Dilution Tubes Research Articles

Estimation of MBC: MIC Ratio of Herbal Extracts against Common Endodontic Pathogens

ABSTRACT Herbal extracts have evoked interest owing to the small number of terpenoids and phenolic compounds, which impart antimicrobial, antioxidant, and anti-inflammatory properties. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC) of four herbal extracts (lemon grass oil, basil oil, peppermint oil, and Obicure tea extract) against endodontic pathogens along with the MIC: MBC/MFC ratio were evaluated. The antimicrobial activity by detecting the MIC of three essential oils and tea extract was evaluated against eight common endodontic pathogens by the broth dilution method, while MBC was detected by subculturing onto blood agar from the first –three to five tubes from the MIC dilution tubes (showing no turbidity), which were plated on blood agar. All herbal extracts proved to be effective antimicrobials against tested endodontic pathogens. Basil oil had a bacteriostatic effect on all the organisms (P < 0.05). Mint oil showed bacteriostatic activity on Enterococcus (E.) faecalis and Peptostreptococcus (P > 0.05). Tea extract had a bacteriostatic effect (P > 0.05) against all tested microbes except Actinomyces, Lactobacilli, Staphylococcus (S.) aureus, and Fusobacterium (F.) nucleatum. Lemon grass oil had a bactericidal effect against all the organisms and a bacteriostatic effect against Peptostreptococcus (P > 0.05). It can be concluded that basil oil showed a strong bactericidal effect on the test organisms. The MIC for the organisms ranged from 0.2 to 50 μg/ml.

High incidence of discrepancies in new Siemens assay- a comparison of cardiac troponin I assays.

Cardiac troponin (cTn) is the biochemical gold standard for diagnosing myocardial infarction (MI). We compared the Siemens ADVIA Centaur High-Sensitivity (hs-cTnI) assay with the Siemens Ultra assay (cTnI-U). Over 3months cTnI-U and hs-cTnI were measured simultaneously at Herlev-Gentofte Hospital. Acute myocardial injury was diagnosed using the 4th universal definition. Disputed cases were adjudicated using clinical data. We compared diagnostic accuracy using area under the curve (AUC) of the receiver operating characteristic. Outliers in between-assay differences were defined as a factor-5 difference and≥1 measurement >40ng/L. Patients with outlier differences were invited for re-sampling and tested with serial dilution and heterophilic blocking tubes. From the 18th January to the 20th April 2019, 4,369 samples on 2,658 patients were included. cTnI-U measured higher concentrations than hs-cTnI (mean 23%,-52-213%), resulting in a higher frequency of acute myocardial injury, 255 (9.6%) vs. 203 (7.6%), p<0.001. This remained significant after adjudication, 212 vs 197, p<0.001. AUC for the prediction of MI for was 0.963 for cTnI-U and 0.959 for hs-cTnI, p=0.001. Outlier differences were seen in 35 (1.2%) patients, primarily with elevated hs-cTnI (n=33, 94%). On two re-samplings (median 144 and 297days since inclusion), 16 of 20 (80%) and 11 of 11 had sustained elevation of hs-cTnI. The samples showed no signs of heterophilic antibodies. Using hs-cTnI resulted in a subset of patients with large, discrepant elevations in concentration. These patients still had elevated hs-cTnI 6-10months post admission but no heterophilic antibodies.

A computational fluid dynamics study of flame gas sampling in horizontal dilution tubes

The performance of horizontal dilution tubes is investigated by Reynolds-averaged Navier–Stokes and large-eddy simulations. The flame gas enters the dilution tube through a pinhole. The orifice flow and the dilution process inside the tube are studied. The volume flow through the orifice is shown to be proportional to the square root of the pressure drop. The discharge coefficient is 0.9 ± 0.3 in the cold air (calibration) case and drops to 0.35 under hot (flame) conditions. The resulting dilution ratio is roughly a factor of five below typical literature data. The gas sample remains in the wall boundary layer and the mixing process is not complete at the end of the dilution tube. Turbulence decays rapidly behind the tube inlet, which shifts the flow into the laminar to turbulent transition regime. Turbulence increases significantly in the outlet section which has much smaller pipe cross-sections. Despite its relatively low Reynolds number, the outlet flow to the particle sizer (or to the gas analyzer) is clearly turbulent, and interactions with the wall are probable. The results are in agreement with previous findings from laminar jets in cross-flow. Guidelines for optimization of the sampling conditions are suggested.

