Concentration Of Colony-forming Units Research Articles

Chlorin photosensitizer photolysis products for treating hospital-acquired pneumonia

Purpose: to develop a technique for therapeutic application of photoactivated photosensitizer «Radachlorin» to be included into the complextherapy of nosocomial pneumonia.Material and methods. Twelve patients with nosocomial pneumonia were taken in the trial. They were treated with a device having a nebulizer witha ring-shaped irradiator and diodes λ ≈ 660 nm for photoactivation of photosensitizer «Radachlorin» (manufactured by OOO «RADA-PHARMA»,Moscow, registration number LS-001868). The photosensitizer was developed according to the technical tasks provided by OOO «New SurgicalTechnologies» (Moscow, Russia). Radachlorin solution photolysis products were inhaled by the patients twice a day for 3 days in combinationwith the standard therapy when they were in ICU. Patients in the control group (n = 12) did not have any inhalations.Results and discussion. On day 3, the number of colony-forming units (CFU) of various pathogens found in swabs from the throat decreasedby 39.45 ± 3.17 % on average; C-reactive protein decreased by 42.38 ± 4.26 %; procalcitonin decreased by 51.83 ± 3.45 %, if to compareto baseline limits. At the same period in the control group, CFU in swabs increased in average by 17.28 ± 4.21 %; C-reactive protein increasedby 27.76 ± 3.52 %; procalcitonin increased by 30.15 ± 2.24 %. On day 10 in the main group, the number of CFU pathogens decreasedby 35.45 ± 3.32 %; C-reactive protein decreased by 72.15 ± 2.56 %; procalcitonin decreased by 55.56 ± 4.23 %, if to compare to baseline limits. On day 10 in the control group, CFU concentration remained elevated by 31.15 ± 2.56 %; C-reactive protein – by 65.32 ± 4.27; procalcitonin – by 30.21 ± 3.45 %, if to compare to baseline values. These findings indicate that the inflammatory process acquired a progressive form which demands switching to other curative modalities and more careful selection of antibiotics. The probability of random origin in statistical processing is p < 0.05. There were no undesirable side effects during procedures.Conclusion. Justified application of the described technique – inhalations of photolysis products of E 6 chloride derivatives – has an effective therapeutic impact at the course of nosocomial pneumonia. It seems promising to continue investigations on the described technique, namely, to search for the most effective ways of its application, to find out an optimal number and frequency of procedures under the control of general clinical and special methods of examination of the studied patients, since the data which have been obtained by the authors us are preliminary and should be supplemented with new findings.

TECHNOLOGY DEVELOPMENT OF PROBIOTIC “SPORO-LEKS” MANUFACTURING

An important element of the probiotics quality is the technology of their production. There is great variability between the results of preclinical and clinical trials of probiotic microorganisms. To a large extent, this is influenced by the technology and conditions of production of probiotics. The purpose of the work was to compare the manufacturing technologies of “Sporo-leks” probiotic. Probiotic “Sporo-leks” is a mixture of probiotic cultures of Bacillus licheniformis VK-25 and Bacillus subtilis MK-3, sorbed on a complex activated with the addition of a natural standardized sorbent. Two versions of the “Sporo-leks” probiotic manufacturing technologies were developed: in a liquid nutrient medium and on a dense nutrient medium. During the tests, intermediate quality controls were developed. As a result of the conducted research, lists of intermediate quality controls were formed during the production of the probiotic “Sporo-leks” in a liquid nutrient medium and on a dense nutrient medium. Dense nutrient media manufacturing has been found to be faster and more cost-effective as it requires only 4 intermediate control stages compared to 5 stages in liquid nutrient media manufacturing. Depending on the stage and production technology, various intermediate studies were carried out, which included microscopy, determination of typicality of growth, determination of contamination by foreign bacterial and fungal microflora, determination of sporulation, determination of the concentration of colony-forming units. Also, during the research, a list of quality control indicators of the finished probiotic “Sporo-leks” was formed and tested. It has been proven that the process of sporulation during the production of the probiotic “Sporo-leks” on a dense nutrient medium occurred faster by 24 hours compared to the technology of production in a liquid nutrient medium. Probiotic production technology on a dense nutrient medium ensured the formation of a more mature spore, which led to the stability of the drug during storage according to the indicator "the number of living cells in 1 g of product." According to the rest of the quality indicators, both technologies equally ensured the stability of the product during storage for 36 months.

