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.     

Influence of various dust sampling and extraction methods on the measurement of airborne endotoxin.

The influence of various filter types and extraction conditions on the quantitation of airborne endotoxin with the Limulus amebocyte lysate test was studied by using airborne dusts sampled in a potato processing plant. Samples were collected with an apparatus designed to provide parallel samples. Data from the parallel-sampling experiment were statistically evaluated by using analysis of variance. In addition, the influence of storage conditions on the detectable endotoxin concentration was investigated by using commercially available lipopolysaccharides (LPS) and endotoxin-containing house dust extracts. The endotoxin extraction efficiency of 0.05% Tween 20 in pyrogen-free water was seven times higher than that of pyrogen-free water only. Two-times-greater amounts of endotoxin were extracted from glass fiber, Teflon, and polycarbonate filters than from cellulose ester filters. The temperature and shaking intensity during extraction were not related to the extraction efficiency. Repeated freeze (-20 degrees C)-and-thaw cycles with commercial LPS reconstituted in pyrogen-free water had a dramatic effect on the detectable endotoxin level. A 25% loss in endotoxin activity per freeze-thaw cycle was observed. Storage of LPS samples for a period of 1 year at 7 degrees C had no effect on the endotoxin level. House dust extracts showed a decrease of about 20% in the endotoxin level after they had been frozen and thawed for a second time. The use of different container materials (borosilicate glass, "soft" glass, and polypropylene) did not result in different endotoxin levels. This study indicates that the assessment of endotoxin exposure may differ considerably between groups when different sampling, extraction, and storage procedures are employed.     

Year-long assessment of airborne endotoxin at a concentrated dairy operation

In this study, we monitored total airborne endotoxins at upwind and downwind sites at a large open-lot dairy each month for a year. At the upwind site, the average airborne concentration was 28.5 endotoxin units (EU) m⁻³, while at the downwind edge of the lot and 200 m from the lot edge, the average concentrations were 169 and 72 EU m⁻³, respectively. At the downwind edge of the lot, there was a significant correlation between the airborne endotoxin concentration and wind speed or air temperature. A comparison between total and inhalable airborne endotoxin concentrations, near the end of the study, revealed no significant differences between the two endotoxin collection methods. Our data suggest that endotoxin exposure can be reduced as one increases their distance from the open-lot dairy.     

Optimization of airborne endotoxin exposure assessment: effects of filter type, transport conditions, extraction solutions, and storage of samples and extracts

Endotoxin exposure occurs in homes and occupational environments and is known to cause adverse health effects. In order to compare results from different studies and establish standards, airborne endotoxin exposures should be assessed using standardized methods. Although the European Committee for Standardization (CEN) developed guidelines for endotoxin exposure assessment, these leave room for individual interpretation. The influence of methods of sampling, extraction, and analysis has never been investigated in a full experimental design. Thus, we sought to fully elucidate the importance of all facets of endotoxin assessment. Inhalable dust samples collected simultaneously were used to investigate the effects on and interactions with airborne endotoxin concentration in two working environments of filter type (glass fiber or Teflon), transport conditions (with/without desiccant), sample storage (-20 or 4 degrees C), extraction solution (pyrogen-free water [PFW] or PFW plus 0.05% Tween 20), extract storage (-20 or 4 degrees C), and assay solution (PFW or PFW plus 0.05% Tween 20). Four hundred samples were collected and randomly distributed over the 20 combinations of treatments. There were no differences found for transport conditions and storage temperature of extracts. Also, no interactions between study variables existed. Sampling on glass-fiber filters, storage of samples in the freezer, and extraction in PFW plus 0.05% Tween 20 resulted in 1.3-, 1.1-, and 2.1-fold-higher estimated endotoxin concentrations, respectively. Use of PFW plus 0.05% Tween 20 in the assay solution had an additive effect. Thus, this study investigated gaps in the CEN protocol and provides data with which to fully specify a protocol for standardization of endotoxin exposure assessment.     