ANTI-MICROBIAL EFFICACY OF AN ETHANOLIC EXTRACT OF MURRAYA KOENIGII AGAINST PERIODONTAL PATHOGENS LIKE PORPHYROMONAS GINGIVALIS AND AGGREGATIBACTER ACTINOMYCETEMCOMITANS: IN VITRO ANALYSIS

Periodontitis is primarily an infectious disease with Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans present in plaque biofilm as the key pathogens, causing destruction of supporting periodontal tissues. Herbal formulations are widely substituting synthetic anti-microbials due to their minimal adverse effects and cost effectiveness. Murraya koenigii (M. koenigii) is a well-known anti-microbial agent used in treating systemic infections. Hence its anti-microbial efficacy against key periodontal pathogens also needs to be tested. The aim of this study is to determine Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), and zone of inhibition of M. koenigii ethanolic extract against P. gingivalis and A. actinomycetemcomitans. Thioglycollate agar was used for culturing both P. gingivalis and A. actinomycetemcomitans. For determination of MIC and MBC broth dilution technique was used. For MIC, serial dilutions of extract were made and culture tubes were incubated in an anaerobic jar and observed for their turbidity. For MBC determination, dilution tubes sensitive to MIC were plated and incubated for the next 24 hours to monitor growth, and colony count was taken. MIC results showed A. actinomycetemcomitans was more sensitive to M. koenigii extract than P. gingivalis and got inhibited at 0.8 μg/ml. Also, MBC results showed extract has strong bactericidal activity towards A. actinomycetemcomitans. Disk diffusion test results showed bactericidal activity against both A. actinomycetemcomitans and P. gingivalis with a larger inhibition zone (15 mm) towards P. gingivalis at a concentration of 75 μl/ml. M. koenigii ethanolic extract is bactericidal against periodontal pathogens like A. actinomycetemcomitans and P. gingivalis and can be used as a safe and effective alternative for synthetic chemotherapeutic agents in the future.

Internal verification of BD BACTEC FX40 instrument for rapid detection of bloodstream infections

Aim: The BD BACTEC FX40 automated instrument was evaluated for use by Ballarat Australian Clinical Laboratory (ACL) to aid in the detection of blood stream infections. Method: 20 organisms were chosen from the reference culture collection to be inoculated into two aerobic, two anaerobic and two paediatric blood culture bottles filled with designated volume of human blood. For each organism, 50 μL of the recommended dilution was aseptically added for each bottle with 5 mL of normal saline in each dilution tubes. There were 12 control bottles injected with sterile water. All bottles were loaded into the instrument. Bottles were removed as they flagged positive. A gram stain and culture were performed on each bottle that flagged positive. Identification by standard laboratory procedures was performed on all isolates. Result: All bottles that were expected to flag positive, flagged positive within the specified time frame listed. The time to detection for each bottle type with the organisms seeded into the blood culture bottles was recorded. Conclusion: All predicted positive blood culture bottles flagged positive except Streptococcus pneumonia in the anaerobic bottle. Controls remained negative. BACTEC FX40 instrument was fit for purpose given that it demonstrated acceptable agreement with inoculated organisms and culture.

Marker Pen Device with Concentration Gradient Nib for Antibiotic Susceptibility Testing.