Impact of needle-point bipolar ionization system in the reduction of bioaerosols in collective transport

The transmission rate of SARS-CoV-2 is higher in collective transport than in other public environments. Transport companies require preventive strategies to mitigate airborne risk of contagion which not imply responsible use at the individual level. Air purification systems, such as UV-C or needle-tip bipolar ionization, are attractive alternatives. However, only a few studies addressing the validation of this technology against bioaerosols in actual operation conditions have been published so far. In this work, the efficiency of a bipolar ionization unit in the Zaragoza Tram has been evaluated. Against environmental bioaerosols, ionization (~25.7 · 109 ions/m3, on average) reduced the concentration of colony-forming units (CFU) by ~46 % and ~69 % after 30 and 90 min. No clear benefits were obtained against microorganisms on inner tram surfaces (seats, grab bars, walls, and windows). ‘Pre-pandemic’ filtration equipment located in the HVAC based on a Coarse 45 %-type filter removed ~73 and ~ 92 % of aerial CFU by itself after 30 and 90 min. Microscopic visualization of the CFUs revealed that they were mostly >1 μm, much larger than the SARS-CoV-2 virion (~100 nm) and SARS-CoV-2-loaded bioaerosols (from 0.25 μm). Then, we studied the filter behavior under normalized laboratory methods. The filters efficiency against submicron particles was limited (between 5 and 12 % against 0.1 to 0.3 μm NaCl particles). Another ionization strategy was to generate aerosol agglomerates to enhance filtration performance, but the combined action of ionization and filtration did not improve substantially. The effect of these technologies was also characterized using the clean air delivery rate (CADR). Relative to untreated air (CADR = 0.299 m3/min), ionization and filtration reduce ambient CFUs (CADR = 5.153 and 9.261 m3/min, respectively; and CADR = 13.208 m3/min, combined) which implies that it has a substantial impact on indoor bioaerosols.

Toxicity of nZVI in the growth of bacteria present in contaminated soil.

The use of nano zero-valent iron (nZVI) for the remediation of degraded areas is a consolidated practice. However, the long-term reactions that occur in the environment remain unknown. This study aimed to evaluate the potential toxic effects on the growth of colony-forming units (CFUs) of Bacillus cereus and Pseudomona aeruginosa present in soil contaminated with hexavalent chromium (Cr6+) and pentachlorophenol (PCP) nanoremediated with nZVI. The treatments were natural soil (control), soil contaminated by Cr6+, soil contaminated by PCP, and soil contaminated by Cr6+ and PCP (Cr6+ and PCP), all in duplicate. The concentration of contaminants used was 100mg/kg of soil. One of the drums of the duplicate received an injection of nZVI solution with a concentration of 50g/kg. Analysis was performed 7, 15, 21, 30, 60, and 90 days after the nZVI injection. Temporary oscillations in the abundance of the microbiological community were observed, characterizing the adaptation of bacteria to the contaminants. The bacteria showed similar behavior. Ninety days after the injection of nZVI, the averages of the CFUs were statistically equal, with the lowest coefficient of variation and the highest concentration of CFUs occurring. The strains of B. cereus and P. aeruginosa were resistant to the concentrations of nZVI, Cr6+, and PCP. The nanoremediation of nZVI in soil contaminated by Cr6+ and PCP had no toxic effects on the population of the bacteria evaluated and did not present major disturbances in temperature, electrical conductivity, pH, and humidity over time.