The effect of the intramammary infusion of Escherichia coli endotoxin on ovulation in lactating dairy cows

The purpose of this experiment was to determine if intramammary inflammation during the periovulatory period affects the occurrence of ovulation in lactating dairy cows. Ten lactating, cyclic, Holstein dairy cows received 2 injections of prostaglandin F2alpha at eleven-day intervals, to synchronize luteolysis. The day of the second injection was designated as day 0. Ovulation was anticipated to occur 3-5 days later (on days 3-5). Beginning at the morning milking on day 1, cows received intramammary infusions of either Escherichia coli endotoxin (10 microg; n=5) or infusion vehicle (pyrogen free Hank's balanced salt solution; n=5) into 2 quarters immediately after milking. The same quarters were infused after each milking through day 4. Venous blood samples were collected daily from day -1 through 13 for determination of progesterone to monitor luteolysis and formation of a new corpus luteum. Blood samples were also collected at 4-h intervals (days -1 to 2), then at 2-h intervals (days 2 to 5) to measure concentrations of luteinizing hormone. Ovaries were examined ultrasonographically on days -1 through 5 and on day 12 to monitor follicular growth and formation of the corpus luteum. Collectively, these observations were used to determine if and when ovulation occurred. Intramammary infusion of E. coli endotoxin induced an immediate increase in the concentration of somatic cells in milk from treated quarters. However, this treatment had no effect on the occurrence or timing of ovulation. Based on ultrasonography and concentrations of progesterone, four of five cows in each treatment group appeared to have ovulated. Preovulatory surges of LH were detected within the intensive bleeding periods for three cows in each treatment group. The magnitude of the LH surge was reduced in cows receiving endotoxin.     

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.     

The effect of storage on whole blood chemiluminescence measurement of equine neutrophils

The aim of this study was to determine the effect of duration and temperature of sample storage on whole blood chemiluminescence measurement results. Venous blood from 18 clinically healthy Polish half‐bred horses aged 4 to 11 years were used in the study. Luminol dependent chemiluminescence (CL) was used to measure neutrophil oxygen metabolism in whole blood. Blood samples were examined for spontaneous CL and stimulated by a surface receptor stimulus as well as extra‐receptor stimulus. The assay was performed in two parallel experimental sets with samples stored at 4 and 22 °C, respectively. Whole blood CL was estimated at 2, 6, 24, 48, 72, 96 and 120 h after collection. The study demonstrated that temperature and duration of sample storage are factors that determine the quality of CL measurements of whole blood in horses. The study concluded that samples should be stored at 4 °C and the assay should be performed as early as possible. It was also shown that the viability period of horse blood for CL assays is relatively long. Material stored at room temperature for 24 h and even up to 48 h at 4 °C did not show any significant decrease in spontaneous or stimulated chemiluminescence. Copyright © 2012 John Wiley & Sons, Ltd.     

The effect of endotoxin and endotoxin tolerance on inflammation induced by mycobacterial adjuvant

Peptidoglycan, the substance in mycobacteria thought to be responsible for inducing adjuvant arthritis, and endotoxin (lipopolysaccharide or LPS) share many inflammatory properties. Since repeated administration of LPS produces tolerance, i.e., resistance to the toxic and inflammatory effects of LPS, we tested whether LPS and/or LPS tolerance might influence inflammation due to mycobacterial adjuvant. Male Sprague-Dawley rats were injected with Escherichia coli LPS or saline intraperitoneally and then challenged with 100 micrograms killed Mycobacteria butyricum (adjuvant) in the footpad. A single dose of 100 micrograms LPS three or 24 hours before adjuvant markedly, but transiently, reduced the local footpad swelling that begins within hours of the adjuvant injection and histologically resembles a sterile abscess. Animals that received multiple doses of LPS and were therefore tolerant or animals that received LPS 72 hours before adjuvant demonstrated adjuvant-induced footpad swelling nearly equal to controls. The anti-inflammatory effect of LPS was transient since footpad swelling in all groups was nearly comparable six days after the adjuvant injection and LPS failed to inhibit consistently the arthritis that develops two or more weeks after adjuvant injection. These studies establish that LPS can markedly inhibit the prodrome of adjuvant arthritis (footpad swelling due to M. butyricum), that inhibition of this prodrome does not prevent the subsequent development of arthritis, and that LPS tolerance diminishes this anti-inflammatory effect of LPS.     

The effect of storage on the biomechanical behavior of articular cartilage--a large strain study.

