This work was financially supported by the National Natural Science Foundation of China (82273669, 82241086, 42207488), the Taishan Scholars Program of Shandong Province (No. tsqn202211121), and the Innovation and Technology Program for the Excellent Youth Scholars of Higher Education of Shandong Province (2022KJ295).

The study received approval from the Medical Ethics Committee of the Institute of Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention (NIOHP201604), with written informed consent obtained from all subjects prior to the study and biological sample collection. For this study, carbon black packers who had been working in a carbon black factory for more than 1 year and were exposed to carbon black aerosols were selected. Waterworks workers in a waterworks factory with no significant occupational exposure to harmful factors were recruited from the local area as a control population to establish the study. The following inclusion and exclusion criteria were selected for the study population: Inclusion criteria for carbon black packers included direct contact with carbon black during most shifts and a minimum of one year&#39;s work in the carbon black exposure area. Waterworks workers were included if they had no occupational exposure to carbon black or other pollutants in their work environment. Exclusion criteria encompassed workers with chronic diseases such as cancer, those who had undergone X-ray exposure in the past three months, individuals with a history of pulmonary tuberculosis, pulmonary surgery, viral myocarditis, congenital heart disease, recent fever, or inflammation, and workers who had taken antibiotics recently. The reagents and the equipment used in the study are listed in the Table of Materials.
1. Sputum preservation
<ol>
	<li>Prepare the fixative solution following the steps below:
	<ol>
		<li>Melt 2% polyethylene glycol (PEG1500) in a water bath. Take 20 mL of melted PEG1500 in a centrifuge tube. Add 20 mL of 50% ethanol to the centrifuge tube and shake the mixture thoroughly to create the mother liquor.</li>
		<li>Gradually pour 40 mL of the mother liquor into 960 mL of 50% ethanol. Shake the solution well to form the Saccomanno fixation solution. Store the solution in a cool, dark place.</li>
	</ol>
	</li>
	<li>Add 20-30 mL of the prepared fixative solution to a centrifuge tube containing approximately 2 mL of sputum. Mix the contents thoroughly. 
	NOTE: Ensure the total volume does not exceed 40 mL.</li>
	<li>Seal the centrifuge tube with a sealing film. Store it in a cool, dark place. Transport the tube to the laboratory for further processing when ready. 
	NOTE: Always melt PEG1500 in a water bath and use as per the prescribed dosage.</li>
</ol>
2. Preparation of cell suspension in sputum
<ol>
	<li>Prepare the digestive solution by dissolving 0.1 g of dithiothreitol in 100&#160;mL of saline as per the actual dosage. Store the prepared solution at 4 &#176;C for up to 48 h.</li>
	<li>Centrifuge the tube containing the sputum at 1,998 x g for 30 min at 4 &#176;C.</li>
	<li>Discard the supernatant. Add an equal volume of sputum digest to the precipitate. Vortex and shake the mixture thoroughly.
	<ol>
		<li>Place the tube in a 37 &#176;C water bath until complete liquefaction (10-15 min). 
		NOTE: Continuously shake the tube during the liquefaction process.</li>
	</ol>
	</li>
	<li>Filter the liquefied mixture with a 70 &#181;m filter membrane.</li>
	<li>Centrifuge the filtered solution at 500 x g for 7 min at 4 &#176;C.</li>
	<li>Discard the supernatant, retaining the cell precipitate.</li>
	<li>Resuspend the cell precipitate in Duchenne phosphate buffer. Centrifuge the resuspended solution at 500 x g for 7 min at 4 &#176;C to obtain a pure cell precipitate.</li>
</ol>
3. Preparation of cell smear
<ol>
	<li>Add 200-600 &#181;L of phosphate buffer to the cell sediment. Mix the contents thoroughly; adjust the buffer volume based on cell counting results (aim for &#62;1.0 &#215; 105 cells).</li>
	<li>Take 20 &#181;L of the cell suspension and create a cell smear using the hematocrit method17.</li>
	<li>Let the smear air-dry naturally (within 48 h). Fix the smear with a commercially available staining solution for 10 s.</li>
	<li>Immerse the smear in A staining solution for 8 s. During staining, gently lift the slide up and down, and rinse off excess stain with running water.</li>
	<li>Stain the smear in B staining solution for 8 s. Lift the slide during staining and rinse off excess stain under running water. 
	NOTE: Staining solutions A and B are available in the commercially available staining kit.</li>
	<li>Dip the slide in anhydrous ethanol twice, each time for 2-3 s.</li>
	<li>Once dry, apply an appropriate amount of neutral gum. Seal the slide with coverslips.</li>
</ol>
4. Quantitative analysis of CCAM
<ol>
	<li>Use a light microscope with a 100x oil lens objective. Capture images of randomly selected, well-stained, and morphologically intact macrophages. Take 50 macrophage pictures randomly for each sample.</li>
	<li>Perform image analysis in the Image J software following the steps below:
	<ol>
		<li>Measure the scale and determine the actual length and pixel-to-pixel conversion (282 pixels = 10 &#181;m). Set the scale in Image J (Analyze &#62; Set scale), input Distance in pixels, actual length, unit of length (&#181;m), and tick Global.</li>
		<li>Use an irregular shape to outline the cells. Remove the background (freehand selections, Edit &#62; Clear outside). Measure the total area of the cells (Analyze &#62; Measurement).</li>
		<li>Cut out the nucleus (Edit &#62; Cut). Convert the grayscale image to black and white (Image &#62; Type &#62; 8 bit).</li>
		<li>Adjust the grayscale value specific to each cell staining for accurate carbon particle counting (Image &#62; Adjust &#62; Threshold &#62; Apply, Analyze &#62;&#62; Measurement).</li>
		<li>Calculate the carbon content of 50 macrophages per sample based on the measured area and image analysis in Image J software.</li>
	</ol>
	</li>
</ol>

