This work was supported by the National Natural Scientific Foundation of China (32102244), the National Agricultural Products Quality and Safety and Risk Assessment Project (GJFP2021001), the Natural Scientific Foundation of Anhui Province (19252002), and the USDA (HAW05020H).

For the present study, the following weed species were collected: Ludwigia prostrata Roxb., Murdannia triquetra (Wall. ex C. B. Clarke) Bruckn., Ageratum conyzoides L., Chenopodium ambrosioides, Trachelospermum jasminoide (L.) Lem., Ageratum conyzoides L., Emilia sonchifolia (L.) DC, Ageratum conyzoides L., and Crassocephalum crepidioides (Benth.) S. Moore. The fresh tea leaves were picked from the variety of Longjing 43# tea trees, and the dried tea samples were commercially available tea processed according to the green tea manufacturing process (see Table of Materials).
1. Sample collection
<ol>
	<li>Collect 40 real samples.
	<ol>
		<li>Collect 10 weeds, 10 soils, and 10 fresh tea leaves randomly from multiple tea gardens. 
		NOTE: For the present study, the soil was sampled at a depth of 20 cm with a sample amount of 200 g.</li>
		<li>Randomly collect 10 dried tea products (250 g) from a supermarket.</li>
	</ol>
	</li>
	<li>Collect samples of weeds, soil, and fresh tea leaves to study the contamination source of PAs in tea.
	<ol>
		<li>Set five sampling points in the same tea garden, with three replicates at each point.</li>
		<li>Collect Ageratum conyzoides L. weed samples with the highest PA content commonly found in tea gardens. 
		NOTE: The sample amount was 250 g for the present study.</li>
		<li>Collect the soil samples. 
		NOTE: The soil samples were A. conyzoides rhizospheric soil at a depth of 20 cm with a sample amount of 200 g.</li>
		<li>Collect the fresh tea leaves from different parts of the tea plants, including one bud with two leaves, one bud with three leaves, one bud with four leaves, and mature leaves. 
		&#8203;NOTE: The sample amount was 250 g.</li>
	</ol>
	</li>
</ol>
2. Sample treatment
<ol>
	<li>Pretreat the samples following the steps below.
	<ol>
		<li>Grind the dried tea and soil samples with a grinder, pass the pulverized samples through a 200-mesh sieve, and store them at &#8722;20 &#176;C. 
		NOTE: The dried tea was a commercially available tea product (see Table of Materials), so it was directly crushed and sieved for storage. The soil samples (200 g) were placed in a ventilated place in the dark to air dry for about one week.</li>
		<li>Homogenize the weed and fresh tea leaves with a homogenizer and store them at &#8722;20 &#176;C.</li>
	</ol>
	</li>
	<li>Perform sample treatment of the dried tea products, fresh tea leaves, and weeds.
	<ol>
		<li>Weigh 1.00 g of each sample (dried tea products, fresh tea leaves, and weeds) and place it in a 50 mL centrifuge tubes.</li>
		<li>Add 10 mL of 0.1 mol/L sulfuric acid solution and vortex for 2 min for solid-phase extraction (using SPE cartridge, see Table of Materials) and 1 min for adsorbent purification. Perform ultrasonic extraction23 for 15 min, and then centrifuge for 10 min at a speed of 9,390 x g at room temperature. 
