This work was partially supported by financial grants from the Department of Science and Technology-Promotion of University Research and Scientific Excellence (DST-PURSE) Scheme of the University of Mumbai. Devika Ghadigaonkar worked as a Project Fellow under the scheme. The technical help provided by Harshali Shinde, Senior Research Fellow under the Department of Science and Technology-Science and Engineering Research Board (DST-SERB) Project no: CRG/2018/003624, is acknowledged.

1. Sample collection and processing
<ol>
	<li>Sample collection
	<ol>
		<li>Collect the appropriate volume of the water sample in sterile sample container(s), ensuring that not more than 3/4 of the container is filled.</li>
		<li>Transport the samples to the laboratory under aseptic conditions as soon as possible after collection and immediately process them.</li>
	</ol>
	</li>
	<li>Sample processing
	<ol>
		<li>Aseptically filter the water sample through a sterile muslin cloth to remove any particulate matter.</li>
		<li>Carry out appropriate serial dilutions of the filtered water for further analysis.</li>
	</ol>
	</li>
</ol>
2. Estimation of the total bacterial load and the antibiotic-resistant bacteria count
<ol>
	<li>Determination of the total bacterial load
	<ol>
		<li>Suspend 18.12 g of R2A Agar, Modified powder in 1,000 mL of double-distilled water, and dissolve the mixture by heating. Autoclave the dissolved mixture at 121 &#176;C, 15 psi for 20 min. Prepare R2A Agar, Modified plates by pouring the appropriate quantity of the autoclaved mixture into sterile Petri plates (e.g., add approximately 20 mL of autoclaved sterile medium to a 90 mm sterile Petri plate).</li>
		<li>Evenly spread 100 &#181;L of the appropriate dilutions of the filtered water sample on the R2A Agar, Modified plate once the medium solidifies. Perform the experiment in duplicate.</li>
		<li>Incubate all the above plates at 35-37 &#176;C for 48 h (vary the temperature and incubation time depending upon the media used for isolation).</li>
		<li>Express the total bacterial load in terms of colony-forming units per milliliter (CFU/mL) using equation (1): 
		<img alt="Equation 1" src="/files/ftp_upload/63934/63934eq01.jpg" style="margin: auto;" />&#160; &#160;(1)</li>
	</ol>
	</li>
	<li>Determination of the AR bacterial count
	<ol>
		<li>Follow steps 2.1.1-2.1.4. However, instead of R2A Agar, Modified plates, use R2A Agar, Modified plates supplemented individually with five different antibiotics, namely cefotaxime (3 &#181;g/mL), ciprofloxacin (0.5 &#181;g/mL), erythromycin (20 &#181;g/mL), kanamycin (15 &#181;g/mL), and vancomycin (3 &#181;g/mL).</li>
		<li>Add the antibiotics separately into tubes containing 20 mL of sterile molten R2A Agar, Modified (with the temperature of the molten R2A Agar, Modified at &#8804;40 &#176;C) to achieve the final antibiotic concentration as mentioned in step 2.2.1.</li>
		<li>Swirl for even mixing and pour onto sterile Petri plates before the agar solidifies. Perform the experiment in duplicate.</li>
		<li>Incubate all the above plates at 35-37 &#176;C for 48 h (if using a different media, the temperature and incubation time may vary).</li>
		<li>For quality control and checking the efficacy of the antibiotics, spread 100 &#181;L of bacterial suspensions of the Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 strains onto their respective antibiotic-containing R2A Agar, Modified plates (ensure that the density of the fresh culture used for the inoculation is OD = 0.5 at 600 nm).</li>
		<li>Determine the antibiotic-resistant bacterial count in terms of CFU/mL as described in step 2.1.4.</li>
	</ol>
	</li>
	<li>Glycerol stocks of the isolates
	<ol>
		<li>Select morphologically distinct AR colonies.</li>
		<li>Suspend a single isolated colony in 2 mL of sterile Luria-Bertani broth containing the respective antibiotic (e.g., if a colony was selected from a plate containing cefotaxime, inoculate the colony from step 2.3.1 in sterile Luria-Bertani broth containing cefotaxime at its respective concentration).</li>
		<li>Incubate the inoculated tubes at 37 &#176;C at 80 rpm until the OD600 reaches 0.5.</li>
		<li>Prepare glycerol stocks of the isolates by mixing 750 &#181;L of the culture suspension from step 2.3.3 into 250 &#181;L of sterile 100% glycerol under aseptic conditions.</li>
		<li>Store the glycerol stocks at &#8722;80 &#176;C until further analysis. 