Quantifying Carbon in Pulmonary Macrophages via Induced Sputum Analysis

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The authors declare no conflicts of interest.

This study presents a detailed experimental protocol for using induced sputum-derived CCAM as a biological marker for internal exposure to atmospheric particulate matter. CCAM can be detected and quantified through optical microscopy, serving as a precise internal exposure biomarker reflecting the relationship with health effects. Therefore, there is a need to establish and optimize a convenient, reliable, and efficient induced sputum preservation method and a CCAM quantification method to standardize the methodological procedures.
The determination of the carbon content of lung macrophages involves several steps, including the collection of lung macrophages, smearing, staining, photographing, and image analysis. Currently, there are two methods for collecting lung macrophages: induced sputum and lung lavage, each with its own advantages and limitations18,19. Lung lavage can yield many homogeneous lung macrophages, but it is a traumatic sampling method that requires anesthesia and other medical conditions. Therefore, it is not suitable for large-scale general population sampling. In contrast, induced sputum is easy to perform, non-invasive, and suitable for large-scale sampling of the general population. It also allows for repeat samples to be obtained. Lung lavage obtains macrophages from alveoli or distal bronchioles, while induced sputum obtains macrophages that may originate from the airways. In this study, we performed induced sputum following the standard protocol published by the European Respiratory Society20.
The success rate of induced sputum is influenced by various factors, including the concentration of nebulized inhaled saline and the presence of respiratory diseases in the patient21,22,23. Sputum samples may also contain saliva, which can interfere with the measurement of macrophage carbon content, potentially reducing the count of airway macrophages in the sample24. The operational procedures in this study were standardized, the methods were straightforward, and the reproducibility was high. It was observed that factors such as the size of the nebulized inhalation flow rate, the adequacy of induction time, and the presence of intervals during induction affected the success rate of induced sputum. These considerations are important for ensuring accurate and reliable measurement of CCAM as a biomarker for particulate matter exposure.
The Saccomanno fixative solution, consisting of 50% ethanol and 2% polyethylene glycol, is typically used as a fixative for preserving biological samples like bronchoalveolar lavage fluid. In this study, for the first time, this fixative solution was utilized to preserve sputum obtained from epidemiological field investigations. The results met the subsequent quantification of carbon in macrophages, akin to preserving sputum cells in the morning to obtain clear cancer cell smears25. The operational procedures were standardized, the methods were straightforward, and the reproducibility was high. The advantages of induced sputum preservation in this study include (1) Easy configuration of fixative, (2) no refrigeration is needed for preservation, which is convenient for large-scale epidemiological surveys, and (3) samples can be preserved in a cool place away from light. Clear view under the microscope after staining, with identifiable nuclei, minimal impurities, and complete cell morphology. This is superior to direct sputum processing and smear application in terms of morphology26.