BackgroundPen-based devices have emerged as useful tools for measuring pH and glucose, and for fabricating microchannels and microarrays. Pen-based devices take advantage of flexible patterning, inexpensive costs, and small volumes, thereby saving time and increasing efficiency. We have developed a gradient nib marker pen device that generated simultaneously different antibiotic concentrations in bacteria antibiotic susceptibility testing (AST).MethodsThe device can deposit on the target surface with the antibiotic gradient. The designed polyester fiber nibs are a highly uniform porosity with unidirectional orientation and produce a visible gradient pattern.ResultsWe have demonstrated and quantitatively analyzed bacterial growth after antibiotic marking. The antibiotic marking produces an inhibition zone of bacterial growth. The inhibition zones of bacterial growth are captured and converted to 8-bit grayscale images, and then quantified by gray values using the Image J program. A profile of the inhibition zone showed different gray values in response to bacterial viability.ConclusionThe gradient nib marker pen device can be used to determine the quantitative antibiotic concentration based on the relationship between gray values and bacterial density conveniently without requiring a series of dilution tubes, including nutrient medium, and diversely diluted antibiotics.

In vitro antimicrobial activity of Spinacia Oleracea against Streptococcus mutans and Lactobacillus acidophilus

Introduction: The use of traditional medicine is well known in India. The high number of bacterial resistance to antibiotics drives much of the current interest in natural antimicrobial molecules. Spinacia oleracea (spinach) possesses medicinal properties. Dental caries is prevalent in about 89% of the Indian population. Streptococcus mutans is the primary causative organism for dental caries and Lactobacillus for further development of caries. Aim: The aim of this study is to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of aqueous and ethanolic extracts of spinach against S. mutans and Lactobacillus. Materials and Methods: About 10 g of freshly harvested spinach was extracted with 95% ethanol and distilled water. The extract was converted into dried powder in desiccator and was dissolved to get 20 mg/ml concentration. The MIC was determined by nine dilutions of each aqueous and ethanolic extracts made with brain–heart infusion. To determine the MBC, the MIC dilution tubes, with no visible growth, and the control tube were subcultured onto the broth media and incubated for 24 h anaerobically and the colonies were counted on the next day. Results: The ethanolic extract exhibited MIC at 12.5 μg/ml against S. mutans and at 25 μg/ml against Lactobacillus. In MBC, the ethanolic extract showed greater bacteriostatic activity against S. mutans compared to Lactobacillus acidophilus. Conclusion: There is preliminary evidence for the antimicrobial activity of spinach extracts against S. mutans and this raises the possibility that spinach may have preventive effect on dental caries.

The Use of Activated Partial Thromboplastin Time As a Surrogate to Predict Residual FVIII Activity in the Bethesda Assay

Introduction: Bethesda assays, used in the quantification of factor VIII inhibitors, are time and labor intensive. They are performed by mixing one part of normal pooled plasma (NPP) with an equal amount of serial dilutions of patient plasma in imadizole buffered saline (IBS), and incubating at 37°C for 2 hours. After the incubation time, most laboratories perform factor VIII (FVIII) activities on each tube. These results are divided by the activity resulted on the control (NPP+IBS) to obtain the percent residual activity. The dilution with a percent residual FVIII activity between 25-75% is then converted to a Bethesda Unit using the Bethesda graph. This value is multiplied by the dilution to calculate final Bethesda units/mL.We propose an alternative method, involving performing partial thromboplastin times (PTTs) on all dilution tubes and only performing FVIII activity testing on a limited number of tubes e.g. 2 tubes rather than all 10 tubes. The study below describes the methodology and provides data to show how the PTT can be used as a surrogate. Using PTTs may reduce cost per test by reducing the amount of FVIII deficient plasma used for FVIII activity testing, especially in the case of very high titer inhibitors.Methods: Serial dilutions of patient plasma with IBS are prepared to the 512 dilution (10 tubes) and incubated with an equal amount of NPP along with a control consisting of IBS and NPP. PTTs are run using Siemens Actin FSL reagent on the BCS coagulometer (Siemens) on all 10 tubes and the control. The tube with a resultant PTT within 5 seconds above the control is identified and then that tube, along with the next higher dilution, are tested for residual factor VIII activity (ACL TOP, Instrumentation Laboratory). These results are used to calculate the percent residual FVIII activity and Bethesda units/mL as described above. To support our hypothesis, we performed PTT and FVIII activities on all dilution tubes.Results: We performed 14 inhibitor assays on 9 patients. 44% (4/9) had congenital hemophilia A with an inhibitor and 56% had acquired hemophilia A. Table 1 shows results from dilution tubes that resulted in residual FVIII activities between 25-75%. The dilutions with residual activity closest to 50% were used in the final interpretation and are bolded. Using the 5 seconds above control PTT to identify dilutions to limit FVIII assays correlated with residual FVIII activities in 12 of the 14 samples tested (86%). Furthermore, this method identified the dilution with the residual FVIII activities closest to 50% in 10 of the 12 samples.Two samples demonstrated no correlation between PTT and FVIII activities. These samples had low titer inhibitors (<5 BU), which could have been predicted by the low PTTs in the first two dilutions. In these cases, serial FVIII activities were used in calculations. More data is needed to definitively say whether this methodology could not be used for low titer inhibitors.Conclusion: An alternative method, using PTT testing to perform targeted FVIII activity testing, may be used in the Bethesda Assay. [Display omitted] DisclosuresSarode:CSL Behring: Consultancy, Honoraria.

