Effect of extraction and assay media on analysis of airborne endotoxin

The measurement of airborne endotoxins is thus far not standardized. Earlier studies reported higher endotoxin yields when Tween 20 was added to the media used for filter extraction and in the Limulus amebocyte lysate (LAL) assay. This study compared four common media and assessed the effects of Tween during extraction and analysis separately. Parallel airborne dust samples from five work environments (n = 250) were used to compare the four media (pyrogen-free water [PFW], PFW-Tween 20, PFW-Tris, and PFW-triethylamine-phosphate [TAP]) and an extraction time of 10 or 60 min. A subset of the extracts in PFW or PFW-Tween (n = 40) were analyzed in parallel LAL assays with PFW or PFW-Tween as the assay medium. The results produced by a shorter extraction time or the presence of Tris were similar to the results for the reference procedure (PFW and 60 min of shaking). The use of PFW-TAP showed overall lower yields and a deviant calibration curve. The presence of Tween in the extraction medium resulted in significantly (P < 0.05) higher endotoxin yields from all dust types, independent of the effect of Tween in the assay. Tween in the LAL assay, however, also strongly inhibited the reactivity of the lipopolysaccharide (LPS) standard, thus shifting the calibration curve to higher values. The inhibition of LPS in test samples was less pronounced and varied between dust sources, resulting in enhanced calculated concentrations. This assay effect could be circumvented by diluting extracts at least 50-fold before the LAL assay. In conclusion, of the media tested, only Tween enhances the efficiency of endotoxin extraction from airborne dust samples in a consistent manner. We recommend extraction in PFW-Tween combined with dilution and LAL analysis in PFW.     

The effect of extraction,storage, and analysis techniques on the measurement of airborne endotoxin from a large dairy

The objective of this study was to fill in additional knowledge gaps with respect to the extraction, storage, and analysis of airborne endotoxin, with a specific focus on samples from a dairy production facility. We utilized polycarbonate filters to collect total airborne endotoxins, sonication as the extraction technique, and 0.05% Tween 20 in pyrogen-free water (PFW) as the extraction solution. Endotoxin concentrations were determined via the Limulus amebocyte lysate (LAL) assay. The endotoxin concentrations in extracts after 15 and 30 min of filter sonication were similar, while the concentration in 60 min extracts was about twofold lower. Rapidly vortexing samples for up to 15 min after sonication did not increase the endotoxin concentration. However, concentrations were 13 and 26% lower in extracts that were centrifuged at 1,000 and 10,000g for up to 15 min, respectively. Field samples and endotoxin standard were also sonicated in glass or polypropylene tubes for up to 120 min. Regardless of the extraction vessel, a decrease in endotoxin concentration occurred when sonicated for >30 min. Samples and endotoxin standard subjected to 12 freeze–thaw cycles at −20°C only showed a slight but not significant decrease in endotoxin concentration. Our results also demonstrate the importance of simultaneously adding LAL reagent to 96-well plates before initiating the LAL assay.     

Evaluation of the efficiency of extraction of ultraviolet-absorbing pollen allergens and organic pollutants from airborne dust samples by capillary electromigration methods

Capillary electromigration methods, zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC), have been used for evaluation of the efficiency of different extraction agents applied to the extraction of pollen allergens and organic pollutants from dust samples collected during different periods (before, during and after pollen seasons) and in different locations in air-filtration devices (car-traffic tunnel in Prague and a metro station in Paris). Water and acetic acid extracts were analyzed by CZE using acetic acid as background electrolyte (BGE). Water and alkaline water-SDS-buffer extracts were analyzed by MEKC in Tris-phosphate BGE with anionic detergent sodium dodecylsulfate (SDS) micellar pseudophase. More material was extracted and more components were found in the water-buffer extracts than in the water extracts, and better resolution of the components was achieved by MEKC than by CZE. Significant differences have been found in the analyses of dust extracts of different origin. More material and more components have been found in the extracts of the dust collected in the pollen-rich period (March, April) than in the pollen-free period (December, January).     

Mutagenicity studies of ambient airborne particles. II. Comparison of extraction methods

Organic materials were extracted with acetone from airborne particles by shaking, soxhletion and sonication for varying durations. 4-h, 1-h and 1/8-min extractions by shaking, soxhletion and sonication, respectively gave maximum his+ revertants with the Ames Salmonella/microsome assay. In a comparative study of extraction methods, sonication gave the highest and soxhletion the lowest mutagenic response. It appears that sonication with acetone is the best procedure for the extraction of mutagens from airborne particles as shown by Ames assay and Arar assay systems in Salmonella typhimurium.     