Regarding experimental methods, the following details were noted: Sputum was periodically shaken during digestion in a 37 &#176;C water bath until completely liquefied. After liquefaction, filtration on a 70 &#181;m membrane was done before centrifugation to remove impurities for clearer staining background. Cellular precipitates were washed with DPBS again before smearing on slides. Excess liquid was removed during staining by washing slides with running water. Slide holders were changed between stain I and stain II to prevent residue. For photography, macrophages with complete morphology and good staining were randomly selected, affecting CCAM quantification27.
Quantitative analysis software options for this study include Image SXM and Image J28. Image SXM requires manual image editing before analysis to enhance accuracy, which significantly extends the required time. It is also limited to processing a single cell type. In contrast, the Image J software used in this study offers better specificity in image processing and broader compatibility. In other disciplines, high-content screening microscopy methods have been employed for the selection and subsequent analysis of fluorescently labeled cells, achieving automated high-throughput operations. The future direction of methodological research may involve developing automated labeling of macrophages in sputum and screening procedures29. During Image J processing, factors influencing CCAM measurement include the measurer&#39;s knowledge of sample information and the determination of grayscale value thresholds. Adopting a blind approach can reduce observer bias, and some samples may need to be repeatedly measured to assess reproducibility. Independent image capture and analysis by two individuals with high-level expertise can enhance the accuracy of macrophage identification.
The study employed standard methods from the National Institute for Occupational Safety and Health (NIOSH) to assess the subjects&#39; exposure to occupational particulate matter. Multiple evaluations were conducted on fine particulate matter (PM2.5) levels and PM2.5-related elemental carbon (EC) content in the carbon black packaging area and the work areas of the control group water plant workers. Up to 99.6% of carbon black (CB) particles had an aerodynamic diameter smaller than 2.5 &#181;m, and 96.7% were smaller than 1.0 &#181;m. In the autumn of 2012, the average PM2.5 level in the CB packing facility was 800 micrograms per cubic meter, with a PM2.5-related EC level of 657.0 micrograms per cubic meter30,31. In the autumn of 2018, the geometric mean of CB was 637.4 &#181;g/m3, and the PM2.5-related EC level was 364.6 &#181;g/m3. In both observations, the EC/TC ratio exceeded 92.5%, consistent with the fact that CB is almost pure EC. Thus, it can be inferred from this study that carbon particles in macrophages are primarily derived from exposure to carbon black30,31. As a result, the PM2.5 concentration in the working environment of the population exposed to carbon black in this study was 637.4 &#181;g/m3, significantly higher than the PM2.5 concentration in the working environment of the control population (130.0 &#181;g/m3) and about five times higher than that of the control population. Elemental carbon levels in the population exposed to carbon black were 364.6 &#181;g/m3, much higher than the 2.0 &#181;g/m3 in the control population.
In summary, this study utilized hypertonic saline-induced sputum to collect samples, obtained lung macrophages, made cell smears, and quantified the carbon content within 50 macrophages, using the median number to represent the level of macrophage carbon content for each individual. This research methodology can be adapted for large-scale population samples and used to accurately evaluate an individual&#39;s level of internal exposure to particulate matter.

