four human pathogens from biocontaminated water and HPW distribution systems. We propose that the described NAD assay and associated methodology could be applied to routine testing of water and HPW distribution systems to assure microbiological safety and high water quality standards.

Electronic supplementary material

The online version of this article (doi:10.1186/s12896-015-0124-1) contains supplementary material, which is available to authorized users.     

A PCR-MPN BASED QUANTITATIVE APPROACH TO ENUMERATE NITRIFYING BACTERIA IN ZEOPONIC SUBSTRATES

Self-sustaining, regenerative life-support systems are required for long duration missions to the Moon and Mars. Improved activity of nitrifying bacteria to convert NH₄⁺ to NO₃^- has been shown to promote plant growth in zeoponic substrates. Due to physiological characteristics, such as slow growth and low yield, nitrifying bacteria are not easily enumerated by traditional microbiological techniques. A method for rapid detection and enumeration of a commercial inoculum of nitrifying bacteria in a zeoponic substrate was developed using a polymerase chain reaction (PCR)-most probable number (MPN) approach. Samples from four-week laboratory incubation studies were processed to extract their total microbial community DNA and the sequences specific to 16s rRNA of Nitrobacter spp. were PCR amplified. The detection limit of the methodology was 2,000 Nitrobacter cells per assay. The quantitative assay demonstrated that the zeoponic substrate was capable of supporting 10⁵ to 10⁶ MPN Nitrobacter cells per gram of substrate. The PCR-MPN method can be an effective and rapid approach to enumerate nitrifying bacteria in zeoponic substrates.     

Force‐ and kinesin‐8‐dependent effects in the spatial regulation of fission yeast microtubule dynamics

Microtubules (MTs) are central to the organisation of the eukaryotic intracellular space and are involved in the control of cell morphology. For these purposes, MT polymerisation dynamics are tightly regulated. Using automated image analysis software, we investigate the spatial dependence of MT dynamics in interphase fission yeast cells with unprecedented statistical accuracy. We find that MT catastrophe frequencies (switches from polymerisation to depolymerisation) strongly depend on intracellular position. We provide evidence that compressive forces generated by MTs growing against the cell pole locally reduce MT growth velocities and enhance catastrophe frequencies. Furthermore, we find evidence for an MT length‐dependent increase in the catastrophe frequency that is mediated by kinesin‐8 proteins (Klp5/6). Given the intrinsic susceptibility of MT dynamics to compressive forces and the widespread importance of kinesin‐8 proteins, we propose that similar spatial regulation of MT dynamics plays a role in other cell types as well. In addition, our systematic and quantitative data should provide valuable input for (mathematical) models of MT organisation in living cells.     

A Partially Automated Radioligand Binding Assay System for use in Clinical and Pharmaceutical Research

Abstract

Using a Tecan robotic sample processor and IBM compatible PCs we have developed a flexible, partially automated radioligand binding assay system. It handles pipetting parameters of up to 16 saturation or competition experiments at a time with up to 24 radioligand- or competitor-concentrations in a range over 4 orders of magnitude per experiment. The system provides enough flexibility so that all pipetting parameters including different tube-, rack-sizes, sample volumina and pipetting sequences may be easily adapted to the large variety of experimental requirements in binding assays. It rationalizes and increases assay throughput (up to 70% spare of working time), improves reliance and reproducibility of results. Radioactive exposure is minimized to the time preparing the radioligand working solution and transferring the sample tubes to and from the sample processor. The system has proven effective in various investigations on binding interactions, as well as in clinical studies on receptor expression under physiologic, pathological and therapeutic conditions.     

Microorganisms in heat supply lines and internal corrosion of steel pipes

In laboratory experiments with batch cultures of thermophilic microorganisms isolated from urban heat supply systems, the growth of sulfate-reducing, iron-oxidizing, and iron-reducing bacteria was found to accelerate the corrosion rate of the steel-3 plates used in the pipelines. In the absence of bacteria and dissolved oxygen, minimal, corrosion was determined. The aforementioned microorganisms, as well as sulfur-oxidizing bacteria, were found to be widespread in water and corrosion deposits in low-alloy steel pipelines (both delivery and return) of the Moscow heat networks, as well as in the corrosion deposits on the steel-3 plates in a testing unit supplied with the network water. The microorganisms were found in samples with water pH ranging from 8.1 to 9.6 and a temperature lower than 90 degrees C. Magnetite, lepidocrocite, goethite, X-ray amorphous ferric oxide were the corrosion products identified on the steel-3 plates, as well as siderite, aragonite, and S0. The effect of microbiological processes on the rate of electrochemical corrosion was evaluated from the accumulation of corrosion deposits and from variation in total and local corrosion of the steel plates in a testing unit.     