191. High-Dose Rifampin-containing Regimens for the Treatment of TB Meningitis

191. High-Dose Rifampin-containing Regimens for the Treatment of TB Meningitis

The Influence of Carpeting, Human Activity and Number of Beds on Airborne Fungi Concentration in Hotel Bedrooms

In urban environments, people spend about 90% of their time indoors, where strong indicators of air-borne contaminants have been found. Currently, there are no reports on the fungal presence and distribution in the air of hotel bedrooms. In this study, we assessed the presence of airborne fungi in bedrooms from three hotels and correlated with room characteristics. We sampled 100 L (L) of air from hotels in Nuevo León, Mexico, then fungi colony forming units (CFU) were measured and identification was made based on morphological features. Variables considered were the presence of carpet, number of beds, cleaning status for the room and floor number. Penicillium, Cladosporium and Aspergillus exhibited the highest CFU concentration and frequency. A slight tendency was observed towards lower fungi concentrations when rooms had been cleaned before sampling. Statistical differences were found between rooms with carpet vs. no carpet, and one vs. two beds. Furthermore, a correlation between floor number and fungi concentration was observed with correspondence to the hotels’ room assignment protocol. These findings offer new variables to take into consideration when designing and implementing preventive or corrective sanitization procedures to improve their efficiency and could be relevant for hotel bedrooms as well as any other type of room.

Legionella and agar cultures in healthcare facilities waters: a monitoring study in North-East Italy

Abstract Background Water healthiness is fundamental in medical facilities. Monitoring Legionella spp. and agar cultures concentration is therefore very important. This study looks into these parameters to examine possible criticalities that could need interventions. Methods We collected 399 Legionella and 82 agar cultures withdrawals between November 2019 and February 2020 from 157 detection points (like taps and showers) in 31 structures of four health districts. Withdrawals referred to the 4 quarters of year 2019 (t1, t2, t3, t4). Legionella values were expressed in colony forming units (CFU)/l; agar cultures in CFU/ml. We performed a non-parametric analysis to compare mean values between districts. We used the statistical software package SAS version 9.4 for Windows, setting the significance level at α = 0.05. Results 45% of structures presented at least one positive withdrawal for Legionella (in six we found positivity in more than 1 quarter). Concerning Legionella concentration, we found significant difference between districts in t2 (p = 0.012) and between wards of one district in t3 (p < 0.05). We observed agar positive values in 11 structures (in four cases in more than 1 quarter). In t4 we found a very significant difference (p < 0.0001) between agar values in the four districts, while in t3 we observed a non significant association (p = 0.067). In t3 we found a significant difference for agar values between wards of one district (p = 0.025). We finally considered the simultaneous presence or absence of Legionella and agar positivities: without considering quarters division, we observed accordance (both present or both absent) in 60 detection points, while in 34 points we didn't find it. Conclusions Data provided by this study show that Legionella is rather present in our districts, revealing an association with concentration of CFU in agar. Disinfecting operations should be implemented considering the differences between districts to provide a safe water in every ward. Key messages Water quality monitoring in healthcare facilities is fundamental to provide a safe and healthy environment. Legionella spp. and agar cultures concentrations depend by quarter and spot considered; focused operations should be taken into account to improve disinfection quality.

On distinguishing the natural and human-induced sources of airborne pathogenic viable bioaerosols: characteristic assessment using advanced molecular analysis

Ambient air consists of bioaerosols that constitute many microbes from biosphere due to natural and anthropogenic activities. Size-dependent ambient measurements of bioaerosols at two seminatural and three anthropogenic coastal sites in southern tropical India were taken during the summer 2017. All the five sites considered in this study considerably contributed to the bioaerosol burden with larger contribution from the dumping yard site followed by the marshland site, wastewater treatment plant, composting site, and Indian Institute of Technology Madras. The colony-forming units concentration for all the sites ranged from 17 to 2750 m−3 for bacteria and 42–2673 m−3 for fungi. Firmicutes and Actinomycetes were the dominant phyla observed in 698 bacterial OTUs obtained, and Ascomycota and Zygomycota were the dominant phyla observed in 159 fungal OTUs obtained in the study. Further, the study revealed the presence of pathogenic and ice-nucleating bacteria and fungi in the bioaerosols that can largely affect the well-being of the human population and vegetation in this region. Moreover, the statistical analysis revealed high bacterial abundance and diversity at the grit chamber of wastewater treatment plant and high fungal abundance and diversity at the dumping yard. Further, principal coordinate analysis of the sites studied inferred that the marshland, wastewater treatment plant, and the dumping yard sites shared similar microbial community composition indicating the existence of similar source materials and activities at the sites. Further, this study evidently brings out the fact that urban locations may play an important role in anthropogenic contribution of both pathogenic and ice-nucleating microorganisms.