Ambient air pollution is associated with deaths due to respiratory and cardiovascular diseases, posing a serious threat to human health1,2. Epidemiological data indicate that chronic exposure to ambient particulate matter less than or equal to 2.5 &#181;m in diameter (PM2.5) is responsible for the premature deaths of between 4 and 9 million people globally. PM2.5 was ranked as the fifth most important risk factor for global mortality in the 2015 Global Burden of Disease, Injuries, and Risk Factors Study (GBD)3,4,5,6. Studies have found that adherence to WHO air pollution guidelines could prevent 51,213 deaths per year from PM2.5 exposure3. Currently, most studies lack the evaluation of intra-individual exposure and are only based on crude evaluations at larger regional monitoring sites, which are far from individual exposure levels. The available biomarkers of internal exposure, such as urinary PAHs and benzo(a)pyrene, do not reflect the associations between particulate matter exposures and health effects7,8,9. This leads to the inability to establish an accurate relationship with health effects. Therefore, the search for markers that reflect the level of particulate matter exposure in an individual is one of the keys to accurate exposure assessment for individuals.
Particles inhaled into the bronchi can be excreted with sputum through ciliary oscillations in the bronchi. The lack of a ciliated mucous flow transport system in the alveoli means that the primary clearance route for particles entering the alveoli is through phagocytosis and translocation by macrophages10,11. Based on the anatomical structure of the lung, its clearance of insoluble particulate foreign matter is slow. This allows particulate matter to interact with lung cells for extended periods and initiate various biological effects, causing damage to lung tissue and other organs12,13. Stimulation by particulate matter leads to macrophage activation, triggering a cascade of inflammatory factors in the lungs that can cause a systemic inflammatory response14. Considering the crucial role of macrophage cytophagy in eliciting cytokine storms in the lungs, it is theorized that carbon particles from lung macrophages could reflect the biologically effective dose of airborne carbon-nucleated particulate exposure15. Furthermore, since there is no aggregation of elemental carbon in mammalian cells and carbon-containing particles can be observed as black particulate matter under a light microscope, the collection of alveolar and bronchial macrophages and measurement of the carbon content in them can serve as a marker for evaluating particulate matter exposure16.
This study identified a method for accurately assessing individual particulate matter exposure levels, known as the Carbon Content of Airway Macrophages (CCAM). Specifically, population sputum samples were collected after participants inhaled hypertonic saline generated by an ultrasonic nebulizer. These samples were then preserved using a fixative solution. Airway macrophages were isolated, stained, and photographed under a light microscope to identify macrophages containing carbon-containing particles, which were then quantified. This method provides a biological marker for accurately assessing individual particulate matter exposure levels. It establishes a methodological foundation for investigating the relationship between particulate matter exposure and health effects, serving as a research basis for exploring associations between PM exposure and health outcomes such as lung diseases.

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		<tr>
			<td>3 mL sterile straws</td>
			<td>Shanghai YEASEN Biotechnology Co., LTD</td>
			<td>84202ES03</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tube</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>339652</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute ethyl alcohol</td>
			<td>Sinopharm Group Chemical Reagent Co. LTD</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Cedar oil</td>
			<td>Shanghai McLean Biochemical Technology Co., LTD</td>
			<td>C805296</td>
			<td></td>
		</tr>
		<tr>
			<td>Diff-quick staining solution</td>
			<td>Shanghai YEASEN Biotechnology Co., LTD</td>
			<td>40748ES76</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Solebo Bio Co., LTD</td>
			<td>D8220</td>
			<td></td>
		</tr>
		<tr>
			<td>Duchenne phosphate buffer (DPBS)</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope camera</td>
			<td>Olympus Corporation of Japan</td>
			<td>DP72</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral tree gum</td>
			<td>Solebo Bio Co., LTD</td>
			<td>G8590</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon filter membrane 70um</td>
			<td>BD Falcon Bioscience, USA</td>
			<td>211755</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical microscope</td>
			<td>Olympus Corporation of Japan</td>
			<td>BX60</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol</td>
			<td>Sinopharm Group Chemical Reagent Co. LTD</td>
			<td>25322-68-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>SL40R</td>
			<td></td>
		</tr>
		<tr>
			<td>Viscous slide</td>
			<td>Jiangsu SHitAI EXPERIMENTAL Equipment Co. LTD</td>
			<td>188105</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring carbon content in airway macrophages exposed to carbon-containing particulate matters

Pulmonary macrophages exhibit a dose-dependent pattern in phagocytizing particles. Following engulfment, these macrophages are subsequently excreted with sputum, rendering macrophages and particles visible and quantifiable under light microscopy. Notably, elemental carbon within the mammalian body originates exclusively from external contaminants. Consequently, the carbon content in airway macrophages (CCAM) serves as a valid exposure biomarker, accurately estimating individual exposure to carbon-containing particulate matter (PM). This article delineates a protocol involving sputum collection, preservation, processing, slide preparation, and staining, as well as macrophage photo acquisition and analysis. After removing the macrophage nuclei, the proportion of cytoplasm area occupied by carbon particles (PCOC) was calculated to quantify carbon content in each macrophage. The results indicate an elevation in CCAM levels after exposure to carbon-containing PM. In summary, this non-invasive, precise, reliable, and standardized method enables the direct measurement of carbon particles within target cells and is utilized for large-scale quantification of individual CCAM through induced sputum.