		NOTE: For the revival of the cultures from the glycerol stocks, thaw the glycerol stocks at 4 &#176;C. Inoculate a loopful of this stock into 2 mL of sterile Luria-Bertani broth containing the respective antibiotic and allow to grow.</li>
	</ol>
	</li>
</ol>
3. Identification of culturable bacteria by 16S rRNA gene sequencing
<ol>
	<li>Preparation of DNA template from the isolates for PCR 
	NOTE: The protocol described for the preparation of DNA template for PCR for the isolation of crude DNA from the bacteria is given by Carlson et al.10.
	<ol>
		<li>Using a sterile toothpick, take a single, isolated, pure colony of the isolate growing on a Petri plate. Suspend the bacterial colony in 100 &#181;L of sterile double-distilled water in a sterile microcentrifuge tube and boil for 10 min.</li>
		<li>Centrifuge the suspension at 10,000 &#215; g for 2 min to pellet the debris, and transfer the supernatant to a fresh sterile microcentrifuge tube for use as the crude DNA template.</li>
	</ol>
	</li>
	<li>Targeted PCR amplification of the V3 region of the 16S rRNA gene and sequencing
	<ol>
		<li>Prepare 40 &#181;L of the reaction mixture in a PCR tube for PCR amplification, as mentioned in Table 1. 
		NOTE: The DNA preparation should be carried out on an ice block while minimizing the chance of contamination (wear gloves while handling the reagents, and clean the work surface thoroughly with 70% ethanol).</li>
		<li>Place the tube in the thermal block, and run the appropriate program in the PCR thermal cycler. See Table 2 for the standardized PCR cycling conditions and the primer information for the amplification of the V3 regions of the 16S rRNA genes.</li>
		<li>For resolving the amplicons and visualization, carry out agarose gel electrophoresis (AGE). Mix 10 &#181;L of the amplified PCR product and 2 &#181;L of 6x gel loading buffer (Table 3), and load this mixture into wells on 1.5% agarose gel (dissolve 1.5 g of agarose powder in 100 mL of 1x TAE buffer [Table 3]) containing 5 &#181;L of 10 mg/mL ethidium bromide (EtBr) to a final concentration of 0.5 &#181;g/mL EtBr in 100 mL of the agarose gel. 
		CAUTION: EtBr is a potent carcinogen. Gloves should be worn at all times while handling EtBr and gels containing EtBr.</li>
		<li>Add DNA ladder for the estimation of the size of the amplicons.</li>
		<li>Carry out electrophoresis of the gel in a TAE tank buffer at 80-100 V.</li>
		<li>Once the tracking dye runs 3/4 of the gel, stop the electrophoresis, and visualize the amplicon bands under a UV transilluminator.</li>
		<li>Use the PCR product (amplicon) for 16S rRNA gene sequencing to identify the isolate.</li>
		<li>Quantify the amplicon by subjecting it to spectrophotometric analysis using equation (2). 
		<img alt="Equation 2" src="/files/ftp_upload/63934/63934eq02.jpg" style="margin: auto;" />&#160; &#160;(2)</li>
		<li>To check the purity of the DNA, calculate the ratio of A260/A280. 
		NOTE: Ideally, this number should be above 1.5 and, preferably, between 1.8 and 2.0.</li>
		<li>To identify the isolates, compare the sequences that are obtained with sequence databases using an appropriate alignment search tool.</li>
	</ol>
	</li>
</ol>
4. Detection of antibiotic resistance in the isolates using antibiotic susceptibility testing
NOTE: This protocol describes the method for antibiotic susceptibility testing (AST) by disc diffusion. The following antibiotic discs were used: cefotaxime (5 &#181;g), ampicillin (10 &#181;g), levofloxacin (5 &#181;g), chloramphenicol (30 &#181;g), tigecycline (15 &#181;g), ceftriaxone (30 &#181;g), imipenem (10 &#181;g), gentamicin (10 &#181;g), neomycin (10 &#181;g), trimethoprim (5 &#181;g), and ciprofloxacin (5 &#181;g).