Monitoring airborne fungal spores in an experimental indoor environment to evaluate sampling methods and the effects of human activity on air sampling.

Aerobiological monitoring was conducted in an experimental room to aid in the development of standardized sampling protocols for airborne microorganisms in the indoor environment. The objectives of this research were to evaluate the relative efficiencies of selected sampling methods for the retrieval of airborne fungal spores and to determine the effect of human activity on air sampling. Dry aerosols containing known concentrations of Penicillium chrysogenum spores were generated, and air samples were taken by using Andersen six-stage, Surface Air System, Burkard, and depositional samplers. The Andersen and Burkard samplers retrieved the highest numbers of spores compared with the measurement standard, an aerodynamic particle sizer located inside the room. Data from paired samplers demonstrated that the Andersen sampler had the highest levels of sensitivity and repeatability. With a carpet as the source of P. chrysogenum spores, the effects of human activity (walking or vacuuming near the sampling site) on air sampling were also examined. Air samples were taken under undisturbed conditions and after human activity in the room. Human activity resulted in retrieval of significantly higher concentrations of airborne spores. Surface sampling of the carpet revealed moderate to heavy contamination despite relatively low airborne counts. Therefore, in certain situations, air sampling without concomitant surface sampling may not adequately reflect the level of microbial contamination in indoor environments.     

Exposure of agricultural workers to airborne microorganisms and endotoxin during handling of various vegetable products

Airborne concentrations of viablemicroorganisms and bacterial endotoxin were determinedduring 10 intramural agricultural activities includinghandling or processing of 6 kinds of vegetableproducts: grain, hay, horticulture seeds, herbs, flaxand potatoes. The median values of the concentrationof total viable microorganisms (bacteria + fungi)were within a range from 43.2 × 10³ CFU/m³at potato processing to 1240.0 × 10³ CFU/m³in small granaries, exceeding in 9 out of 10 cases thelevel of 10⁵ CFU/m³ suggested as aoccupational exposure limit (OEL). Many of theisolated bacteria and fungi were reported asallergenic and/or immunotoxic agents. The medianvalues of the endotoxin concentration ranged from0.0125 g/m³ at handling hay to54.9 g/m³ at crushing grain, exceeding in 5out of 7 cases the suggested OEL of0.1 g/m³. The high levels of exposure toairborne microorganisms and endotoxin found by thisstudy indicate a potential risk of occupationalrespiratory disorders among agricultural workers, mostly in those handling grain.     

Influence of different cell extraction methods on cytometric features.

The purpose of this study was to investigate the influence of different cell extraction procedures on relative nuclear DNA content (IOD), nuclear area, and texture features of Feulgen-stained nuclei. In imprints and smears of fine-needle aspirates and suspensions from one human liver specimen, 50 diploid, 50 tetraploid, and 25 octaploid nuclei were measured from each slide. In addition, for DNA measurements, the progressive mean of IOD and tetraploid/diploid and octaploid/diploid ratios was calculated. The results show that the progressive mean of the IOD is constant after measuring 25-30 nuclei. For the three types of specimens, the IOD of diploid nuclei varied slightly. The average coefficient of variation was about 5% for the fine-needle aspirates, imprints, and suspensions. For all tissue sampling methods, the 99% confidence limits of the tetraploid/diploid ratio and octaploid/diploid ratio were within the range of 1.9-2.1 and 3.7-4.3, respectively. The nuclear area and most of the texture features showed a significant difference (p less than 0.01) between the three sampling methods in all nuclear populations. In conclusion, different tissue sampling methods have little or no effect on DNA-related IOD measurements, whereas the outcome of nuclear area and texture features is very dependent of the cell extraction procedure.     