The sputum, preserved and processed with the fixative solution, displayed intact macrophage morphology under an optical microscope during morphological examination. The macrophages exhibited clear, round, or kidney-shaped, easily stainable cell nuclei. Following staining, the cell nuclei appeared bluish-purple, while the cytoplasm was light pink or light blue. The microscopic field showed minimal impurities, which facilitated easy cell identification. Within the cells, black carbon particles were distinctly visible in small aggregates, enabling quantitative analysis of carbon particles.
Representative images of sputum macrophages are presented in Figure 1, depicting varying numbers of carbon particles in different cells. For instance, Figure 1A shows no carbon particles, while Figure 1D exhibits the highest concentration. The values of CCAM were measured as 0.00%, 2.24%, 6.64%, and 34.61% for macrophages (A-D), respectively. During the CCAM parameter determination process, a quantitative assessment of carbon content in each macrophage was conducted to mitigate biases arising from variations in macrophage size and the overlap of carbon particles with cell nuclei. CCAM calculation and analysis were performed for 50 macrophages per study subject, as shown in Figure 2.
The study selected carbon black packers (CBP) from a specific acetylene carbon black factory as the exposed group and recruited water pump workers from local water plants, who had no apparent occupational exposure to harmful factors, as the control group (Non-CBP). The results presented in Figure 2 indicate a higher CCAM in carbon black packaging workers compared to the control group.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66781/66781fig01.jpg" /> 
Figure 1: Optical microscopic image showing carbon particles within airway macrophages. The black color in the cytoplasm represents carbon particles. Panels (A-D) depict representative images of airway macrophages, each exhibiting varying levels of CCAM. The CCAM values for macrophages in (A-D) were 0.00%, 2.24%, 6.64%, and 34.61%, respectively. The highest CCAM level was observed in (D). Scale bars: 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66781/66781fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66781/66781fig02.jpg" /> 
Figure 2: Quantitative levels of CCAM in the population. Fifty cytoplasmically intact and well-stained macrophages were randomly selected for analysis and quantification per study subject. Data were analyzed using Student&#39;s t-test: *P &#60; 0.05. CBP: carbon black packers; Non-CBP: control group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66781/66781fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Measuring Carbon Content in Airway Macrophages Exposed to Carbon-Containing Particulate Matters

This article describes a detailed experimental protocol for measuring the carbon content of airway macrophages with the aim of assessing the internal exposure dose at the level of individual particulate matter exposure.

Pulmonary macrophages exhibit a dose-dependent pattern in phagocytizing particles. Following engulfment, these macrophages are subsequently excreted with ...

measuring-carbon-content-airway-macrophages-exposed-to-carbon

Nanyang Technological University

Research

JoVE Journal

Environment

219 Views.  Qingdao University. This article describes a detailed experimental protocol for measuring the carbon content of airway macrophages with the aim of assessing the internal exposure dose at the level of individual particulate matter exposure.

Video: Measuring Carbon Content in Airway Macrophages Exposed to Carbon-Containing Particulate Matters

Analysis of Fatty Acid Content and Composition in Microalgae

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (&omega;-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes.
With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of.
This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other.
The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.

A method for the determination of fatty acid content and composition in microalgae based on mechanical cell disruption, solvent based lipid extraction, transesterification, and quantification and identification of fatty acids using gas chromatography is described. A tripentadecanoin internal standard is used to compensate for the possible losses during extraction and incomplete transesterification.

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of ...

Measuring Carbon-based Contaminant Mineralization Using Combined CO2 Flux and Radiocarbon Analyses

A method is described which uses the absence of radiocarbon in industrial chemicals and fuels made from petroleum feedstocks which frequently contaminate the environment. This radiocarbon signal &#8212; or rather the absence of signal &#8212; is evenly distributed throughout a contaminant source pool (unlike an added tracer) and is not impacted by biological, chemical or physical processes (e.g., the 14C radioactive decay rate is immutable). If the fossil-derived contaminant is fully degraded to CO2, a harmless end-product, that CO2 will contain no radiocarbon. CO2 derived from natural organic matter (NOM) degradation will reflect the NOM radiocarbon content (usually &#60;30,000 years old). Given a known radiocarbon content for NOM (a site background), a two end-member mixing model can be used to determine the CO2 derived from a fossil source in a given soil gas or groundwater sample. Coupling the percent CO2 derived from the contaminant with the CO2 respiration rate provides an estimate for the total amount of contaminant degraded per unit time. Finally, determining a zone of influence (ZOI) representing the volume from which site CO2 is collected allows determining the contaminant degradation per unit time and volume. Along with estimates for total contaminant mass, this can ultimately be used to calculate time-to-remediate or otherwise used by site managers for decision-making.