		NOTE: The power of the ultrasonic oscillator was 290 W, the oscillation frequency was 35 kHz, and the temperature was set to 30 &#176;C.</li>
		<li>Transfer the supernatant into a 50 mL centrifuge tube with a plastic-tip dropper.</li>
		<li>Follow the steps above to repeat the extraction once. Combine the two supernatants.
		<ol>
			<li>Activate the SPE cartridges with 5 mL of methanol and 5 mL of deionized water. Add 10 mL of supernatant to the pre-activated cartridge and perform sample clean-up.</li>
			<li>After the sample solution level reached the the upper layer of cartridges, elute the analytes with with 5 mL of 1% formic acid solution and then 5 mL of methanol. Discard the eluate.</li>
			<li>Elute with 5 mL of methanol (containing 0.5% ammonia water), filter the eluate through a 0.22 &#181;m membrane filter, and analyze by UPLC-MS/MS (see Table of Materials).</li>
		</ol>
		</li>
		<li>Perform sample clean-up using adsorbents.
		<ol>
			<li>Take 2 mL of the supernatant (step 2.2.4) into a 10 mL centrifuge tube filled with the adsorbents of GCB:PSA:C18 (10 mg:20 mg:15 mg, see Table of Materials), vortex for 1 min, and centrifuge at 9,390 x g for 8 min at room temperature.</li>
			<li>Pass 1 mL of the supernatant through a 0.22 &#181;m membrane filter before analysis by UPLC-MS/MS.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Perform treatment of the soil samples.
	<ol>
		<li>Weigh a 1.00 g soil sample. Place it in a 50 mL centrifuge tube, and add 0.1 mL of 0.1 mol/L trisodium citrate solution (see Table of Materials) to adjust the soil pH value to 6.0.</li>
		<li>Allow to stand for 2 min, and then add 10 mL of 0.1 mol/L sulfuric acid methanol solution, vortex for 2 min and shake for 30 min, and then perform ultrasonic extraction for 30 min.</li>
		<li>Centrifuge at 9,390 x g for 10 min, and transfer the supernatant into a 50 mL centrifuge tube with a plastic-tip dropper.</li>
		<li>Follow the above steps to repeat the extraction and combine the supernatant twice. 
		&#8203;NOTE: The purification method was the same as in step 2.2.5.1 and step 2.2.5.2.</li>
	</ol>
	</li>
</ol>
3. Instrumental analysis
<ol>
	<li>Detect the 15 PAs in dried tea samples, fresh tea leaves, weed, and soil (samples from step 2) using a commercially available UPLC-MS/MS system (2.1 mm x 100 mm, 1.8 &#181;m) (see Table of Materials).</li>
	<li>Set the column temperature to 40 &#176;C, the flow rate to 0.250 mL/min, and the injection volume to 3 &#181;L.</li>
	<li>Set the mobile phase A: methanol (containing 0.1% formic acid + 1 mmol/L ammonium formate), and mobile phase B: water (containing 0.1% formic acid + 1 mmol/L ammonium formate).</li>
	<li>Set a gradient elution procedure: 10% A from 0.0 min to 0.25 min, 10%-30% A from 0.25 min to 6.0 min, 30%-40% A from 6.0 min to 9.0 min, 40%-98% A from 9.0 min to 9.01 min that was held for 1.9 min, and 98%-100% A from 11.0 min to 11.1 min that was held for 2.9 min.</li>
	<li>Set the mass spectrometer parameters: ionization mode, electrospray positive ion source (ESI+); atomizer pressure, 7.0 bar; capillary voltage, 4.0 kV; taper hole back blowing flow, 150 L/h; solvent gas flow, 800 L/h; dissolvent temperature, 400 &#176;C; impact gas flow, 0.25 mL/min.</li>
</ol>