Rare occurrence of airborne bioparticles in a speleotherapy site: the case study of the Sant’Aloisio disused siderite mine (Brescia, Italy)

Caves and mines have a suitable microclimate in terms of temperature, humidity and air purity that could be exploited for speleotherapy. The aim of this study was to evaluate the current load of allergenic airborne bioparticles of a subterranean environment being considered as a potential site for treating respiratory allergies. Sampling was performed over 12 months (2015–2016) inside and outside the Sant'Aloisio mine by: (i) continuous aerobiological sampling with Hirst-type samplers and (ii) monthly air sampling with an on-plate volumetric sampler using three different selective culture media. The following protocols were applied for the two methodologies: (i) Samples were processed following the European standard (UNI CEN/TS 16868:2015) and analyzed by light microscopy to identify and count pollen grains and fungal spores; (ii) plates were incubated, colony-forming units (CFU) were counted and concentration of CFU was calculated. Spore and pollen load, as evaluated by the continuous monitoring, resulted in very low values inside the mine, with a total of 106 pollen grains and 41 fungal spores from two species (Alternaria and Cladosporium) collected over the entire year; the pollen and spore concentration in the subterranean environment was negligible when compared to the open-air sampling (0.2% for pollen; 0.02% for spores). Viable airborne microorganisms were less represented in the mine than in the open air (median: 7 and 83 CFU*m−3, respectively). The indoor air of the disused mine was confirmed to be suitable for hosting respiratory disease sufferers from an aerobiological point of view. The residual low quantity of pollen grains could be the result of unintentional human transport. The indoor fungal component of the viable airborne microorganisms was mostly derived from non-allergenic wood-decaying fungi.

Relación entre el estrés y las bacterias entomopatógenas Pantoea (Erwinia) agglomerans (herbicola) y Bacillus cereus en jobotos (Col: Melolonthidae) (Phyllophaga spp., Anomala spp. y Cyclocephala spp.), en Costa Rica.

Concentrations of Colony Forming Units (CFU) were determined for two entomopathogenic bacteria (Pantoea agglomerans and Bacillus cereus), at the egg, larval, pupal, and adult stages of agriculturally important Phyllophaga and Cyclocephala white grubs, which were collected in five agroecosystems in Costa Rica. L2 and L3 larvae of Phyllophaga elenans collected in all regions where the study was conducted were extensive carriers of Pantoea agglomerans and Bacillus cereu. L2 and L3 larvae of Phyllophaga obsoleta, Phyllophaga menetriesi, Cyclocephala sanguinicollis and Cyclocephala castaniella found in the Central Valley and Central Pacific regions were carriers of Pantoea agglomerans and Bacillus cereus bacteria. In 60% to 90% of larvae in all white grub varieties studied, Pantoea agglomerans showed greater concentrations of CFU than Bacillus cereu, which showed the lowest CFU concentration. Egg, pupal, and adult mortality in all Phyllophaga species was due to Pantoea agglomerans in 62%, 80% and 22.5% of the cases, respectively. A possible antagonistic interaction between Pantoea agglomerans and Bacillus cereus is also discussed. In general, it was noted that light and larval manipulation were the main stress factors affecting these scarabids.