Measuring Carbon-based Contaminant Mineralization Using Combined CO2 Flux and Radiocarbon Analyses

A protocol is described wherein CO2 mineralized from organic contaminant (derived from petroleum feedstocks) biodegradation is trapped, quantified, and analyzed for 14C content. A model is developed to determine CO2 capture zone's spatial extent. Spatial and temporal measurements allow integrating contaminant mineralization rates for predicting remediation extent and time.

A method is described which uses the absence of radiocarbon in industrial chemicals and fuels made from petroleum feedstocks which frequently contaminate ...

Assessment of Labile Organic Carbon in Soil Using Sequential Fumigation Incubation Procedures

Management practices and environmental changes can alter soil nutrient and carbon cycling. Soil labile organic carbon, a readily decomposable C pool, is highly sensitive to disturbance. It is also the primary substrate for soil microorganisms, which is fundamental to nutrient cycling. Due to these attributes, labile organic carbon (LOC) has been identified as an indicator parameter for soil health. Quantifying the turnover rate of LOC also aids in understanding changes in soil nutrient cycling processes. A sequential fumigation incubation method has been developed to estimate soil LOC and potential C turnover rate. The method requires fumigating soil samples and quantifying CO2-C respired during a 10 day incubation period over a series of fumigation-incubation cycles. Labile organic C and potential C turnover rate are then extrapolated from accumulated CO2 with a negative exponential model. Procedures for conducting this method are described.

Labile organic carbon (LOC) and the potential carbon turnover rate are sensitive indicators of changes in soil nutrient cycling processes. Details are provided for a method based on fumigating and incubating soil in a series of cycles and using the CO2 accumulated during the incubation periods to estimate these parameters.

Management practices and environmental changes can alter soil nutrient and carbon cycling. Soil labile organic carbon, a readily decomposable C pool, is ...

Biofilm Removal Using Carbon Dioxide Aerosols without Nitrogen Purge

Biofilms can cause serious concerns in many applications. Not only can they cause economic losses, but they can also present a public health hazard. Therefore, it is highly desirable to remove biofilms from surfaces. Many studies on CO2 aerosol cleaning have employed nitrogen purges to increase biofilm removal efficiency by reducing the moisture condensation generated during the cleaning. However, in this study, periodic jets of CO2 aerosols without nitrogen purges were used to remove Pseudomonas putida biofilms from polished stainless steel surfaces. CO2 aerosols are mixtures of solid and gaseous CO2 and are generated when high-pressure CO2 gas is adiabatically expanded through a nozzle. These high-speed aerosols were applied to a biofilm that had been grown for 24 hr. The removal efficiency ranged from 90.36% to 98.29% and was evaluated by measuring the fluorescence intensity of the biofilm as the treatment time was varied from 16 sec to 88 sec. We also performed experiments to compare the removal efficiencies with and without nitrogen purges; the measured biofilm removal efficiencies were not significantly different from each other (t-test, p &gt; 0.55). Therefore, this technique can be used to clean various bio-contaminated surfaces within one minute.

Biofilms on surfaces can be effectively and rapidly removed by using a periodic jet of carbon dioxide aerosols without a nitrogen purge.

Biofilms can cause serious concerns in many applications. Not only can they cause economic losses, but they can also present a public health hazard. ...

Production and Measurement of Organic Particulate Matter in a Flow Tube Reactor

Organic particulate matter (PM) is increasingly recognized as important to the Earth's climate system as well as public health in urban regions, and the production of synthetic PM for laboratory studies have become a widespread necessity. Herein, experimental protocols demonstrate approaches to produce aerosolized organic PM by &#945;-pinene ozonolysis in a flow tube reactor. Methods are described for measuring the size distributions and morphology of the aerosol particles. The video demonstrates basic operations of the flow tube reactor and related instrumentation. The first part of the video shows the procedure for preparing gas-phase reactants, ozonolysis, and production of organic PM. The second part of the video shows the procedures for determining the properties of the produced particle population. The particle number-diameter distributions show different stages of particle growth, namely condensation, coagulation, or a combination of both, depending on reaction conditions. The particle morphology is characterized by an aerosol particle mass analyzer (APM) and a scanning electron microscope (SEM). The results confirm the existence of non-spherical particles that have grown from coagulation for specific reaction conditions. The experimental results also indicate that the flow tube reactor can be used to study the physical and chemical properties of organic PM for relatively high concentrations and short time frames.