Influence of various factors on the measurement of multifrequency bioimpedance

Body impedance values at various frequencies (from 1 to 100 kHz) were determined in 104 subjects on seven separate days over a two-week period. The variability of body impedance in different measurement conditions was studied. In particular, the effects of the electrode locations, the ingestion of some substances (sugar, alcohol, mineral salts), body spatial geometry, the time spent in the supine position and the menstrual cycle were assessed. Under standardized conditions (in the morning, in the fasting state, with an empty bladder and with the body in a standardised spatial position), the within-subject day-to-day variability was 3-14 Ohms. Under different experimental conditions, the within-subject variability was generally much higher. This was particularly evident for female subjects. We observed significant mean variations in relation to the different experimental factors introduced one at a time, with the exception of the menstrual cycle. For example, half an hour after the intake of various substances, body impedance had generally increased by 6-17 Ohms in comparison with values in the fasting state. Changes in body impedance during the menstrual cycle, however, were small and never statistically significant. The impedance variations obviously caused significant changes in estimated parameters of body composition. It is concluded that controlled conditions and standardization of multifrequency bioimpedance analysis (MBIA) methods are indispensable for the application of this technique.     

Monitoring airborne fungal spores in an experimental indoor environment to evaluate sampling methods and the effects of human activity on air sampling

[This corrects the article on p. 226 in vol. 59.].     

A comparative study on the extraction of membrane-bound bilirubin from erythrocyte membranes using various methods.

In this study, we used three different methods for the extraction of membrane-bound bilirubin (EMB) from erythrocyte membranes. Use of 2.5% albumin, pH 7.4, for elution of EMB resulted in only 34% of the total EMB which was estimated after the solubilization of bilirubin-loaded erythrocyte membranes (BLEMs) with 1% SDS. On the other hand, incubation of BLEMs with 38 mM sodium carbonate solution containing 5 mM EDTA, pH 11.0, yielded 77% of the total EMB. Application of Fog's reaction for the estimation of EMB directly on the BLEMs resulted in the estimation of 75% of the total EMB. These results suggest that either of the above methods, i.e. use of albumin or high pH, or direct Fog's reaction cannot estimate the total EMB correctly. Increase in ionic strength from 0.15 to 0.45 did not release any EMB from erythrocyte membranes. Therefore, the best method for the estimation of total EMB is the solubilization of membrane with 1% SDS followed by Fog's reaction method.     

A comparative study on the airborne fungi in Athens,Greece, by viable and non-viable sampling methods

Airborne fungi were studied in the city of Athens using two complementary methods in which 136 concurrent samplings were carried out during the 12-month period from January until December 1998. A portable Burkard air sampler for agar plates was used for trapping the culturable portion of the mycobiota. Nineteen genera of fungi were identified and assessed in terms of total numbers and fluctuations in concentration (Alternaria, Arthrinium, Aspergillus, Aureobasidium, Botrytis, Chrysonilia, Cladosporium, Drechslera, Epicoccum, Fusarium, Mucor, Nigrospora, Paecilomyces, Penicillium, Rhizopus, Sclerotinia, Scopulariopsis, Trichoderma and Ulocladium), with the exception of those included in the Sphaeropsidales, the yeasts, and the non-sporulating fungi, which were counted as groups. A volumetric Burkard air sampler for glass slides was operating simultaneously for detecting the total mycobiota, including the non-culturable and the non-viable portion. Ascospores, basidiospores, spores of Myxomycetes, Ustilaginales, Uredinales and Erysiphales, teliospores of Puccinia, as well as conidia of the genera Curvularia, Helminthosporium, Periconia, Pestalotiopsis, Pithomyces, Polythrincium, Stachybotrys, Stemphylium and Torula were also recorded. Only seven of the genera were recovered by both samplers. The total numbers of fungal spores, which had a maximum concentration of 3,175 spores/m³, as well as the spore concentrations of the genera Cladosporium (2,565 spores/m³) and Alternaria (280 spores/m³) were underestimated by the viable method (2,435 CFU/m³ for the total, 2,169 CFU/m³ for Cladosporium and 180 CFU/m³ for Alternaria). The non-viable method fails to resolve the identification of the genera Penicillium and Aspergillus, which are major components of the airborne mycobiota (1,068 CFU/m³ and 204 CFU/m³, respectively) based on recovery by the viable method.     