Efficacy of 3D conforming nickel titanium rotary instruments in eliminating canal wall bacteria from oval-shaped root canals

ObjectivesTo evaluate the effectiveness of TRUShape® 3D Conforming Files, compared with Twisted Files, in reducing bacteria load from root canal walls, in the presence or absence of irrigant agitation. MethodsExtracted human premolars with single oval-shaped canals were infected with Enterococcus faecalis. Teeth in Group I (N=10; NaOCl and QMix® 2in1 as respective initial and final irrigants) were subdivided into 4 subgroups: (A) TRUShape® instrumentation without irrigant activation; (B) TRUShape® instrumentation with sonic irrigant agitation; (C) Twisted Files without irrigant agitation; (D) Twisted Files with sonic irrigant agitation. To remove confounding factor (antimicrobial irrigants), teeth in Group II (N=10) were irrigated with sterile saline, using the same subgroup designations. Specimens before and after chemomechanical débridement were cultured for quantification of colony-forming units (CFUs). Data from each group were analyzed separately using two-factor ANOVA and Holm-Sidak multiple comparison (α=0.05). Canal wall bacteria were qualitatively examined using scanning electron microscopy (SEM) and light microscopy of Taylor-modified Brown and Brenn-stained demineralised sections. ResultsCFUs from subgroups in Group I were not significantly different (P=0.935). For Group II, both file type (P<0.001) and irrigant agitation (P<0.001) significantly affected log-reduction in CFU concentrations. The interaction of these two factors was not significant (P=0.601). Although SEM showed reduced canal wall bacteria, bacteria were present within dentinal tubules after rotary instrumentation, as revealed by light microscopy of longitudinal root sections. ConclusionsTRUShape® files removed significantly more canal wall bacteria than Twisted Files when used without an antibacterial irrigant; the latter is required to decontaminate dentinal tubules. Clinical significanceRoot canal disinfection should not be focused only on a mechanistic approach. Rather, the rational choice of a rotary instrumentation system should be combined with the use of well-tested antimicrobial irrigants and delivery/agitation techniques to establish a clinically realistic chemomechanical débridement protocol.

Cover crop and conidia delivery system impacts on soil persistence of Metarhizium anisopliae (Hypocreales: Clavicipitaceae) in sugarbeet

The sugarbeet root maggot, Tetanops myopaeformis (Röder), is a major North American pest of sugarbeet, Beta vulgaris L. Previous research suggests that moderate T. myopaeformis control is possible with the entomopathogen Metarhizium anisopliae (Metch.) Sorok. We conducted a three-year (2002–2004) experiment to assess impacts of oat, Avena sativa L. and rye, Secale cereale L., cover crops on persistence of corn grit-based granular or spray formulations of M. anisopliae isolate ATCC 62176 (i.e. MA 1200) applied at 8×1012 viable conidia/ha in sugarbeet. More colony forming units (CFUs) were detected immediately after application [0 days after treatment (DAT)] in spray plots than granule-treated plots. However, 76–92% declines in CFUs per gram of soil occurred in spray plots within 30 DAT. Substantially (i.e. 83–560%) more rainfall occurred in June 2002 than during June of any other year. Subsequently, 71–670% increases in CFU concentrations occurred by 60 DAT in M. anisopliae granule-treated plots with oat or rye cover crops that year. CFU density increases were higher in cover crops in 2002, but no significant cover crop effects were detected. Conidia persisted for up to 30 DAT in M. anisopliae spray plots and 60 DAT in granule-treated plots in 2002; however, no increases occurred in the years with less June rainfall. Trends suggest that M. anisopliae aqueous sprays result in greater conidia concentrations than granules at sugarbeet plant bases in June during T. myopaeformis oviposition and larval establishment on host plants. Increases are possible when delivering conidia via granules, but high post-application rainfall could be necessary for conidia production.

Contamination level and location of recreational freshwater influence the ability to predict Escherichia coli concentration by qPCR targeting Bacteroides

Fecal bacteria are common microbial contaminants in freshwater with the potential to cause human illness. Detection of these microbes have traditionally relied on microbial plating to enumerate colonies of fecal indicator bacteria (FIB) such as Escherichia coli (E. coli), which can take 24 h or longer to complete. Quantitative PCR (qPCR) is a rapid and sensitive method for detection of FIB in recreational water that could compliment or potentially substitute for microbial plating. In this study, we have isolated DNA from the beach water on the shoreline at three different locations of Lake Erie and subjected these samples to qPCR to examine the relative abundance of Bacteroides. These values were compared to colony forming units (CFU) of E. coli. The resultant linear regressions between these different measurements of microbe concentration were used to determine the efficacy of qPCR targeting Bacteroides at predicting E. coli concentrations that are relevant for decision making by recreational water managers. Our findings indicate that the ability of Bacteroides to serve as an early predictive tool for E. coli CFU concentration depends on sample location and level of bacterial contamination, but can be used in some cases to supplement recreational water quality measurement and consequential management.