Persistence of Perchlorate and the Relative Numbers of Perchlorate- and Chlorate-Respiring Microorganisms in Natural Waters, Soils, and Wastewater

Cell numbers of perchlorate (PRM)- and chlorate (CRM)-reducing microorganisms and the persistence of perchlorate were determined in samples of soils, natural waters, and wastewater incubated under laboratory conditions. Complete perchlorate reduction in raw wastewater and creek water was achieved in 4 to 7 days and 8 to 29 days, respectively, depending on the individual growth substrate (acetate, lactate, citric acid, or molasses) employed. Perchlorate persisted in most mixed cultures developed with 2 g of “pristine” soil, but declined in mixed cultures developed with 100 g of soil. Less than seven days were required to completely reduce perchlorate in cultures started with 10 g of a perchlorate-contaminated soil obtained from a site in Texas. The concentration of PRM was estimated using a 5-tube most probable number (MPN) procedure. To account for discrepancies due to differences in the total number of bacteria (per mass of sample) in the samples, difficulty in removing bacteria from soil samples, and the lack of an unequivocal method to measure total viable cells in these different systems, we normalized our MPN results on the basis of 10⁶ or 10⁹ total bacteria counted using acridine orange direct counts (AODC). There were more PRM in wastewater samples on a per-cell basis (15 to 350 PRM/10⁶-AODC) than in water samples (0.02 to 0.4 PRM/10⁶-AODC). There were also more PRM in soils from sites exhibiting direct evidence of perchlorate contamination (100 to 200 PRM/10⁹-AODC) than from other sites (nondetectable to 0.77 PRM/10⁹-AODC). These results demonstrate that perchlorate-reducing bacteria are present at perchlorate-contaminated sites, and that perchlorate can be degraded by these microorganisms through the addition of different electron donors, such as acetate and lactate.     

Automated measurement and quantification of heterotrophic bacteria in water samples based on the MPN method

Quantification of heterotrophic bacteria is a widely used measure for water analysis. Especially in terms of drinking water analysis, testing for microorganisms is strictly regulated by the European Drinking Water Directive, including quality criteria and detection limits. The quantification procedure presented in this study is based on the most probable number (MPN) method, which was adapted to comply with the need for a quick and easy screening tool for different kinds of water samples as well as varying microbial loads. Replacing tubes with 24-well titer plates for cultivation of bacteria drastically reduces the amount of culture media and also simplifies incubation. Automated photometric measurement of turbidity instead of visual evaluation of bacterial growth avoids misinterpretation by operators. Definition of a threshold ensures definite and user-independent determination of microbial growth. Calculation of the MPN itself is done using a program provided by the US Food and Drug Administration (FDA). For evaluation of the method, real water samples of different origins as well as pure cultures of bacteria were analyzed in parallel with the conventional plating methods. Thus, the procedure described requires less preparation time, reduces costs and ensures both stable and reliable results for water samples.     

ASSESSMENT OF IMPEDANCE MICROBIOLOGICAL METHOD FOR THE DETECTION OF ESCHERICHIA COLI IN FOODS

This study was conducted to determine the sensitivity and specificity of the impedance-based microbiological method for the detection of Escherichia coli in foods within 24 h of testing. A Malthus Microbiological Analyzer system (Malthus System V, Malthus Instruments Ltd., Bury, United Kingdom), and a modified Malthus Coliform Broth Medium (MCBM), and an incubation temperature of 44C were used. The sensitivity of the impedance method was determined by testing E. coli-negative food samples spiked with different concentrations of E. coli. The specificity of the method was determined by testing E. coli -negative food samples spiked with Klebsiella pneumoniae, Enterobacter cloacae and Pseudomonas aeruginosa. The test results were compared with those obtained by the Most Probable Number (MPN) method. Milk, milk products, raw and ready-to-eat meats, and vegetables were tested for the presence of E. coli by both methods. The sensitivity of the impedance method and the MPN method for the detection of foods containing 10¹ CFU/g was 100% and 84.4%, respectively. Both methods had a specificity of 100% for food samples spiked with 10¹ CFU/g E. coli. The specificity of the impedance and the MPN methods for the detection of E. coli in naturally contaminated milk and meat samples was 100% and 95.7% respectively. E. coli was detected in foods by the impedance method within 4–24 h of testing at a detection limit of 1 CFU/mL. These results demonstrate that the impedance method can be used as a rapid and sensitive method for the detection of E. coli in foods.     