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The authors have nothing to disclose.

The present work was designed to develop an effective, sensitive method to explore the contamination routes and sources of PAs in tea samples as well as the distribution of PAs in different parts of the tea plants. However, in this study, only 15 PAs were successfully separated on the chromatographic column, which is a very small number in comparison to the large number of alkaloids in plant species3,4. This was not only related to the packing properties of the c...

As secondary metabolites, pyrrolizidine alkaloids (PAs) protect plants against herbivores, insects, and pathogens1,2. Up to now, over 660 PAs and PA-N-oxides (PANOs) with different structures have been found in more than 6,000 plant species worldwide3,4. PA-producing plants are mainly found in the families Asteraceae, Boraginaceae, Fabaceae, and Apocynaceae5,6. PAs are easily oxidized to unstable dehydropyrrolizidine alkaloids, which have strong electrophilicity and can attack nucleophiles such as DNA and proteins, resulting in liver cell necrosis, venous occlusions, cirrhosis, ascites, and other symptoms7,8. The main target organ of PA toxicity is the liver. PAs can also cause lung, kidney, and other organ toxicity and mutagenic, carcinogenic, and developmental toxicity9,10.
Cases of human and animal poisoning have been reported in many countries from the ingestion of traditional herbs, supplements, or teas containing PAs or the indirect contamination of foods such as milk, honey, or meat (toxic from ingestion of pasturage containing PAs)11,12,13. The European Food Safety Authority (EFSA) findings indicate that substances such as (herbal) tea are an important source of human exposure to PAs/PANOs14. Tea samples do not produce PAs, whereas PA-producing plants are commonly found in tea gardens (e.g., Emilia sonchifolia, Senecio angulatus, and Ageratum conyzoides)15. It was previously suspected that the tea could be contaminated with PAs from their producing plants during picking and processing. However, PAs were also detected in some hand-picked tea leaves (i.e., no PA-producing plants), suggesting there must be other routes or sources of contamination16. A co-cultivation experiment of ragwort (Senecio jacobaea) with melissa (Melissa officinalis), peppermint (Mentha piperita), parsley (Petroselinum crispum), chamomile (Matricaria recutita), and nasturtium (Tropaeolum majus) plants was conducted, and the results showed that PAs were detected in all these plants17. It has been verified that PAs are indeed transferred and exchanged between living plants via soil18,19. Van Wyk et al.20 found that rooibos tea (Aspalathus linearis) was severely contaminated in weed-rich sites and contained PAs of the same type and proportion. However, no PAs were detected in rooibos tea in weed-free sites.
At present, ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) with high selectivity and sensitivity has been widely used in the qualitative and quantitative analysis of PAs in agricultural products and food21,22. The sample treatment method is usually comprised of either solid phase extraction (SPE) or the QuEChERS (Quick Easy Cheap Effective Rugged Safe) clean-up of complex food matrices extracts, which can obtain the highest possible sensitivity12,19. However, robust analytical methods allowing the detection and quantification of PAs in complex matrices like soil, weeds, and fresh tea leaves are still missing.
This study analyzed 15 PAs in dried tea samples, fresh tea leaves, weeds, and weed rhizospheric soil samples with UPLC-MS/MS combined with an adsorbent purification method. Furthermore, 15 paired weed and weed rhizospheric soil samples and 60 fresh tea leaf samples were collected from five sampling sites in Jinzhai tea garden in Anhui Province, China, and were analyzed for 15 PAs. These results can provide a survey method and some information on the source and route of PAs (contamination) in tea samples to ensure the quality and safety of tea.