Comparison of antiaspergillar activity of extracts of Tectona grandis Linn according to two antifungal susceptibility testing

Plants used as antimicrobials in the treatment of infectious diseases in folk medicine are regularly evaluated in different Laboratories. And, the processes of evaluation of antifungal susceptibility are often different to one laboratory to another. This study was undertaken in the goal to compare parameters obtained (MIC and MFC) with two antifungal susceptibility testing. The first one is Agar slant double dilution tubes method (M1) and the second is disc diffusion method /broth dilution according document CLSI M38 -A2 supplemented to 2% glucose (M2). Ethanol (EtOH 70%), Methanol (MeOH) and n-Hexane (n-HexTG) extracts of leaves of Tectona grandis have been tested in comparison to Itraconazole against two clinicals strains of Aspergillus fumigatus 896/AB and Aspergillus flavus 1006/OM isolated to HIV patients. Results showed that on A. flavus, agar slant method (M1) gave MIC ranging between 200 µg/mL and 800 µg/mL while disc diffusion meth/broth dilution (M2) showed growth inhibition between 100 µg/mL and 400 µg/mL. On A. fumigatus, inhibition was observed between 200µg/mL and 400 µg/mL with M2 in contrary M1 gave MIC located between 400 µg/mL and 800 µg/mL. For each strain tested, according method M2, MIC and MFC are identical and with method M1, these parameters were different. Evaluation of Itraconazole on each strain gave with M2, MIC= 50 µg/mL while inhibition has been showed at 100 µg/mL and 200µg/mL with M1. Also considering extracts, hydroalcoholic extract of T. grandis (EtOHTG) showed antifungal susceptibility testing less 2 to 4 efficiency than Itraconazole. Disc diffusion method/broth dilution (M2) according CLSI M38-A2 to evaluate Tectona grandis antifungal susceptibility is the process of evaluation which allo wed having lo west antifungal parameters (MIC and MFC) on A. flavus and A. fumigatus.

Evaluation of Petrifilm™ aerobic count plates as an equivalent alternative to drop plating on R2A agar plates in a biofilm disinfectant efficacy test.