Evaluation and Quantification of Heat Treatments Applied to Food Preserves

The quantitative study of heat treatments for sterilisation uses the Bigelow model to calculate the sterilising value (F). Calculation of F requires the previous determination of parameters D (decimal reduction time at experimental temperature) and Z (thermal-death time parameter), obtained from the thermal-death kinetics. Herein we compare two different methods, namely the Bigelow model and a predictive-type statistical method, to calculate the sterilisation effect against Bacillus coagulans spores when heat was applied to runner bean preserves (variety: Helda). Samples were subjected to various autoclave treatments at working temperatures (T ai) of 105, 107, 110, and 115°C for periods from 3 to 35 min. The microorganism used was B. coagulans. Sterilisation achieved by these autoclave treatments was determined by using the equation based on the Bigelow model (n probe = F z Ti/D Ti) where n is the fractional concentration of colony-forming units (or some quality factor), F z Ti is F at temperature T i, and D Ti is D at temperature T i. The Bigelow model can be used to obtain Z (thermal-death time parameter), which is needed to calculate the traditional sterilisation factor F, but not to determine the reduction factor n for the heat treatments, particularly when microbial indicators with low decimal reduction times (D) are studied. The thermokinetic parameters for B. coagulans in runner bean solution resulted to be Z = 10.64°C and D 121 = 0.0264 min (Af = 1.04). Treatment at 115°C for 20 min resulted in the most efficient sterilisation effect for B. coagulans.

Mirasol PRT system inactivation efficacy evaluated in platelet concentrates by bacteria-contamination model

Bacterial contamination of blood components, primarily platelet concentrates (PCs), has been identified as one of the most frequent infectious complications in transfusion practice. PC units have a high risk for bacterial growth/multiplication due to their storage at ambient temperature (20 +/- 2 degrees C). Consequences of blood contamination could be effectively prevented or reduced by pathogen inactivation systems. The aim of this study was to determine the Mirasol pathogen reduction technology (PRT) system efficacy in PCs using an artificial bacteria-contamination model. According to the ABO blood groups, PC units (n = 216) were pooled into 54 pools (PC-Ps). PC-Ps were divided into three equal groups, with 18 units in each, designed for an artificial bacteria-contamination. Briefly, PC-Ps were contaminated by Staphylococcus epidermidis, Staphylococcus aureus or Escherichia coli in concentrations 10(2) to 10(7) colony forming units (CFU) per unit. Afterward, PC-Ps were underwent to inactivation by Mirasol PRT system, using UV (lambda = 265-370 nm) activated riboflavin (RB). All PC-Ps were assayed by BacT/Alert Microbial Detection System for CFU quantification before and after the Mirasol treatment. Samples from non-inactivated PC-P units were tested after preparation and immediately following bacterial contamination. Samples from Mirasol treated units were quantified for CFUs one hour, 3 days and 5 days after inactivation. Results. A complete inactivation of all bacteria species was obtained at CFU concentrations of 10(2) and 10(3) per PC-P unit through storage/investigation period. The most effective inactivation (10(5) CFU per PC-P unit) was obtained in Escherichia coli setting. Contrary, inactivation of all the three tested bacteria species was unworkable in concentrations of > or = 10(6) CFU per PC-P unit. Efficient inactivation of investigated bacteria types with a significant CFU depletion in PC-P units was obtained--3 Log for all three tested species, and 5 Log for Escherichia coli. The safety of blood component therapy, primarily the clinical use of PCs can be improved using the Mirasol PRT system.