Nuclear volume estimation using different sampling, measurement and calculation methods

OBJECTIVE: To study the reliability of volume parameter measured on tissue sections through different sampling, measurement and calculation methods. STUDY DESIGN: The largest nuclear profile image under a 100x, NA 1.30 oil immersion objective of primary spermatocytes and spherical spermatoblasts on 11-micron-thick seminiferous tubule sections and tissue images, under a 20x objective, on 4-micron sections were captured. Their volumes were measured and calculated by the five methods provided by the Technology for Image and Graphics Engineering Research cell image analysis system. RESULTS: The nuclear volumes obtained by nucleator and area equivalent diameter on the largest nuclear profile image were almost the same, including binary images by automated and manual interactive nucleator and grey scale images only by the latter. Nuclear volumes, calculated by random Feret diameter and equivalent diameter of the perimeter, the minimal circumference of the largest nuclear profile binary image, were obviously larger than those of the nucleator and area equivalent diameter. Due to different-sized nuclear slices entrapped in the same section, those nuclear volumes from the seminiferous tubule tissue images were strikingly lower than that of the largest nuclei profile image. The shape factors of primary spermatocytes and spherical spermatoblast nuclei under 100x and 20x objectives were approximately the same. CONCLUSION: The sample preparation, sampling methods and calculation formulas suitable to nuclear form are necessary to obtain reproducible volume parameters.     

Influence of perinatal factors and sampling methods on TSH and thyroid hormone levels in cord blood.

To evaluate the effect of perinatal factors and sampling methods on thyroid stimulating hormone (TSH) and thyroid hormone levels in cord blood, serum TSH, free thyroxine (FT4) and free triiodothyronine (FT3) concentrations were measured in 124 healthy term neonates. Eighty-eight infants were born in normal vaginal deliveries, 25 were delivered by vacuum extractor and 11 by Cesarean section. There was no significant difference among the three infant groups in the mean TSH levels. Birth weight, the infant's sex, duration of labor and uterotonic agents had no effect on cord serum TSH and free thyroid hormone levels in the neonates born by normal vaginal delivery. To assess the adequacy of specimen collection, mixed cord blood samples, obtained by a direct application of cord on a filter paper, and venous blood withdrawn with a plastic syringe were collected in another 200 infants. There was a significant linear correlation in the TSH concentration in mixed cord blood and cord venous serum from the same individuals, while a poor correlation was found in T4 values from two specimens. Our results suggest that the TSH value in cord blood is less influenced by perinatal factors, including the sampling method, and the mixed cord blood collected by this technique might be a feasible alternative specimen for a TSH screening program with cord blood which is useful in countries where neonatal blood is not available.     

Airborne environmental endotoxin: a cross-validation of sampling and analysis techniques.

A standard method for measurement of airborne environmental endotoxin was developed and field tested in a fiberglass insulation-manufacturing facility. This method involved sampling with a capillary-pore membrane filter, extraction in buffer using a sonication bath, and analysis by the kinetic-Limulus assay with resistant-parallel-line estimation (KLARE). Cross-validation of the extraction and assay method was performed by comparison with methanolysis of samples followed by 3-hydroxy fatty acid (3-OHFA) analysis by gas chromatography-mass spectrometry. Direct methanolysis of filter samples and methanolysis of buffer extracts of the filters yielded similar 3-OHFA content (P = 0.72); the average difference was 2.1%. Analysis of buffer extracts for endotoxin content by the KLARE method and by gas chromatography-mass spectrometry for 3-OHFA content produced similar results (P = 0.23); the average difference was 0.88%. The source of endotoxin was gram-negative bacteria growing in recycled washwater used to clean the insulation-manufacturing equipment. The endotoxin and bacteria become airborne during spray cleaning operations. The types of 3-OHFAs in bacteria cultured from the washwater, present in the washwater and in the air, were similar. Virtually all of the bacteria cultured from air and water were gram negative composed mostly of two species, Deleya aesta and Acinetobacter johnsonii. Airborne countable bacteria correlated well with endotoxin (r2 = 0.64). Replicate sampling showed that results with the standard sampling, extraction, and Limulus assay by the KLARE method were highly reproducible (95% confidence interval for endotoxin measurement +/- 0.28 log10). These results demonstrate the accuracy, precision, and sensitivity of the standard procedure proposed for airborne environmental endotoxin.     

Influence of endotoxin on experimental post-alcoholic liver injury.