The transplantation of stored shell osteochondral allografts is a potentially useful alternative to total joint replacements for the treatment of joint ailments. The maintenance of normal cartilage properties of the osteochondral allografts during storage is important for the allograft to function properly and survive in the host joint. Since articular cartilage is normally under large physiological stresses, this study was conducted to investigate the biomechanical behavior under large strain conditions of cartilage tissue stored for various time periods (i.e., 3, 7, 28, and 60 days) in tissue culture media. A biphasic large strain theory developed for soft hydrated connective tissues was used to describe and determine the biomechanical properties of the stored cartilage. It was found that articular cartilage stored for up to 60 days maintained the ability to sustain large compressive strains of up to 40 percent or more, like normal articular cartilage. Moreover, the equilibrium stress-strain behavior and compressive modulus of the stored articular cartilage were unchanged after up to 60 days of storage.     

Isolation, extraction, and measurement of acetylcholine from Lactobacillus plantarum.

The isolation, extraction, and spectrophotometric determination of acetylcholine from Lactobacillus plantarum ATCC 10241 is described. Acetylcholine was extracted with a mixture of sodium tetraphenylboron-butylethylketone-acetonitrile and was measured enzymatically at 340 nm.     

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.     

The effect of endotoxin on murine stem cells

Previous studies showed that after 5 μg of Salmonella typhosa endotoxin there was an increase in colony stimulating factor temporally related to a fall in murine marrow in vitro colony forming cells (CFC). This was followed by differentiation along the marrow granulocytic pathway. The present studies showed that after 5 μg of endotoxin the peripheral blood CFC fell by approximately 50% at one hour, rose to a level ten fold that of control at six hours and then returned to control values by 48 hours. There was a progressive increase in the number of splenic CFC to ten fold that of control from 24 to 72 hours after endotoxin. These data imply a migration of CFC from the marrow to the spleen along with an in-situ increase in splenic CFC. Thus, either migration or differentiation may explain the fall in marrow CFC after endotoxin. Spleen colony forming units (CFU) in the marrow were measured by a transplantation technique and the transplantation fraction (f Fx) determined. A decrease in marrow CFU at 24 hours after endotoxin was secondary to a change in the f Fx. from 11.1% to 7.6%. There was however, an increased percentage of CFU in DNA synthesis in the interval of 6–48 hours after endotoxin, as judged by the hydroxyurea technique. As the marrow CFC fell within 20 minutes of endotoxin administration, the data suggest the CFC may be affected initially and that changes in the generative cycle of the CFU may be of a secondary nature.     

The effects of saliva collection,handling and storage on salivary testosterone measurement

Several endocrine parameters commonly measured in plasma, such as steroid hormones, can be measured in the oral fluid. However, there are several technical aspects of saliva sampling and processing that can potentially bias the validity of salivary testosterone measurement. The aim of this study was to evaluate the effects caused by repeated sampling; 5 min centrifugation (at 2000, 6000 or 10,000g); the stimulation of saliva flow by a cotton swab soaked in 2% citric acid touching the tongue; different storage times and conditions as well as the impact of blood contamination on salivary testosterone concentration measured using a commercially available ELISA kit. Fresh, unprocessed, unstimulated saliva samples served as a control. Salivary testosterone concentrations were influenced neither by repeated sampling nor by stimulation of salivary flow. Testosterone levels determined in samples stored in various laboratory conditions for time periods up to 1 month did not differ in comparison with controls. For both genders, salivary testosterone levels were substantially reduced after centrifugation (men F = 29.1; women F = 56.17, p < 0.0001). Blood contamination decreased salivary testosterone levels in a dose-dependent manner (men F = 6.54, p < 0.01, F = 5.01, p < 0.05). Salivary testosterone can be considered A robust and stable marker. However, saliva processing and blood leakage can introduce bias into measurements of salivary testoterone using ELISA. Our observations should be considered in studies focusing on salivary testosterone.     