This paper describes the operation procedure for the flow tube reactor and related data collection. It shows the protocols for setting the experiments, recording data and generating the number-diameter distribution as well as the particle mass information, which gives useful information about chemical and physical properties of the organic aerosols.

Organic particulate matter (PM) is increasingly recognized as important to the Earth's climate system as well as public health in urban regions, and the ...

Production and Measurement of Organic Particulate Matter in the Harvard Environmental Chamber

The production and the evolution of atmospheric organic particulate matter (PM) are insufficiently understood for accurate simulations of atmospheric chemistry and climate. The complex production mechanisms and reaction pathways make this a challenging research topic. To address these issues, an environmental chamber, providing enough residence time and close-to-ambient concentrations of precursors for secondary organic materials, is needed. The Harvard Environmental Chamber (HEC) was built to serve this need, simulating the production of gas and particle phase species from volatile organic compounds (VOCs). The HEC has a volume of 4.7 m3 and a mean residence time of 3.4 h under typical operating conditions. It is operated as a completely mixed flow reactor (CMFR), providing the possibility of indefinite steady-state operation across days for sample collection and data analysis. The operation procedures are described in detail in this article. Several types of instrumentation are used to characterize the produced gas and particles. A High-Resolution Time-of-Fight Aerosol Mass Spectrometer (HR-ToF-AMS) is used to characterize particles. A Proton-Transfer-Reaction Mass Spectrometer (PTR-MS) is used for gaseous analysis. Example results are presented to show the use of the environmental chamber in a wide variety of applications related to the physicochemical properties and reaction mechanisms of organic atmospheric particulate matter.

This paper describes operation procedures for the Harvard Environmental Chamber (HEC) and related instrumentation for measuring gaseous and particle species. The environmental chamber is used to produce and study secondary organic species produced from the organic precursors, especially related to atmospheric organic particulate matter.

The production and the evolution of atmospheric organic particulate matter (PM) are insufficiently understood for accurate simulations of atmospheric ...

Measurement of the Rheology of Crude Oil in Equilibrium with CO2 at Reservoir Conditions

A rheometer system to measure the rheology of crude oil in equilibrium with carbon dioxide (CO2) at high temperatures and pressures is described. The system comprises a high-pressure rheometer which is connected to a circulation loop. The rheometer has a rotational flow-through measurement cell with two alternative geometries: coaxial cylinder and double gap. The circulation loop contains a mixer, to bring the crude oil sample into equilibrium with CO2, and a gear pump that transports the mixture from the mixer to the rheometer and recycles it back to the mixer. The CO2 and crude oil are brought to equilibrium by stirring and circulation and the rheology of the saturated mixture is measured by the rheometer. The system is used to measure the rheological properties of Zuata crude oil (and its toluene dilution) in equilibrium with CO2 at elevated pressures up to 220 bar and a temperature of 50 &#176;C. The results show that CO2 addition changes the oil rheology significantly, initially reducing the viscosity as the CO2 pressure is increased and then increasing the viscosity above a threshold pressure. The non-Newtonian response of the crude is also seen to change with the addition of CO2.

Measurement of the Rheology of Crude Oil in Equilibrium with CO2 at Reservoir Conditions

A method for measuring the rheology of crude oil in equilibrium with carbon dioxide at reservoir conditions is presented.

A rheometer system to measure the rheology of crude oil in equilibrium with carbon dioxide (CO2) at high temperatures and pressures is described. The ...

Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by environmental organochlorine pesticides (OCPs) in hepatocytes. HepG2 cells were exposed to &#946;-hexachlorocyclohexane (&#946;-HCH) for 24 h at an equivalent dose of internal exposure in general population. Ultrastructure in hepatocytes was examined by transmission electron microscopy (TEM) to show the damage of mitochondria. Mitochondrial function was further evaluated by mitochondrial fluorescence intensity, adenosine 5'-triphosphate (ATP) levels, oxygen consumption rate (OCR) and mitochondrial membrane potential (MMP) in HepG2 cells incubated with &#946;-HCH. The mitochondria fluorescence intensity after stained by mitochondrial green fluorescent probe was observed with a fluorescence microscopy. The luciferin-luciferase reaction was used to determine ATP levels. The MMP was detected by the cationic dye JC-1 and analyzed under flow cytometry. OCR was measured with an extracellular flux analyzer. In summary, these protocols were used in detecting mitochondrial function in hepatocytes with to investigate mitochondria damages.