<ol>
	<li>Preparation of the inoculum for the AST
	<ol>
		<li>Aseptically inoculate a single, isolated, purified AR colony using a sterile loop in 2 mL of a sterile non-selective medium, such as Luria-Bertani broth (without any antibiotic), and incubate at 37 &#176;C at 80 rpm overnight.</li>
		<li>Resuspend by taking 100-150 &#181;L of the overnight grown culture (approximately, OD600 = 1.8-2.0) in 2 mL of fresh non-selective Luria-Bertani broth medium and incubate for 2-4 h (until the OD600 reaches 0.4-0.5).</li>
		<li>Dilute this freshly grown culture suspension using sterile 0.85% saline solution such that the density of the culture is equal to 0.5 McFarland standard (approximately, OD600 = 0.1), which roughly corresponds to 1-2 &#215; 108 cells/mL.</li>
		<li>Gently mix the bacterial suspension for an even cell distribution.</li>
		<li>Use the above suspension within 15 min of dilution.</li>
	</ol>
	</li>
	<li>Inoculation of the agar plates
	<ol>
		<li>Prepare Mueller-Hinton Agar (MHA) plates for performing the AST by mixing 38 g of MHA in 1,000 mL of double-distilled water, and dissolve the mixture by heating. Autoclave the dissolved mixture at 121 &#176;C, 15 psi for 15 min.</li>
		<li>Ensure that the depth of MHA in the plates is 4 mm (25 mL of medium per plate).</li>
		<li>Simultaneously, remove the antibiotic discs from the freezer and warm them to room temperature. 
		NOTE: The antibiotic discs should be gradually thawed by initially thawing the discs at 4 &#176;C and later at room temperature to reduce any potential danger of condensation on the discs, which may subsequently affect the zone of inhibition (ZOI).</li>
		<li>Under aseptic conditions, dip a sterile cotton swab into the inoculum prepared in step 4.1, and remove excess suspension to avoid over-inoculation of the plates.</li>
		<li>Spread the culture evenly on the plates, starting from the top of the MHA plate and going back and forth from edge to edge. Rotate the plate by 60&#176; while swabbing.</li>
	</ol>
	</li>
	<li>Application of the antibiotic discs
	<ol>
		<li>With the help of flame-sterilized forceps, aseptically transfer the antibiotic discs onto the inoculated MHA plates, and press the discs gently to ensure complete level contact with the agar. 
		NOTE: This procedure has to be done within 15 min of the inoculation of the culture on the plates.</li>
		<li>Place the appropriate number of antibiotic discs on the agar plate by taking into account the organism, the antibiotic used, and the size of the plate to avoid overlapping of the zones of inhibition. 
		NOTE: Four to five discs can be accommodated on a 90 mm circular plate.</li>
	</ol>
	</li>
	<li>Incubation of the plates
	<ol>
		<li>Within 15 min of the application of the antibiotic discs, invert the plates and incubate at 37 &#176;C overnight.</li>
	</ol>
	</li>
	<li>Interpretation of the results
	<ol>
		<li>Measure the ZOI diameter in millimeters (mm), and interpret according to the breakpoint values given by EUCAST11. See the two examples given below.
		<ol>
			<li>The zone diameter breakpoint (mm) for a ciprofloxacin antibiotic disc (5 &#181;g) for Enterobacterales is S &#8805; 25 and R &#60; 22, which means that it is considered to be sensitive (S) if ZOI &#8805; 25 mm, while it is resistant (R) if ZOI &#60; 22 mm. If the ZOI diameter falls between 22 and 25, the isolate is considered to be intermediate (I).</li>
			<li>The zone diameter breakpoint (mm) for a chloramphenicol antibiotic disc (30 &#181;g) for Staphylococcus spp. is S &#8805; 18 and R &#60; 18, which means that it is considered to be sensitive if ZOI &#8805; 18 mm, while it is resistant if ZOI &#60; 18 mm.</li>
		</ol>
		</li>
		<li>Determine the multiple antibiotic resistance (MAR) index by finding the ratio of the number of antibiotics to which the isolate is resistant to the total number of antibiotics to which the isolate is exposed. 