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	<tbody>
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			<td>120-400 MESH, 10g. per box&#160;</td>
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			<td>HERMLE</td>
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			<td>30000&#160;rpm (min:10 rpm), Dimensions (W x H x D):&#160;71.5 cm&#215; 42 cm &#215; 51 cm</td>
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			<td>Lvming, Qingshan, Luyuchun, Changling, Huixing, Wuyunjian, Heshengchun</td>
			<td>loose tea</td>
			<td>Green tea</td>
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			<td>CNW</td>
			<td>D2110060</td>
			<td>40-63 &#956;m,100g.per box</td>
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			<td>BioCrick</td>
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			<td>cence</td>
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			<td>Max:16000 r/min, 330 &#215; 390 &#215; 300 mm (L &#215; W &#215; H), Capacity: 6 &#215; 50 mL</td>
		</tr>
		<tr>
			<td>HSS T3 column</td>
			<td>Waters</td>
			<td>186004976</td>
			<td>ACQUITY UPLC HSS T3 (2.1 &#215; 100 mm 1.8 &#956;m)</td>
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			<td>BioCrick</td>
			<td>340066</td>
			<td>CAS No:95462-14-9</td>
		</tr>
		<tr>
			<td>Jacobine (Jb) (98.0%)</td>
			<td>BioCrick</td>
			<td>132282048</td>
			<td>CAS No:6870-67-3</td>
		</tr>
		<tr>
			<td>Jacobine-N-oxide (JbNO) (98.0%)</td>
			<td>ChemFaces</td>
			<td>CFN00461</td>
			<td>CAS No:38710-25-7</td>
		</tr>
		<tr>
			<td>Methyl Alcohol (99.9%)</td>
			<td>Tedia Company,Inc.</td>
			<td>21115100</td>
			<td>CAS No:67-56-1</td>
		</tr>
		<tr>
			<td>PSA</td>
			<td>Agela</td>
			<td>P19-00833</td>
			<td>40-60 &#956;m, 60 &#197; 100g.per box</td>
		</tr>
		<tr>
			<td>Retrorsine (Re) (98.0%)</td>
			<td>BioCrick</td>
			<td>5281743</td>
			<td>CAS No:480-54-6</td>
		</tr>
		<tr>
			<td>Retrorsine-N-oxide (ReNO) (98.0%)</td>
			<td>BioCrick</td>
			<td>5281734</td>
			<td>CAS No:15503-86-3</td>
		</tr>
		<tr>
			<td>Senecionine (Sc) (98.0%)</td>
			<td>BioCrick</td>
			<td>5280906</td>
			<td>CAS No:130-01-8</td>
		</tr>
		<tr>
			<td>Senecionine-N-oxide (ScNO) (98.0%)</td>
			<td>BioCrick</td>
			<td>5380876</td>
			<td>CAS No:13268-67-2</td>
		</tr>
		<tr>
			<td>Seneciphylline N-oxid (SpNO) (98.0%)</td>
			<td>BioCrick</td>
			<td>6442619</td>
			<td>CAS No:38710-26-8</td>
		</tr>
		<tr>
			<td>Seneciphylline (Sp) (98.0%)</td>
			<td>BioCrick</td>
			<td>5281750</td>
			<td>CAS No:480-81-9</td>
		</tr>
		<tr>
			<td>Senkirkine (Sk) (98.0%)</td>
			<td>BioCrick</td>
			<td>5281752</td>
			<td>CAS No:2318-18-5</td>
		</tr>
		<tr>
			<td>SPE PCX</td>
			<td>Agilent Technologies</td>
			<td>12108206</td>
			<td>Cation Mixed Mode, 6 mL</td>
		</tr>
		<tr>
			<td>Sulfuric acid (97%)</td>
			<td>Wuxi Zhanwang Chemical Reagent Co., Ltd.</td>
			<td>1003019</td>
			<td>CAS No:7664-93-9</td>
		</tr>
		<tr>
			<td>Trisodium citrate</td>
			<td>Sinpharm Chemical Reagent Co., Ltd.</td>
			<td>20121009</td>
			<td>CAS No:6132-04-3</td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner</td>
			<td>Supmile</td>
			<td>KQ-600B</td>
			<td>Inner slot size: 500 &#215; 300 &#215; 150 mm; Capacity: 22.5 L</td>
		</tr>
		<tr>
			<td>UPLC-xevoTQMS</td>
			<td>Waters</td>
			<td>ZPLYY-003</td>
			<td>Triple four-stage rod mass analyzer, Waters Alliance 2695/Waters ACQUITY UPLC Liquid Phase System</td>
		</tr>
		<tr>
			<td>Water bath thermostat oscillator</td>
			<td>Guoyu instrument</td>
			<td>SHY-2AHS</td>
			<td>Oscillation times:&#160; 60-300 times/min, Constant temperature range: room temperature to 100 &#176;C</td>
		</tr>
	</tbody>
</table>

source and route of pyrrolizidine alkaloid contamination in tea samples

Toxic pyrrolizidine alkaloids (PAs) are found in tea samples, which pose a threat to human health. However, the source and route of PA contamination in tea samples have remained unclear. In this work, an adsorbent method combined with UPLC-MS/MS was developed to determine 15 PAs in the weed Ageratum conyzoides L., A. conyzoides rhizospheric soil, fresh tea leaves, and dried tea samples. The average recoveries ranged from 78%-111%, with relative standard deviations of 0.33%-14.8%. Fifteen pairs of A. conyzoides and A. conyzoides rhizospheric soil&#160;samples and 60 fresh tea leaf samples were collected from the Jinzhai tea garden in Anhui Province, China, and analyzed for the 15 PAs. Not all 15 PAs were detected in fresh tea leaves, except for intermedine-N-oxide (ImNO) and senecionine (Sn). The content of ImNO (34.7 &#181;g/kg) was greater than that of Sn (9.69 &#181;g/kg). In addition, both ImNO and Sn were concentrated in the young leaves of the tea plant, while their content was lower in the old leaves. The results indicated that the PAs in tea were transferred through the path of PA-producing weeds-soil-fresh tea leaves in tea gardens.