Rapid Screening ofAlicyclobacillus acidoterrestrisSpoilage of Fruit Juices by Electronic Nose: A Confirmation Study

Early screening ofAlicyclobacillus spp.in fruit juices is a major applicative goal for the food industry, since juice contamination can lead to considerable loss of quality, and subsequently, to economic damages for juice producers. This paper presents an accurate study to assess and confirm the EOS507 electronic nose's (EN) ability of diagnosingAlicyclobacillus acidoterrestrisspoilage in artificially contaminated fruit juices. The authors experimental results have shown that the EOS507 can early identify, just after 24 hours from inoculation, contaminated orange and pear juices with an excellent classification rate close to 90% and with a detection threshold as low as 103 cfu/ml. In apple juice the detection threshold was about 105 cfu/ml, thus requiring longer incubation times (72 hours). PLS regression of EOS507 data can be also used to predict with fair accuracy the colony-forming units concentration of the bacteria. These results were supported by the GC/MS/MS measurements of specific chemical markers, such as guaiacol.

Transpedicular aspiration of osteoprogenitor cells from the vertebral body: progenitor cell concentrations affected by serial aspiration

Background context Spinal fusion is facilitated when the fusion site is augmented with autograft bone. Iliac crest, long the preferred source of autograft material, is the site of frequent complications and pain. Connective tissue progenitor cells (CTPs) aspirated from marrow provide a promising alternative to traditional autograft harvest. The vertebral body represents an even larger potential reservoir of progenitor cells than the ilium. Purpose To test the hypothesis that a suitable concentration of osteoprogenitor cells can be aspirated from different depths of the vertebral body, maintaining progenitor cell concentrations comparable to the “gold standard,” the iliac crest, even after sequential aspirations along the same transpedicular axis. Study design Prospective clinical investigation quantifying CTP concentrations within the vertebral body relative to depth of sequential aspirations. Patient sample Adult men and women undergoing elective posterior lumbar fusion and pedicle screw instrumentation (six men and seven women, mean age 56 years [range 40–74 years]). Outcome measures Cell count, CTP concentration (CTPs/cc marrow), and CTP prevalence (CTPs/million cells) were calculated for both individual and pooled aspirate samples. Methods Thirteen patients were enrolled into an institutional review board–approved protocol studying transpedicular aspiration of marrow progenitor cells. Connective tissue progenitor cells were aspirated from four depths along the transpedicular axis of the vertebral body and quantified according to cell concentration and CTP prevalence. Histochemical analysis of alkaline phosphatase–positive colony-forming units (CFUs) provided the prevalence of vertebral CTPs relative to depth of aspiration, vertebral level, age, and gender. Results Four 2.0 cc aspirations were obtained from each pedicle of lumbar vertebrae selected for pedicle screw fixation (four 2.0 cc aspirates from each of four pedicles). Aspirates of vertebral marrow demonstrated comparable or greater concentrations of CFUs compared with standards previously established for the iliac crest. Overall, the 208 aspirations from 26 vertebral bodies provided a mean CTP concentration of 741.5±976.2 CTPs per cubic centimeter of marrow, ranging from a mean concentration of 1316±1473 CTPs per cubic centimeter of marrow at superficial (30 mm) aspirations to 439±557 CTPs per cubic centimeter marrow at deepest (45 mm) aspiration depths (p<.00002). There were no significant differences relative to vertebral body level, side aspirated, or gender. An age-related decline in cellularity was suggested for vertebral body aspirates. Conclusions The vertebral body is a potential marrow reservoir for aspiration of autograft osteogenic CTPs that can be used to augment spinal fusion. The cancellous bone within that portion of the vertebral body routinely cannulated during pedicle screw placement allows serial aspirations with only modest depletion of progenitor cell concentrations or dilution with peripheral blood. Connective tissue progenitor cell concentrations from all depths were comparable to the mean levels previously established for the iliac crest. The ability to simultaneously harvest progenitor cells for graft augmentation while preparing the pilot hole for pedicle screw fixation will expand the potential for cell harvest techniques for fusion augmentation and reduce the need for iliac crest harvest.