		&#8203;NOTE: For quality control, E. coli ATCC 25922 and S. aureus ATCC 29213 are used as reference strains following the protocol as in step 4.</li>
	</ol>
	</li>
</ol>
5. PCR-based detection of antibiotic resistance genes in the isolates
<ol>
	<li>Use a standard PCR protocol for the identification of ARGs in the isolates. Prepare the DNA template using the protocol given in step 3.1. 
	NOTE: The PCR cycling conditions used in this study were 94 &#176;C for 10 min, followed by 35 cycles of 94 &#176;C for 30 s, annealing for 30 s at the appropriate temperature (as standardized for each primer set), extension at 72 &#176;C for 40 s, and a final extension at 72 &#176;C for 5 min. The reaction mixture is described in Table 4. The list of ARGs, primers, and annealing temperatures is given in Table 5.</li>
	<li>To resolve, visualize, and check the purity of the amplicons, follow steps 3.2.3-3.2.10.</li>
</ol>
6. Whole metagenomic DNA analysis for the identification of the total bacterial diversity and the detection of ARGs in the metagenome
<ol>
	<li>Extraction of the total DNA (metagenome) from the water sample
	<ol>
		<li>Extract the metagenomic DNA from the filtered water samples. 
		NOTE: In the current study, the metagenomic DNA (total DNA) was extracted from the filtered water samples using the referenced DNA isolation kit following the manufacturer&#39;s protocol (see the Table of Materials).</li>
		<li>Check the quality of the DNA by loading 3 &#181;L of the extracted metagenomic DNA onto a 0.8% agarose gel, and run the gel at 80-110 V for approximately 30 min.</li>
		<li>Check for the presence of a single intact band.</li>
		<li>Check the DNA concentration using a fluorometer.</li>
	</ol>
	</li>
	<li>Determination of bacterial diversity and detection of ARGs using whole metagenomic DNA sequencing
	<ol>
		<li>Library preparation and PCR amplification:
		<ol>
			<li>Prepare a paired-end sequencing library using the referenced DNA library prep kit (see the Table of Materials).</li>
			<li>Ready the DNA for adapter ligation by taking 200 ng of DNA and mechanically shearing it into smaller fragments, followed by a continuous step of end-repair in which an &#34;A&#34; is added to the 3&#39; ends.</li>
			<li>Depending on the platform used for sequencing, ligate specific adapters to both ends of the DNA fragments. 
			NOTE: Sequences crucial for binding dual-barcoded libraries to a flow cell for sequencing are present in these adapters. This allows for PCR amplification of the adapter-ligated fragments and binding the standard sequencing primers.</li>
			<li>To check the quality and quantity, analyze the amplified library using a high-sensitivity DNA chip as per the manufacturer&#39;s instructions.</li>
		</ol>
		</li>
		<li>Cluster generation and sequencing:
		<ol>
			<li>Load the amplified library onto the appropriate sequencing platform for cluster generation and subsequent sequencing. 
			NOTE: The library molecules bind to the complementary adapter oligos on the paired-end flow cell. During sequencing, the forward strands are selectively cleaved after the resynthesis of the reverse strand. This copied reverse strand is then sequenced from the opposite end of the fragment.</li>
		</ol>
		</li>
		<li>Bioinformatic analysis:
		<ol>
			<li>Generate scaffolds from the high-quality data using the appropriate platform.</li>
			<li>Subject these scaffolds to bioinformatics analysis for the taxonomic classification and identification of the ARGs. 
			NOTE: The workflow for the whole metagenomic DNA analysis for the identification of the total bacterial diversity and the detection of ARGs in the metagenome is given in Figure 1. A flowsheet of the complete methodology described in the manuscript is given in Figure 2.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors have no conflicting interests to disclose.