An optimized DNA extraction and purification method from dairy manure compost for genetic diversity analysis

An unbiased DNA extraction protocol is necessary for analysis of genetic diversity, particularly, of genes in complex environmental samples by nucleic acid techniques. In the present study, three manual extraction methods and two commonly used commercial kits, which were accompanied by two DNA purification strategies, were compared based on cell lysis efficiency, DNA and humic acid yields, PCR amplification and denaturing gradient gel electrophoresis (DGGE) analysis. The results show that in spite of higher cell lysis efficiencies of the two commercial kits, the purified DNA yields were only one-third of that obtained by the two manual methods of FTSP (Freeze–thaw–SDS–Protein K) and FTSPP (Freeze–thaw–SDS–Protein K-Polyvinylpolypyrrolidone). The purified DNA from all five methods was pure enough for successful PCR and real-time PCR amplifications in the presence of 1 μg μL^?1 BSA. However, the FTSPP extraction method with DNA purification by a Wizard^® kit yielded the largest number of 16S rRNA gene copies and ribotypes or bands in DGGE profiles, which indicated a superiority over the other four methods. The development of this optimized DNA extraction and purification method may provide a valuable tool for further molecular analysis of compost.     

The effect of a surfactant in media for the enumeration, growth and identification of airborne fungi

Air sampling on to culture media for viable fungi is complicated by the presence of fast-growing species. A non-ionic surfactant (Triton N-101) incorporated into the medium effectively reduced the spread of such species and thus facilitated the enumeration and identification of fungal colonies collected by an Andersen sampler. The concentration of surfactant from 250 to 8000 ppm was not critical. Sixteen species of common moulds inoculated on surfactant media showed restricted radial growth but never total inhibition. Surfactant did not prevent sporulation and did not greatly affect morphological characteristics. Rose bengal media performed poorly in comparative tests. These observations indicate the practical value of this surfactant in aeromycological studies.     

The effect of a surfactant in media for the enumeration, growth and identification of airborne fungi

Air sampling on to culture media for viable fungi is complicated by the presence of fast-growing species. A non-ionic surfactant (Triton N-101) incorporated into the medium effectively reduced the spread of such species and thus facilitated the enumeration and identification of fungal colonies collected by an Andersen sampler. The concentration of surfactant from 250 to 8000 ppm was not critical. Sixteen species of common moulds inoculated on surfactant media showed restricted radial growth but never total inhibition. Surfactant did not prevent sporulation and did not greatly affect morphological characteristics. Rose bengal media performed poorly in comparative tests. These observations indicate the practical value of this surfactant in aeromycological studies.     

The effect of anticoagulants, enzyme inhibitors and long term storage on the extraction of known concentrations of methionine enkephalin in human plasma

The stability of methionine enkephalin (M-E) during long term storage was investigated using various anticoagulants and enzyme inhibitors, eg EDTA, heparin, trasylol, citric acid. Plasma was stored for different lengths of time up to six weeks. High pressure liquid chromatography (HPLC) was used to separate and quantify M-E. We found that EDTA, heparin or trasylol per se are ineffective in preserving M-E for short term extraction. Blood collected in chilled heparin tubes with citric acid crystals and the plasma further acidified with hydrochloric acid gave the highest recovery. With storage times up to six weeks further degradation was marked in samples taken in plain tubes but did not occur with tubes containing citric acid crystals and hydrochloric acid.     

The effect of surface charge, negative and bipolar ionization on the deposition of airborne bacteria

Aims:  A series of experiments were conducted to evaluate the effect of surface charge and air ionization on the deposition of airborne bacteria.
Methods and Results:  The interaction between surface electrostatic potential and the deposition of airborne bacteria in an indoor environment was investigated using settle plates charged with electric potentials of 0, ±2·5kV and ±5kV. Results showed that bacterial deposition on the plates increased proportionally with increased potential to over twice the gravitational sedimentation rate at +5kV. Experiments were repeated under similar conditions in the presence of either negative or bipolar air ionization. Bipolar air ionization resulted in reduction of bacterial deposition onto the charged surfaces to levels nearly equal to gravitational sedimentation. In contrast, diffusion charging appears to have occurred during negative air ionization, resulting in an even greater deposition onto the oppositely charged surface than observed without ionization.
Conclusions:  Static charges on fomitic surfaces may attract bacteria resulting in deposition in excess of that expected by gravitational sedimentation or simple diffusion. Implementation of bipolar ionization may result in reduction of bacterial deposition.
Significance and Impact of Study:  Fomitic surfaces are important vehicles for the transmission of infectious organisms. This study has demonstrated a simple strategy for minimizing charge related deposition of bacteria on surfaces.