This paper compares Petrifilm™ aerobic count (AC) plates to drop plating on R2A agar plates as an alternative method for biofilm bacteria enumeration after application of a disinfectant. A Pseudomonas aeruginosa biofilm was grown in a Centers for Disease Control and Prevention biofilm reactor (ASTM E2562) and treated with 123 ppm sodium hypochlorite (as free chlorine) according to the Single Tube Method (ASTM E2871). Aliquots from the same dilution tubes were plated on Petrifilm™ AC plates and drop plated on R2A agar plates. The Petrifilm™ AC and R2A plates were incubated for 48 and 24 h, respectively, at 36 ± 1 °C. After nine experimental runs performed by two technicians, the mean difference in biofilm log densities [log biofilm density (LD) = log10(CFU/cm(2))] between the two methods for control coupons, treated coupons, and log reduction (LR) was 0.052 (p = 0.451), -0.102 (p = 0.303), and 0.152 (p = 0.313). Equivalence testing was used to assess equivalence of the two plating methods. The 90 % confidence intervals for the difference in control and treated mean LDs between methods were (-0.065, 0.170) and (-0.270, 0.064), both of which fall within a (-0.5, +0.5) equivalence criterion. The 90 % confidence interval for the mean LR difference (-0.113, 0.420) also falls within this equivalence criterion. Thus, Petrifilm™ AC plates were shown to be statistically equivalent to drop plating on R2A agar for the determination of control LDs, treated LDs, and LR values in an anti-biofilm efficacy test. These are the first published results that establish equivalency to a traditional plate counting technique for biofilms and for a disinfectant assay.

Optimization of mangroveleaf extraction by mixture design and investigation of its antimicrobial effect on Listeria innocua, Enterococcus faecium and Escherichia coli

در این پژوهش اثر حلال های آب، اتانول، متانول و گلیسیرین هر کدام در پنج سطح (صفر، 25/31، 33/83، 125 و250 میلی لیتر) با استفاده از‌ هندسه مخلوط برای استخراج عصاره برگ گیاه حرا استفاده شد. از مدل چند جمله ای شف و بهینه‌ سازی عددی به منظور مدل سازی و بهینه سازی عصاره استفاده گردید. مدل چند جمله ای شف به طور معنی‌داری قادر به پیش بینی بازده حاصل از استخراج عصاره برگ حرا می باشد (میزان ضریب تبین و ضریب تبین اصلاح شده به ترتیب 94/0 و84/0 وغیر معنی دار بودن آزمون ضعف برازش و مقدار اندیس F ( 62/14) نشان دهنده صحت مدل برای پیش بینی بازدهی عصاره می باشد). به علاوه میزان ضریب تغییرات 6/14% محاسبه شد که نشان دهنده تکرار پذیری قابل قبول برای داده های آزمایشی است. میزان تابع مطلوبیت بهینه سازی 94/0 محاسبه شد که نشانگر صحت عملیات بهینه سازی می باشد. بر این اساس، فرمولاسیون بهینه حاوی گلیسیرین (صفر میلی لیتر)، آب (22/28 میلی لیتر)، متانول ( 83/59 میلی لیتر) و اتانول (95/161 میلی لیتر) بود. اثر ضد میکروبی عصاره های برگ گیاه حرا بر سه باکتری 33090 Listeria innocua ATTC، Enterococcus faecium ATTC 51559 وEscherichia coli ATTC 25992 به سه روش (انتشار در آگار به کمک دیسک، حداقل غلظت ممانعت کننده رشد و حداقل غلظت کشندگی) در هشت غلظت مختلف بررسی شد. نتایج نشان داد بیشترین اثر بازدارندگی بر Listeria innocua و بالاترین میزان مقاومت در برابر عصاره، مربوط به Escherichia coli بود، همچنین با افزایش غلظت عصاره ها قطر هاله باز دارندگی به طور معنی داری (05/0p

Isolation and characterisation of the marine ultramicrobacteriumSphingomonassp. strain RB2256