The sample collection and processing play a significant role and might affect the results and interpretation of the study. Hence, to rule out variability in the samples, it is important to carry out sampling at multiple locations of the freshwater body being studied. Maintaining proper aseptic environmental conditions when handling such samples can prevent contamination. Moreover, to prevent changes in the bacterial composition that may influence the quality and quantity of extracted nucleic acids, the transit conditions...

Antimicrobial resistance (AMR) has been identified as one of the most pressing global problems. The rapid evolution of AMR and its worldwide spread are one of the greatest threats to human health and the global economy in terms of the health costs associated with it1. The overuse and misuse of antibiotics have led to an increase in AR. This has been highlighted by the COVID-19 pandemic, during which the treatment of associated secondary infections, in many cases, was hugely compromised due to AMR in the affected patients2. Besides the direct use/misuse of antibiotics by humans, the overuse and misuse of antibiotics in agriculture and animal husbandry and their inappropriate discharge into the environment, including water bodies, are a major concern3. The rise of new resistance traits and multidrug resistance in bacteria urgently highlights the need for a better understanding of the factors leading to the development of AR and its dissemination. Multiple antibiotic-resistant bacteria, which often carry multiple AR genes (ARGs) on mobile genetic elements such as plasmids, can transfer these resistance genes to non-resistant microorganisms, including potential human pathogens, thus leading to the emergence of superbugs that are untreatable with even last-resort antibiotics4. These multiple antibiotic-resistant bacteria, if present in water ecosystems, can directly enter the human gut via the consumption of contaminated water-based foods such as fish, crabs, and mollusks. Previous studies have shown that the spread of AR bacteria in naturally occurring water systems can also reach other water supplies, including drinking water, and, thus, can enter the human food chain5,6,7.
The aim of the present study is to provide a comprehensive protocol using a combination of culture-based and non-culture-based (whole metagenomic analysis) techniques to obtain complete information about the total bacterial diversity and the total pool of different ARGs present in a water body in Mumbai, India. Conventionally, culture-based techniques have been used to study the bacterial diversity in water bodies. As culturable microorganisms constitute only a small percentage of the total microbiota in any niche, to have a better understanding of the overall status of bacterial diversity and the various resistant traits prevalent in any sample, various culture-based and culture-independent techniques must be used in tandem. One such robust and reliable culture-independent technique is whole metagenomic DNA analysis. This high-throughput method has been successfully utilized in various studies on bacterial diversity or the functional annotations of various ARGs8,9. This technique uses the metagenome (the total genetic material in a sample) as the starting material for various analyses and, hence, is culture-independent. The protocols in the present study can be used for whole metagenomic DNA analysis to obtain information about the total bacterial diversity and various ARGs (resistome) in water samples.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 bp DNA ladder</td>
			<td>Himedia</td>
			<td>MBT049-50LN</td>
			<td>For estimation of size of the amplicons</td>
		</tr>
		<tr>
			<td>2x PCR Taq mastermix</td>
			<td>HiMedia</td>