Binding of nontarget microorganisms from food washes to anti-Salmonella and anti-E. coli O157 immunomagnetic beads: minimizing the errors of random sampling in extreme dilute systems

For most applications, 3-5 observations, or samplings (n), are utilized to estimate total aerobic plate count in an average population (μ) that is greater than about 50 cells, or colony forming units (CFU), per sampled volume. We have chosen to utilize a 6 × 6 drop plate method for bacterial colony selection because it offers the means to rapidly perform all requisite dilutions in a 96-well format and plate these dilutions on solid media using minimal materials. Besides traditional quantitative purposes, we also need to select colonies which are well-separated from each other for the purpose of bacterial identification. To achieve this goal using the drop plate format requires the utilization of very dilute solutions (μ < 10 CFUs per sampled drop). At such low CFU densities the sampling error becomes problematic. To address this issue we produced both observed and computer-generated colony count data and divided a large sample of individual counts randomly into N subsamples each with n = 2-24 observations (N × n = 360). From these data we calculated the average total mean-normalized (x⁻(tot), n = 360) deviation of the total standard deviation (s (tot)) from each jth subsample's estimate (s ( j )), which we call Δ. When either observed or computer-generated Δ values were analyzed as a function of x⁻(tot), a set of relationships (∞ ₋₂√ ⁻x(tot)) were generated which appeared to converge at an n of about 18 observations. This finding was verified analytically at even lower CFU concentrations (⁻x(tot) ≈ 1 − 10 CFUs per observation). Additional experiments using the drop plate format and n = 18 samplings were performed on food samples along with most probable number (MPN) analyses and it was found that the two enumeration methods did not differ significantly.

Improved Culturing Techniques for Campylobacter

ABSTRACT Improved techniques for culturing and enumerating Campylobacter jejuni were developed. Parameters included (1) growth vessel type, (2) growth atmosphere, (3) incubation time, and (4) frequency of subcul‐turing. Improved growth and consistent morphology were obtained using a tissue culture flask compared with an Erlenmeyer flask or a test tube as well as exposure to modified atmosphere (10% CO2, 85% N2, and 5% O2). When cultures of C. jejuni were incubated more than 24 h, transformation to the coccoid increased, and fewer colony‐forming units were detected. Excessive subculturing (&gt;2 times) resulted in increased formation of coc‐coid forms. Decreased concentrations of colony‐forming units of C. jejuni commonly attributed to the production of a viable, nonculturable form also may be due to cellular clumping. These techniques should alleviate difficulty and reduce variation when working with Campylobacter.

Effect of brassicaceous plants on the survival and infectivity of insect pathogenic fungi

Studies were conducted to investigate potentialinteractions between brassicaceous plants, theisothiocyanates they produce and insectpathogenic fungi. Studies in vitro showedthat 100 ppm of 2-phenylethyl isothiocyanatecompletely inhibited growth of Metarhiziumanisopliae and Tolypocladiumcylindrosporum. T. cylindrosporum wassignificantly inhibited by 10 ppm 2-phenylethylisothiocyanate, but not by 1 ppm. M.anisopliae was not inhibited by 10 or 1 ppm2-phenylethyl isothiocyanate. The effect offreshly grated rutabaga on virulence of M.anisopliae to Galleria mellonella larvaewas also tested, resulting in reduced infectionby M. anisopliae. To study the effect ofplants in the Brassicaceae under morefield-like conditions fungi were added to soilwithout plants or at the bases of two differentplant species, Barbarea vulgaris and Eruca vesicaria sativa. Soil was sampled at 5,10 and 15 days to determine the concentrationof colony forming units (CFUs). Treatments withplants did not have fewer CFUs than thecontrol. Instead significantly fewer CFUsresulted when M. anisopliae was added topots with soil only than to pots with plants.There were no significant differences betweenthe two plant species in the number of CFUs atday 5. For days 10 and 15, however,significantly more M. anisopliae CFUswere found in pots containing E. vesicariasativa than B. vulgaris. T.cylindrosporum was less affected by thedifferent treatments than M. anisopliae.Our studies demonstrated that whileisothiocyanates can inhibit insect pathogenicfungi in Petri dishes, when using a morerealistic fungus/plant/soil microcosm no fungalinhibition was found.