Indigenous heterotrophic marine bacteria are of great importance to global nutrient cycling. Predominant native bacteria are of ultramicrobacterial dimensions, are not associated with aggregates and must have truly remarkable abilities for substrate capture. Agar media are unsuited for the isolation of the dominant oceanic bacterioplankton. In contrast, the dilution culture technique [Button et al. (1993) Appl. Environ. Microbiol. 59, 881–891; Schut et al. (1993) Appl. Environ. Microbiol. 59, 2150–2160] leads to a successful enrichment of the dominant cell types. Up to 50% of the indigenous bacterial population in water obtained from Resurrection Bay was able to grow in dilution tubes containing only filtered, autoclaved natural sea water (FAS). Ultramicrobacteria (UMB), very small bacteria with small genomes, predominate in such cultures. Generally, dilution factors that resulted in inocula of approximately 2 cells per tube were optimal and prevented outgrowth of atypical large bacteria. Strain RB2256 [Schut et al. (1993) Appl. Environ. Microbiol. 59, 2150–2160], one of the UMB isolated by this dilution culture technique and tentatively identified as a marine Sphingomonas sp., was investigated in more detail. Although reverted from obligately oligotrophic to facultatively oligotrophic upon isolation, this strain possessed a number of traits assigned to a ‘model oligotroph’ and some unpredicted novel properties. The cells showed no miniaturisation upon starvation but consistently exhibited low cell volumes. They had a very low DNA content, were rich in protein, and contained only one copy of the rRNA operon. The cells were well adapted to the simultaneous utilisation of mixed substrates. A constitutive, high-affinity and binding protein-dependent uptake system for mixed amino acids was found that would allow realistic in situ generation times at the prevailing amino acid concentrations. Further studies on this same organism revealed that the cells appeared to be extremely resistant to various stress-inducing agents. High survival rates were observed after high-intensity heat shocks, treatments with H2O2 or with ethanol. Moreover, no marked differences were observed between starved or actively growing cells in this respect, particularly when cells were grown in chemostat. Application of the dilution culture technique to the field of subsurface microbiology could be adopted to study the occurrence of UMB in groundwater with a comparable and stable ‘low-nutrient-conditioned’ phenotype.

Phenol- and toluene-degrading microbial populations from an aquifer in which successful trichloroethene cometabolism occurred

We characterized the bacterial populations that grew in a Moffett Field, Calif., aquifer following three sequential field tests of phenol- or toluene-driven cometabolism of trichloroethene (TCE). Reducing the toluene and phenol concentrations in most-probable-number (MPN) tubes from 50 to 5 ppm increased the population density measured for these degraders by 1.5 and 1 log units, respectively, suggesting that natural populations might be quite sensitive to these substrates. Phenol and toluene degraders were isolated from the terminal MPN dilution tubes; 63 genetically distinct strains were identified among the 273 phenol- and toluene-degrading isolates obtained. TCE was cometabolized by 60% of the genetically distinct strains. Most strains (57%) grew on both phenol and toluene, and 78% of these strains hybridized to the toluene ortho-monooxygenase (TOM) probe. None of the strains hybridized to probes from the four other toluene oxygenase pathways. Gram-positive strains comprised 30% of the collection; all of these grew on phenol, and 47% of them also grew on toluene, but none hybridized to the TOM probe. Among the gram-negative strains, 86% of those that grew on both toluene and phenol hybridized to the TOM probe, while only 5% of those that were TOM-positive grew on toluene alone. A larger proportion of TCE degraders was found among gram-negative than gram-positive strains and among organisms that grew on phenol than those that grew on toluene. Hybridization of strains to the TOM probe was somewhat predictive of their TCE-cometabolizing ability, especially for strains isolated on toluene, but there was also a significant number (20%) of strains that hybridized to the TOM probe but were poor TCE cooxidizers. No Moffett Field isolates were as effective as Burkholderia cepacia G4 in cooxidizing TCE. Most of the aquifer strains ranged from moderately effective to ineffective in TCE cooxidation. Such populations, however, apparently accounted for the successful phenol- and toluene-stimulated TCE removal that occurred during the field assessment of this remediation process. This suggests that naturally occurring communities of only moderate TCE-cooxidizing ability may support successful TCE bioremediation as long as the phenol or toluene present is not limiting. This activity, however, may not be sustainable for the long term, because TCE-inactive populations that consumed toluene at rates equal to that of the best TCE degraders were present and hence would be expected to eventually dominate the community.