The optimized adsorbent purification and analysis method of 15 PAs in dried tea samples, fresh tea leaves, weeds, and soil was established and compared with the commonly used purification method using the SPE cartridge. The results showed that the recoveries of the 15 PAs in dried tea samples, weed, and fresh tea leaves using the SPE cartridge were 72%-120%, while that using adsorbent purification was 78%-98% (Figure 1). The recoveries of the 15 PAs in soil using adsorbent purification were ...

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Source and Route of Pyrrolizidine Alkaloid Contamination in Tea Samples

The present protocol describes the contamination of pyrrolizidine alkaloids (PAs) in tea samples from PA-producing weeds in tea gardens.

Toxic pyrrolizidine alkaloids (PAs) are found in tea samples, which pose a threat to human health. However, the source and route of PA contamination in ...

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1.7K Views.  Anhui Agricultural University. The present protocol describes the contamination of pyrrolizidine alkaloids (PAs) in tea samples from PA-producing weeds in tea gardens.

Video: Source and Route of Pyrrolizidine Alkaloid Contamination in Tea Samples

Tracking Microbial Contamination in Retail Environments Using Fluorescent Powder - A Retail Delicatessen Environment Example

Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne illness. Ready-to-eat (RTE) processed foods such as deli meats, cheese, and in some cases fresh produce, have been involved in foodborne disease outbreaks due to contamination with pathogens such as Listeria monocytogenes. With respect to L. monocytogenes, deli slicers are often the main source of cross contamination. The goal of this study was to use a fluorescent compound to simulate bacterial contamination and track this contamination in a retail setting. A mock deli kitchen was designed to simulate the retail environment. Deli meat was inoculated with the fluorescent compound and volunteers were recruited to complete a set of tasks similar to those expected of a food retail employee. The volunteers were instructed to slice, package, and store the meat in a deli refrigerator. The potential cross contamination was tracked in the mock retail environment by swabbing specific areas and measuring the optical density of the swabbed area with a spectrophotometer. The results indicated that the refrigerator (i.e. deli case) grip and various areas on the slicer had the highest risk for cross contamination. The results of this study may be used to develop more focused training material for retail employees. In addition, similar methodologies could also be used to track microbial contamination in food production environments (e.g.&#160;small farms), hospitals, nursing homes, cruise ships, and hotels.

The overall goal of this study was to demonstrate the potential cross contamination mechanism of foodborne pathogen Listeria monocytogenes in a retail deli setting. This methodology may be applied to a variety of different environments to track pathogen contamination. &#160;

Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes &#8804;4 L or &gt;4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m3) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

We describe a protocol for filtration of water samples with a filter cartridge and extraction of environmental DNA (eDNA) without having to cut open the housing to remove the filter. This protocol is developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has relevant health and economic consequences. Many cyanobacterial strains are toxin producers, and only a few cells are necessary to induce irreparable damage to the environment. Therefore, water-body authorities and administrations require rapid and efficient early-warning systems providing reliable data to support their preventive or curative decisions. This manuscript reports an experimental protocol for the in-field detection of toxin-producing cyanobacterial strains by using an antibody microarray chip with 17 antibodies (Abs) with taxonomic resolution (CYANOCHIP). Here, a multiplex fluorescent sandwich microarray immunoassay (FSMI) for the simultaneous monitoring of 17 cyanobacterial strains frequently found blooming in freshwater ecosystems, some of them toxin producers, is described. A microarray with multiple identical replicates (up to 24) of the CYANOCHIP was printed onto a single microscope slide to simultaneously test a similar number of samples. Liquid samples can be tested either by direct incubation with the antibodies (Abs) or after cell concentration by filtration through a 1- to 3-&#956;m filter. Solid samples, such as sediments and ground rocks, are first homogenized and dispersed by a hand-held ultrasonicator in an incubation buffer. They are then filtered (5 - 20 &#956;m) to remove the coarse material, and the filtrate is incubated with Abs. Immunoreactions are revealed by a final incubation with a mixture of the 17 fluorescence-labeled Abs and are read by a portable fluorescence detector. The whole process takes around 3 h, most of it corresponding to two 1-h periods of incubation. The output is an image, where bright spots correspond to the positive detection of cyanobacterial markers.