Sulfate Reduction at a Lignite Seam: Microbial Abundance and Activity

In a combined isotope geochemical and microbiological investigation, a setting of multiple aquifers was characterized. Biologically mediated redox processes were observed in the aquifers situated in marine sands of Tertiary age and overlying Quaternary gravel deposits. Intercalated lignite seams define the aquitards, which separate the aquifers. Bacterial oxidation of organic matter is evident from dissolved inorganic carbon characterized by average carbon isotope values between ?18.4 per thousand and ?15.7 per thousand (PDB). Strongly positive sulfur isotope values of up to +50 per thousand (CTD) for residual sulfate indicate sulfate reduction under closed system conditions with respect to sulfate availability. Both, hydrochemical and isotope data are thus consistent with the recent activity of sulfate-reducing bacteria (SRB). Microbiological investigations revealed the presence of an anaerobic food chain in the aquifers. Most-probable-number (MPN) determinations for SRB and fermenting microorganisms reached highest values at the interface between aquifer and lignite seam (1.5 x 103 cells/g sediment dry mass). Five strains of SRB were isolated from highest MPN dilutions. Spore-forming bacteria appeared to dominate the SRB population. Sulfate reduction rates were determined by the 35S-radiotracer method. A detailed assessment indicates an increase in the reduction rate in proximity to the lignite seam, with a maximum turnover of 8.4 mM sulfate/a, suggesting that lignite-drived compounds represent the substrate for sulfate reduction.     

Validation of the BacT/ALERT?3D automated culture system for the detection of microbial contamination of epithelial cell culture medium

Living tissue engineering for regenerative therapy cannot withstand the usual pharmacopoeia methods of purification and terminal sterilization. Consequently, these products must be manufactured under aseptic conditions at microbiologically controlled environment facilities. This study was proposed to validate BacT/ALERT(?)3D automated culture system for microbiological control of epithelial cell culture medium (ECCM). Suspensions of the nine microorganisms recommended by the European Pharmacopoeia (Chap. 2.6.27: "Microbiological control of cellular products"), plus one species from oral mucosa and two negative controls with no microorganisms were prepared in ECCM. They were inoculated in FA (anaerobic) and SN (aerobic) culture bottles (Biomérieux, Lyon, France) and incubated in a BacT/ALERT(?)3D automated culture system. For each species, five sets of bottles were inoculated for reproducibility testing: one sample was incubated at the French Health Products Agency laboratory (reference) and the four others at Cell and Tissue Bank of Lyon, France. The specificity of the positive culture bottles was verified by Gram staining and then subcultured to identify the microorganism grown. The BacT/ALERT(?)3D system detected all the inoculated microorganisms in less than 2 days except Propionibacterium acnes which was detected in 3 days. In conclusion, this study demonstrates that the BacT/ALERT(?)3D system can detect both aerobic and anaerobic bacterial and fungal contamination of an epithelial cell culture medium consistent with the European Pharmacopoeia chapter 2.6.27 recommendations. It showed the specificity, sensitivity, and precision of the BacT/ALERT(?)3D method, since all the microorganisms seeded were detected in both sites and the uncontaminated medium ECCM remained negative at 7 days.     

Automated soft agar assay for the high-throughput screening of anticancer compounds

We have established an automated soft agar colony formation assay that can be used as a potent tool in experimental tumor therapy studies as well as anticancer compound screening. It allows the direct and simultaneous comparison of the effects of a high number of anticancer compounds on the anchorage-independent growth of a variety of tumor cell lines. By making use of a commercially available automated pipetting system, the user gets results of excellent quality within 1 week and does not need special cell culture practice.     

Factors That Influence Microbial Contamination of Fluids Associated with Hemodialysis Machines

Studies were conducted on the microbiological quality of fluids associated with different types of dialysis systems located in six dialysis centers and 14 homes. Included were (i) single-pass systems employing either parallel flow (Kiil or Gambro) or capillary cartridge dialyzers and (ii) recirculating single-pass and batch recirculating systems using coil dialyzers. Microbiological assays were performed on the water used to prepare dialysis fluid, the concentrated dialysate, and either pre- and postdialyzer dialysate (single-pass systems) or the dialysate contained in storage reservoirs and recirculating cannisters (recirculating systems). The levels of microbial contamination consisting of gram-negative bacteria were directly related to the type of dialysis system, method of water treatment, distribution system, and in some instances, the type of dialyzer. Recirculating single-pass and batch recirculating systems consistently contained significantly higher levels of contamination than single-pass systems. These results were directly related to the design of recirculating systems which permits carbon- and nitrogen-containing waste products dialyzed from the patient to accumulate, be used as nutrients by microorganisms, and subsequently allow for 2- to 4-log increases in contamination levels during a dialysis treatment. In contrast, levels of contamination in single-pass machines were related more to the quality of the water used to prepare dialysis fluid and the adequacy of cleaning and disinfection procedures than to the design of the system.     