Understanding the influence of environmental organochlorine pesticides (OCPs) on mitochondrial function in hepatocytes is important in exploring the mechanism of OCPs causing metabolic disorders. This paper presents detailed methods on detecting hepatic mitochondrial function.

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by ...

The Calibration and Use of Capacitance Sensors to Monitor Stem Water Content in Trees

Water transport and storage through the soil-plant-atmosphere continuum is critical to the terrestrial water cycle, and has become a major research focus area. Biomass capacitance plays an integral role in the avoidance of hydraulic impairment to transpiration. However, high temporal resolution measurements of dynamic changes in the hydraulic capacitance of large trees are rare. Here, we present procedures for the calibration and use of capacitance sensors, typically used to monitor soil water content, to measure the volumetric water content in trees in the field. Frequency domain reflectometry-style observations are sensitive to the density of the media being studied. Therefore, it is necessary to perform species-specific calibrations to convert from the sensor-reported values of dielectric permittivity to volumetric water content. Calibration is performed on a harvested branch or stem cut into segments that are dried or re-hydrated to produce a full range of water contents used to generate a best-fit regression with sensor observations. Sensors are inserted into calibration segments or installed in trees after pre-drilling holes to a tolerance fit using a fabricated template to ensure proper drill alignment. Special care is taken to ensure that sensor tines make good contact with the surrounding media, while allowing them to be inserted without excessive force. Volumetric water content dynamics observed via the presented methodology align with sap flow measurements recorded using thermal dissipation techniques and environmental forcing data. Biomass water content data can be used to observe the onset of water stress, drought response and recovery, and has the potential to be applied to the calibration and evaluation of new plant-level hydrodynamics models, as well as to the partitioning of remotely sensed moisture products into above- and belowground components.

The hydraulic capacitance of biomass is a key component of the vegetation water budget, which serves as a buffer against short and long-term drought stresses. Here, we present a protocol for the calibration and use of soil moisture capacitance sensors to monitor water content in the stems of large trees.

Water transport and storage through the soil-plant-atmosphere continuum is critical to the terrestrial water cycle, and has become a major research focus ...

Assessing the Particulate Matter Removal Abilities of Tree Leaves

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on collecting various sized particulate matter (PM) retained on leaf surfaces. We further characterized the retention efficiency of leaves to various sized PM, which will help to assess the abilities of urban trees to remove PM from ambient air quantitatively. 
Taking three broadleaf tree species (Ginkgo biloba, Sophora japonica, and Salix babylonica) and two needleleaf tree species (Pinus tabuliformis and Sabina chinensis) as the research objects, leaf samples were collected 4 days (short PM retention period) and 14 days (long PM retention period) after the latest rainfall. PM retained on the leaf surfaces was collected by means of WC, BC, and UC in sequence. Then, retention efficiencies of leaves (AEleaf) to three types of the various sized PM, including easily removable PM (ERP), difficult-to-remove PM (DRP), and totally removable PM (TRP), were calculated. Only around 23%-45% of the total PM retained on leaves could be cleaned off and collected by WC. When the leaves were cleaned through WC+BC, the underestimation of the PM retention capacity of different tree species was in the range of 29%-46% for various sized PM. Almost all PM retained on leaves could be removed if UC was supplemented to WC+BC. 
In conclusion, if the UC was complemented after the conventional cleaning methods, more PM on leaf surfaces could be eluted and collected. The procedure developed in this study can be used for assessing the PM removal abilities of different tree species.

The ultrasonic cleaning method was applied to elute the particulate matter (PM) retained on leaf surfaces after PM was eluted by the conventional cleaning methods (water cleaning only or water cleaning plus brush cleaning). The methodology can help to improve the estimation accuracy for PM retention capacity of leaves.

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on ...

Measuring Carbon Content in Airway Macrophages Exposed to Carbon-Containing Particulate Matters

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