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has ...

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial communities, a relatively precise but effort-intensive technique to assay microbial community composition is phospholipid fatty acid (PLFA) analysis. PLFA was developed to analyze phospholipid biomarkers, which can be used as indicators of microbial biomass and the composition of broad functional groups of fungi and bacteria. It has commonly been used to compare soils under alternative plant communities, ecology, and management regimes. The PLFA method has been shown to be sensitive to detecting shifts in microbial community composition.
An alternative method, fatty acid methyl ester extraction and analysis (MIDI-FA) was developed for rapid extraction of total lipids, without separation of the phospholipid fraction, from pure cultures as a microbial identification technique. This method is rapid but is less suited for soil samples because it lacks an initial step separating soil particles and begins instead with a saponification reaction that likely produces artifacts from the background organic matter in the soil. 
This article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples. The method combines the accuracy achieved through PLFA profiling by extracting and concentrating soil lipids as a first step, and a reduction in effort by saponifying the organic material extracted and processing with the MIDI-FA method as a second step.

The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples.

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial ...

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several limitations that have resulted in the exclusion of many species. Advances in the field over the last two decades have led occupational health researchers to turn to molecular-based approaches for identifying fungal hazards. These methods have resulted in the detection of many species within indoor and occupational environments that have not been detected using traditional methods. This protocol details an approach for determining fungal diversity within air samples through genomic DNA extraction, amplification, sequencing, and taxonomic identification of fungal internal transcribed spacer (ITS) regions. ITS sequencing results in the detection of many fungal species that are either not detected or difficult to identify to species level using culture or microscopy. While these methods do not provide quantitative measures of fungal burden, they offer a new approach to hazard identification and can be used to determine overall species richness and diversity within an occupational environment.

Determining the fungal diversity within an environment is a method utilized in occupational health studies to identify health hazards. This protocol describes DNA extraction from occupational air samples for amplification and sequencing of fungal ITS regions. This approach detects many fungal species that can be overlooked by traditional assessment methods.

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several ...

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ocean temperatures and ocean acidification, corals are increasingly under threat. To tackle these challenges, advances in cell and molecular biology have proven to be crucial for diagnosing the health of corals. Modifying some of the techniques commonly used in human medicine could greatly improve researchers&#39; ability to treat and save corals. To address this, a protocol for in situ hybridization used primarily in human medicine and evolutionary developmental biology has been adapted for use in adult corals under stress.
The purpose of this method is to visualize the spatial expression of an RNA probe in adult coral tissue that has been embedded in paraffin and sectioned onto glass slides. This method focuses on removal of the paraffin and rehydration of the sample, pretreatment of the sample to ensure permeability of the sample, pre-hybridization incubation, hybridization of the RNA probe, and visualization of the RNA probe. This is a powerful method when using non-model organisms to discover where specific genes are expressed, and the protocol can be easily adapted for other non-model organisms. However, the method is limited in that it is primarily qualitative, because expression intensity can vary depending on the amount of time used during the visualization step and the concentration of the probe. Furthermore, patience is required, as this protocol can take up to 5 days (and in many cases, longer) depending on the probe being used. Finally, non-specific background staining is common, but this limitation can be overcome.

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

The goal of this protocol is to perform in situ hybridization on adult coral samples that have been embedded in paraffin and sectioned onto glass slides. This is a qualitative method used to visualize the spatial expression of an RNA anti-sense probe in paraffin-embedded tissues.

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ...

Choice and No-Choice Bioassays to Study the Pupation Preference and Emergence Success of Ectropis grisescens

Many insects live above the ground as larvae and adults and as pupate below the ground. Compared to the above-ground stages of their life cycles, less attention has been paid on how environmental factors affect these insects when they pupate within the soil. The tea looper, Ectropis grisescens Warren (Lepidoptera: Geometridae), is a severe pest of tea plants and has caused huge economic losses in South China. The protocols described here aim to investigate, through multiple-choice bioassays, whether mature last-instar E. grisescens larvae can discriminate soil variables such as the substrate type and moisture content, and determine, through no-choice bioassays, the impact of the substrate type and moisture content on pupation behaviors and the emergence success of E. grisescens. The results would enhance the understanding of the pupation ecology of E. grisescens and may bring insights into soil-management tactics for suppressing E. grisescens populations. In addition, these bioassays can be modified to study the influences of various factors on the pupation behaviors and survivorship of soil-pupating pests.