The role of microorganisms in the formation of pitch deposits in pulp and paper mills

The cause of pitch deposit formation seems still not fully understood. The work reported here demonstrates that microorganisms effect the agglomeration of emulgated resin droplets and the formation of sticky precipitates.

Pitch deposits from mills consist mainly of ethanol-soluble resins. It is also the ethanol-soluble fraction of wood resins which forms stable emulsions and which is easily agglomerated by microorganisms.

Pitch deposits, collected from various pulp and paper mills, were all found to contain large amounts of microorganisms. Sterile resin emulsions prepared from pitch deposits remained stable over long periods. After inoculation with microorganisms the emulsions were destabilized and the resins completely precipitated as sticky lumps.

Various bacteria and fungi are capable of agglomerating the resins, but species isolated from water, pulp and slime in paper mills were usually most effective. Resins from fresh wood were precipitated at a faster rate than aged resins.

Problems of pitch formation can be considerably reduced when microbial growth is kept under control in the production system. To be effective, the control measures, e.g. slimicides, must be applied at the right place and time, and in the correct concentrations. This presupposes a thorough knowledge of the plant's microbiological condition which can only be obtained by microbiological examination. Practical cases of the appropriate application of biocides in pulp and paper mill systems are discussed.     

A comparison of ready-to-use systems for evaluating the microbiological quality of acidic fruit juices using non-pasteurized orange juice as an experimental model.

Several alternative analytical methods are currently available for the rapid microbiological testing of food. Due to their many advantages, particularly their convenience of use, the popularity of ready-to-use systems for the enumeration of hygiene indicator microorganisms is increasing. However, the ability of these systems to enumerate stressed microorganisms, such as those that may be found growing in acidic foods, is unknown. Therefore, the aim of this study was to evaluate the performance of Petrifilm(tm) and SimPlate(tm) plates for the enumeration of total aerobes and fungi (yeasts and molds) in acidic fruit juices, using non-pasteurized orange juice as an experimental model. The samples were analyzed before and after neutralization of pH, and the results were compared with those obtained using conventional procedures, i.e. pour-plates containing Standard Methods Agar, acidified potato dextrose agar, or dichloran-glycerol agar. The results obtained with Petrifilm and SimPlate for counts of mesophilic aerobes as well as for yeast and mold correlated well with those obtained using conventional procedures. Although no statistically significant differences were observed between counts of non-neutralized and neutralized samples (alpha >/== 0.05), better correlation indexes were observed in the neutralized samples. Both Petrifilm and SimPlate proved to be good alternative methods for testing the microbiological quality of acidic fruit juices.     

Evaluation of negative results of BacT/Alert 3D automated blood culture system

Although automated continuous-monitoring blood culture systems are both rapid and sensitive, false-positive and false-negative results still occur. The objective of this study, then, was to evaluate negative results occurring with BacT/Alert 3D blood culture systems. A total of 1032 samples were cultured with the BacT/Alert 3D automated blood culture system, using both aerobic (FA) and anaerobic (FN) [corrected] media, and 128 of these samples yielded positive results. A total of 904 negative blood samples were then subcultured in 5% sheep blood agar, eosin methylene blue, chocolate agar, and sabouraud-dextrose agar. Organisms growing on these subcultures were subsequently identified using both Vitek32 (bioMerieux, Durham, NC) and conventional methods. Twenty four (2.6%) of the 904 subcultures grew on the subculture media. The majority (83.3%) of these were determined to be gram-positive microorganisms. Fourteen (58.3%) were coagulase-negative staphylococci, two (8.3%) were Bacillus spp., one (4.2%) was Staphylococcus aureus, and one (4.2%) was identified as Enterococcus faecium. Streptococcus pneumoniae and Neisseria spp. were isolated together in two (8.3%) vials. Gram-negative microorganisms comprised 12.5% of the subcultures, of which two (8.3%) were found to be Pseudomonas aeruginosa, and one (4.2%) was Pseudomonas fluorescens. The other isolate (4.2%) was identified as Candida albicans. We conclude that the subculture of negative results is valuable in the BacT/Alert 3D system, especially in situations in which only one set of blood cultures is taken.