Research Note: A Modified Most Probable Number Technique to Enumerate Total Aerobes, Enterobacteriaceae, and Salmonella on Poultry Carcasses After the Whole Carcass Rinse Procedure

A modified most probable number (MPN) technique utilizing a single 10-fold dilution series was tested to enumerate the microbiological population from the whole carcass rinse procedure for poultry. Diluent for the MPN series was a general nutrient broth. Resuscitation of damaged cells and pre-enrichment of low numbers of microorganisms was accomplished by the MPN media. The MPN dilution tubes positive for growth were used to calculate the total aerobic bacterial population. Subculturing with tetrathionate enrichment broth and subsequent plating to differential media were used to identify and enumerate Salmonella. Enterobacteriaceae were enumerated by subculture of the MPN positive tubes in brilliant green (BG) bile 2% broth supplemented with 1% dextrose that were then observed for gas production. The aerobic bacterial counts obtained by the modified MPN were similar, yet significantly higher than those obtained with the conventional plate count techniques (P < .05), and both were significantly higher than the three tube MPN counts.

Adaptation to Temperature of Nitric Oxide-Producing Nitrate-Reducing Bacterial Populations in Soil

The population size of nitrate-reducing bacteria in an Egyptian soil and in two German soils was determined by most probable number (MPN) counts using different incubation temperatures between 8 and 50 °C. The highest numbers were observed at 25–30 °C irrespectively from where the soil originated. Bacteria were randomly isolated from the MPN dilution tubes using the same temperature as in the MPN, and were then morphologically and physiologically characterized. At temperatures >30 °C practically all of the isolates were Gram+ and 75–100% of them formed spores. At all the temperatures and soils tested, most (about 75%) of the isolates were denitrifiers, but some of the isolates also belonged to the nitrate-respiring or nitrate-ammonifying bacteria. Up to 80% of the isolated nitrate reducers formed NO during growth. The following species were identified among 11 randomly isolated Gram- denitrifying bacteria: Pseudomonas corrugata (4 isolates), P. glathei (2 isolates), P. fluorescens subgroup B (2 isolates), P. fluorescens subgroup A (1 isolate), P. aurantiaca (1 isolate), and P. chlororaphis (1 isolate). Three bacterial strains which were isolated at 8, 25 and 40 °C., respectively, also preferred these temperatures for optimum denitrification, N2O formation and NO formation. Our results demonstrate that denitrifying populations that are adapted to a particular temperature (psychrophilic and mesophilic) can be isolated from various soils, although the bulk of the soil nitrate reducers grew best at temperatures of 25–30 °C.

Factors Influencing the Emissions of Nitrated-Polynuclear Aromatic Hydrocarbons(Nitro-PAH) from Diesel Engines

The potential problem of 1-nitropyrene (1-NP) formation during filter sampling of diesel emissions from dilution tubes is assessed. Rates of formation are calculated using data generated from several independent filter exposure studies. A portion of the 1-NP (12% average) found in participates collected from light-duty (LDD) and heavy-duty diesels (HOD) was found to be due to formation on the filter under average sampling conditions of 10-15/1 dilution at 44°C with 3 ppm NO2 for 23 min. On the average, the concentration of 1-NP in participates emitted from HDD is 16 times less than that found in LDD run under similiar transient conditions. The average emission rate of 1-NP generated from the LDD and HDD was 4.7 μg/km and 1.5 μg/km, respectively for the vehicles operating under Federal Test Procedure (FTP)-transient conditions. Under operating conditions which increase exhaust temperature (increased speed and load), the concentration of nitro-PAH is significantly reduced with a concurrent increase in the concentration of partially oxidized nitro-PAH and dinitro-PAH. The concentration of 1-NP in the particulates measured from HDD in these dilution tube studies (0.33-0.95 ppm, depending upon engine load) is comparable to that measured in highway tunnel experiments (0.54 ppm average).