			<td>MBT061-50R</td>
			<td>For making PCR reaction mixture</td>
		</tr>
		<tr>
			<td>37 &#176;C Incubator</td>
			<td>GS-192, Gayatri Scientific</td>
			<td>NA</td>
			<td>For incubation of bacteria</td>
		</tr>
		<tr>
			<td>6x Gel Loading Buffer</td>
			<td>HiMedia</td>
			<td>ML015-1ML</td>
			<td>Loading and Tracking dye which helps to weigh down the DNA sample and track the progress of electrophoresis</td>
		</tr>
		<tr>
			<td>Agarose powder</td>
			<td>Himedia</td>
			<td>MB229-50G</td>
			<td>For resolving amplicons during Agarose Gel Electrophoresis (AGE)</td>
		</tr>
		<tr>
			<td>Ampicillin antibiotic disc</td>
			<td>HiMedia</td>
			<td>SD002</td>
			<td>For performing AST</td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>Equitron</td>
			<td>NA</td>
			<td>Required for sterilization of media, glass plates, test tubes, etc</td>
		</tr>
		<tr>
			<td>Bioanalyzer 2100</td>
			<td>Agilent Technologies</td>
			<td>NA</td>
			<td>To check the quality and quantity of the amplified library</td>
		</tr>
		<tr>
			<td>Bisafety B2 Cabinet</td>
			<td>IMSET</td>
			<td>IMSET BSC-Class II Type B2</td>
			<td>Used for microbiological work like bacterial culturing, AST etc.</td>
		</tr>
		<tr>
			<td>Cefotaxime antibiotic disc</td>
			<td>HiMedia</td>
			<td>SD295E-5VL</td>
			<td>For performing AST</td>
		</tr>
		<tr>
			<td>Cefotaxime antibiotic powder</td>
			<td>HiMedia</td>
			<td>TC352-5G</td>
			<td>For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria</td>
		</tr>
		<tr>
			<td>Ceftriaxone antibiotic disc</td>
			<td>HiMedia</td>
			<td>SD065</td>
			<td>For performing AST</td>
		</tr>
		<tr>
			<td>Centrifuge Minispin</td>
			<td>Eppendorf</td>
			<td>Minispin Plus-5453</td>
			<td>Used to pellet the debris during crude DNA preparation</td>
		</tr>
		<tr>
			<td>Chloramphenicol antibiotic disc</td>
			<td>HiMedia</td>
			<td>SD006-5x50DS</td>
			<td>For performing AST</td>
		</tr>
		<tr>
			<td>Ciprofloxacin antibiotic disc</td>
			<td>HiMedia</td>
			<td>SD060-5x50DS</td>
			<td>For performing AST</td>
		</tr>
		<tr>
			<td>Ciprofloxacin antibiotic powder</td>
			<td>HiMedia</td>
			<td>TC447-5G</td>
			<td>For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria</td>
		</tr>
		<tr>
			<td>Colorimeter</td>
			<td>Quest</td>
			<td>NA</td>
			<td>For checking the OD of culture suspensions</td>
		</tr>
		<tr>
			<td>Comprehensive Antibiotic Resistance Database (CARD) database</td>
			<td></td>
			<td></td>
			<td>functional annotation of ARGs; https://card.mcmaster.ca/</td>
		</tr>
		<tr>
			<td>Cooling Shaker Incubator</td>
			<td>BTL41 Allied Scientific</td>
			<td>NA</td>
			<td>For incubation of media plates for culturing bacteria</td>
		</tr>
		<tr>
			<td>Deep Freezer (-40 &#176;C)&#160;</td>
			<td>Haier</td>
			<td>DW40L, Haier Biomedicals</td>
			<td>For storage of glycerol stocks</td>
		</tr>
		<tr>
			<td>DNA Library Prep Kit</td>
			<td>NEB Next Ultra DNA Library Prep Kit for Illumina</td>
			<td>NA</td>
			<td>Paired-end sequencing library preparation</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>HiMedia</td>
			<td>GRM1195-100G</td>
			<td>For preparation of Gel running buffer for Agarose Gel Electrophoresis (AGE)</td>
		</tr>
		<tr>
			<td>Electrophoresis Apparatus</td>
			<td>TechResource</td>
			<td>15 cm gel casting tray</td>
			<td>For making the agarose gel&#160; and carrying out electrophoresis&#160;</td>
		</tr>
		<tr>
			<td>Electrophoresis Power pack with electrodes</td>
			<td>Genei</td>
			<td>NA</td>
			<td>For running the AGE&#160;</td>
		</tr>
		<tr>
			<td>Erythromycin antibiotic disc</td>
			<td>HiMedia</td>
			<td>SD222-5VL</td>
			<td>For performing AST</td>
		</tr>
		<tr>
			<td>Erythromycin antibiotic powder</td>
			<td>HiMedia</td>
			<td>CMS528-1G</td>