Choice and No-Choice Bioassays to Study the Pupation Preference and Emergence Success of Ectropis grisescens

Here, we present a protocol to investigate the pupation preference of mature larvae of Ectropis grisescens in response to soil factors (e.g., substrate type and moisture content) using choice bioassays. We also present a protocol of no-choice bioassays to determine the factors that affect the pupation behaviors and survivorship of E. grisescens.

Many insects live above the ground as larvae and adults and as pupate below the ground. Compared to the above-ground stages of their life cycles, less ...

BtM, a Low-cost Open-source Datalogger to Estimate the Water Content of Nonvascular Cryptogams

Communities of nonvascular cryptogams, such as mosses or lichens, are an important part of the Earth's biodiversity, contributing to the regulation of the carbon and nitrogen cycles in many ecosystems. Being poikilohydric organisms, they do not actively control their internal water content and need a humid environment to activate their metabolism. Therefore, studying water relationships of nonvascular cryptogams is crucial to understand both their diversity patterns and their functions in the ecosystems. We present the BtM datalogger, a low-cost open-source platform for the study of the water content of nonvascular cryptogams. The datalogger is designed to measure ambient temperature, humidity, and conductance from up to eight samples simultaneously. We provide a design for a printed circuit board (PCB), a detailed protocol to assemble the components, and the required source code. All this makes the assembly of the BtM datalogger accessible to any research group, even to those without previous specialized knowledge. Therefore, the design presented here has the potential to help popularize the use of this type of device among ecologists and field biologists.

We present a simple and cost-effective method to build an open-source datalogger that measures the conductance of nonvascular cryptogams together with the environmental temperature and humidity. We describe the hardware design of the datalogger and provide step-by-step assembly instructions, the list of required open-source logging software, the code to run the datalogger, and a calibration protocol.

Communities of nonvascular cryptogams, such as mosses or lichens, are an important part of the Earth's biodiversity, contributing to the regulation of the ...

Measuring the Shape and Size of Activated Sludge Particles Immobilized in Agar with an Open Source Software Pipeline

Experimental bioreactors, such as those treating wastewater, contain particles whose size and shape are important parameters. For example, the size and shape of activated sludge flocs can indicate the conditions at the microscale, and also directly affect how well the sludge settles in a clarifier.
Particle size and shape are both misleadingly &#39;simple&#39; measurements. Many subtle issues, often unaddressed in informal protocols, can arise when sampling, imaging, and analyzing particles. Sampling methods may be biased or not provide enough statistical power. The samples themselves may be poorly preserved or undergo alteration during immobilization. Images may not be of sufficient quality; overlapping particles, depth of field, magnification level, and various noise can all produce poor results. Poorly specified analysis can introduce bias, such as that produced by manual image thresholding and segmentation.
Affordability and throughput are desirable alongside reproducibility. An affordable, high throughput method can enable more frequent particle measurement, producing many images containing thousands of particles. A method that uses inexpensive reagents, a common dissecting microscope, and freely-available open source analysis software allows repeatable, accessible, reproducible, and partially-automated experimental results. Further, the product of such a method can be well-formatted, well-defined, and easily understood by data analysis software, easing both within-lab analyses and data sharing between labs.
We present a protocol that details the steps needed to produce such a product, including: sampling, sample preparation and immobilization in agar, digital image acquisition, digital image analysis, and examples of experiment-specific figure generation from the analysis results. We have also included an open-source data analysis pipeline to support this protocol.

The size and shape of particles in activated sludge are important parameters that are measured using varying methods. Inaccuracies arise from non-representative sampling, suboptimal images, and subjective analysis parameters. To minimize these errors and ease measurement, we present a protocol specifying every step, including an open source software pipeline.

Experimental bioreactors, such as those treating wastewater, contain particles whose size and shape are important parameters. For example, the size and ...