Sampling of Vehicle Emissions for Chemical Analysis and Biological Testing

Representative dilution tube sampling techniques for particulate and gas phase vehicle emissions are described using Teflon filter media and XAD-2 resin. More than 90% of the total gas (C8-C18) and particulate direct acting Ames assay mutagenicity (TA 98) was found in the particulate phase. The gas and particulate phase material was fractionated by HPLC into nonpolar, moderately polar and highly polar chemical fractions. The moderately polar chemical fraction of the particulates contained more than 50% of the direct acting Ames assay mutagenicity for the total extract. The concentration of oxygenated polynuclear aromatic hydrocarbons (oxy-PAH) and nitrated PAH (nitro-PAH) identified in the moderately polar particulate fractions are given. Nitro-PAH account for most of the direct-acting (TA 98) Ames assay mutagenicity in these moderately polar fractions. Reactions and kinetic expressions for chemical conversion of PAH are presented. Chemical conversion of PAH to nitro-PAH during dilution tube sampling of particulates on Teflon filters and gases on XAD-2 resin is a minor problem (representing 10-20%, on the average, of the 1-nitropyrene found in extracts) at short (46 min) sampling times, at low sampling temperatures (42 degrees C), and in diluted exhaust containing 3 ppm NO2. Particulate emissions collected from dilution tubes on filter media appear to be representative of what is emitted in the environment as based upon a comparison of highway and laboratory studies.

Sampling of vehicle emissions for chemical analysis and biological testing.

Representative dilution tube sampling techniques for particulate and gas phase vehicle emissions are described using Teflon filter media and XAD-2 resin. More than 90% of the total gas (C8-C18) and particulate direct acting Ames assay mutagenicity (TA 98) was found in the particulate phase. The gas and particulate phase material was fractionated by HPLC into nonpolar, moderately polar and highly polar chemical fractions. The moderately polar chemical fraction of the particulates contained more than 50% of the direct acting Ames assay mutagenicity for the total extract. The concentration of oxygenated polynuclear aromatic hydrocarbons (oxy-PAH) and nitrated PAH (nitro-PAH) identified in the moderately polar particulate fractions are given. Nitro-PAH account for most of the direct-acting (TA 98) Ames assay mutagenicity in these moderately polar fractions. Reactions and kinetic expressions for chemical conversion of PAH are presented. Chemical conversion of PAH to nitro-PAH during dilution tube sampling of particulates on Teflon filters and gases on XAD-2 resin is a minor problem (representing 10-20%, on the average, of the 1-nitropyrene found in extracts) at short (46 min) sampling times, at low sampling temperatures (42 degrees C), and in diluted exhaust containing 3 ppm NO2. Particulate emissions collected from dilution tubes on filter media appear to be representative of what is emitted in the environment as based upon a comparison of highway and laboratory studies.

Resolution of block neutralization test curves into components of the foot-and-mouth disease virus system.

Classic neutralization studies by Fazekas de St. Groth and Webster (8) on mixtures of influenza viruses and mixtures of rabbit antisera are reinterpreted in terms of a percentage contaminant in the stock used for the dilution series. A very small amount of a different virus changes the shape of quantal assay curves considerably, but even a large amount of a different antiserum has negligible effect on the shape and merely shifts the curve along the serum dilution axis. These conclusions are the reverse of the authors, who only considered an absolute amount of another component in all dilution tubes. An artificial mixture of 2.7 percent O8 in O1 foot-and-mouth disease virus strains was tested against anti-O1 serum assaying in suckling mice. The small amount of O8 virus greatly altered the shape of the neutralization curve in the direction expected from the reanalysis of the influenza literature data. Results from artificial mixtures are used to explain what were hitherto anomalously broad neutralization curves for some other foot-and-mouth disease strains given by Booth et al. (1) Many of the virus stocks studied can now be postulated as a natural mixture of related virus strains. In fact, the O1 and O8 stocks used might also be of themselves mixtures. These virus strains also exhibit still a further test complication in that the virus-antibody reaction appeared to shift away from complexes on dilution immediately prior to assay.

A simplified method for estimating ammonium oxidising bacteria

Most probable number of ammonium oxidising bacteria in soil has been estimated without the need to carry out chemical tests to determine the production of nitrite or nitrate in dilution tubes. The medium used contained phenol red which changes colour from pink to yellow as the oxidation of ammonium to nitrate decreases the pH. This makes it possible to determine growth visually. An incubation period of 8 weeks at 25°C was sufficient to obtain maximum estimates of ammonium oxidising bacteria in the soils tested.