The morphological and biochemical changes of the liver after endotoxin intake were analyzed in rats receiving 20% ethanol during 60 days. Besides morphological changes, concentration of serotonin and histamine in liver homogenates, the activity of asparagine and alanine aminotransferases (AspAT, ALAT), gamma-glutamyltranspeptidase (GGTP) and alcohol dehydrogenase (ADH) in blood serum were determined, too. The most extensive morphologic changes of the liver were seen in group of animals intoxicated with 20% ethanol during 60 days and single dose of endotoxin E. coli 0127:B8 intraperitoneally. These changes included necrosis most hepatocytes, focal steatosis of liver parenchyma, considerable hyperemia and parenchymatous degeneration of the liver cells. The cells lining liver sinuses showed considerable swelling as well as necrotic changes. Figures of cell division and haemorrhagic focuses were seen, too. The clusters of mononuclear cells, surrounding necrotically changed hepatocytes were seen in the central part of the liver lobule. Among the inflammatory mediators estimated in liver homogenate only serotonin reached a high level in the group of experimental animals receiving only endotoxin. Increased activity of aminotransferases AspAt and ALAT were associated with these morphologic and biochemical changes in liver tissue observed in animals receiving ethanol and endotoxin.     

Immunochemical characteristics and serologic properties of lipopolysaccharides isolated from various Brucella species using various extraction methods

The results of the comparative analysis of LPS isolated by different methods of extraction from the cultures of several Brucella species differing in their virulence are presented. Purified LPS preparations have been obtained from Brucella virulent and vaccine strains by using such methods as water-phenol extraction, Boivin's method, mild alkaline hydrolysis of the antigen according to Boivin's procedure. The presence of certain relationship between the method used for the extraction of Brucella LPS to be compared and their chemical composition, immunological characteristics and serological activity has been established. As shown in this investigation, in the process of the preparation of a highly sensitive diagnosticum for the passive hemagglutination test the use of LPS obtained from Brucella virulent strains, but not from the vaccine strain, by the method of mild alkaline hydrolysis according to Boivin's procedure is expedient. The data presented in this work indicate that the soluble complex of lipid A obtained from Brucella LPS has been found to possess serological activity. The results of the study of the serological properties of lipid A indicate that the lipid component may also play a certain role in the manifestation of the serological activity of Brucella LPS.     

不同DNA抽提方法对普通PCR和实时荧光定量PCR方法检测家蚕微孢子虫的影响

为了优选快速、 灵敏、 特异的家蚕微孢子虫Nosema bombycis分子检测方法和DNA抽提方法, 本文通过对家蚕微孢子虫TaqMan探针荧光定量PCR检测方法和SYBR Green荧光定量PCR检测方法的建立以及反应体系优化, 并与普通PCR方法进行比较; 再采用4种不同DNA抽提方法分别对PCR和实时荧光定量PCR方法检测家蚕微孢子虫悬浮液的效果评价。结果显示: 不经过DNA抽提, 直接将家蚕微孢子虫发芽液进行PCR反应的效果优于其他方法, 检测灵敏度由高到低依次为直接法、 酚/氯仿抽提法、 动物组织DNA试剂盒抽提法和植物组织DNA试剂盒抽提法; TaqMan探针法检测家蚕微孢子虫发芽液的灵敏度和SYBR Green法相近, 达到微孢子10²个/mL, 两者均优于普通PCR方法。实验表明, 直接采用发芽液结合荧光定量PCR方法检测家蚕微孢子虫最为简便、 快速、 灵敏。该研究结果将有助于提高家蚕微粒子病监控技术和检疫能力, 对家蚕微粒子病的检疫和防治具有积极意义。     

Bacterial endotoxin testing: a report on the methods, background, data, and regulatory history of extraction recovery efficiency

Since the mid-1970s the Limulus Amebocyte Lysate (LAL) assay has been used to test medical devices for bacterial endotoxins. The Association for the Advancement of Medical Instrumentation (AAMI) recently published a standard designated ANSI/AAMI ST 72: 2002, Bacterial Endotoxins--Test methodologies, routine monitoring, and alternatives to batch testing, which addresses LAL testing and associated issues. In order to perform the bacterial endotoxins test (BET), the test article must be extracted in an aqueous medium, with the extract being used as the test solution. In the early years of testing, and periodically throughout LAL test history, questions have arisen about validation of the extraction efficiency of endotoxins from medical devices. The AAMI Microbiological Methods Committee appointed a Task Group to thoroughly research the issue of extraction efficiency and to recommend whether validation of extraction efficiency is necessary for LAL testing of medical devices.