			<td>For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria</td>
		</tr>
		<tr>
			<td>Erythromycin antibiotic powder</td>
			<td>HiMedia</td>
			<td>TC024-5G</td>
			<td>For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria</td>
		</tr>
		<tr>
			<td>Escherichia coli ATCC 25922&#160;&#160;&#160;&#160;</td>
			<td>HiMedia</td>
			<td>0335X-1</td>
			<td>Used as a control while performing AST</td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>HiMedia</td>
			<td>MB071-1G</td>
			<td>Intercalating agent and visualizaion of DNA after electrophoresis under Gel Documentation System</td>
		</tr>
		<tr>
			<td>Fluorometer</td>
			<td>Qubit 2.0</td>
			<td>NA</td>
			<td>For determining concentration of extracted metagenomic DNA</td>
		</tr>
		<tr>
			<td>Gel Documentation System</td>
			<td>BioRad</td>
			<td></td>
			<td>Used for visualizing PCR amplicons after electrophoresis</td>
		</tr>
		<tr>
			<td>Gentamicin antibiotic disc</td>
			<td>HiMedia</td>
			<td>SD170-5x50DS</td>
			<td>For performing AST</td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>HiMedia</td>
			<td>AS119-500ML</td>
			<td>For preparation of Gel running buffer for Agarose Gel Electrophoresis (AGE)</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>HiMedia</td>
			<td>GRM1027-500ML</td>
			<td>For making glycerol stocks</td>
		</tr>
		<tr>
			<td>Imipenem antibiotic disc</td>
			<td>HiMedia</td>
			<td>SD073</td>
			<td>For performing AST</td>
		</tr>
		<tr>
			<td>Kaiju Database</td>
			<td>NA</td>
			<td>NA</td>
			<td>For taxonomical classification of reads; https://kaiju.binf.ku.dk/</td>
		</tr>
		<tr>
			<td>Kanamycin antibiotic disc</td>
			<td>HiMedia</td>
			<td>SD017-5x50DS</td>
			<td>For performing AST</td>
		</tr>
		<tr>
			<td>Kanamycin antibiotic powder</td>
			<td>HiMedia</td>
			<td>MB105-5G</td>
			<td>For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria</td>
		</tr>
		<tr>
			<td>Levofloxacin antibiotic disc</td>
			<td>HiMedia</td>
			<td>SD216-5VL</td>
			<td>For performing AST</td>
		</tr>
		<tr>
			<td>Luria Bertani broth</td>
			<td>Himedia</td>
			<td>M1245-500G</td>
			<td>For enrichment of cultures</td>
		</tr>
		<tr>
			<td>McFarland Standards</td>
			<td>Himedia</td>
			<td>R092-1No</td>
			<td>To compare density of culture suspension</td>
		</tr>
		<tr>
			<td>Molecular Biology water</td>
			<td>HiMedia</td>
			<td>TCL018-500ML</td>
			<td>For making PCR reaction mixture</td>
		</tr>
		<tr>
			<td>Mueller-Hinton Agar (MHA)&#160;</td>
			<td>HiMedia</td>
			<td>M173-500G</td>
			<td>For performing Antibiotc Susceptibility Testing (AST)</td>
		</tr>
		<tr>
			<td>Neomycin antibiotic disc</td>
			<td>HiMedia</td>
			<td>SD731-5x50DS</td>
			<td>For performing AST</td>
		</tr>
		<tr>
			<td>PCR Gradient Thermal Cycler</td>
			<td>Eppendorf</td>
			<td>Mastercycler Nexus Gradient 230V/50-60 Hz&#160;</td>
			<td>Used for performing PCR for amplification of 16S rRNA region and various Antibiotic Resistance genes</td>
		</tr>
		<tr>
			<td>Primers&#160;</td>
			<td>Xcelris</td>
			<td>NA</td>
			<td>For PCR amplication&#160;</td>
		</tr>
		<tr>
			<td>R2A Agar, Modified</td>
			<td>HiMedia</td>
			<td>M1743</td>
			<td>For preparation of media plates for isolation of total and antibiotic resistant (AR) bacterial load</td>
		</tr>
		<tr>
			<td>Scaffold generation</td>
			<td>CLC Genomics Workbench 6.0</td>
			<td>NA</td>
			<td>For generation of scaffolds</td>
		</tr>
		<tr>
			<td>Sequencer</td>
			<td>Illumina platform (2 x 150 bp chemistry)</td>
			<td>NA</td>
			<td>Sequencing of amplified library</td>
		</tr>
		<tr>
			<td>Sodium Chloride&#160;</td>
			<td>HiMedia</td>
			<td>TC046-500G</td>
			<td>For preparation of 0.85% saline for serially diluting the water sample</td>
		</tr>
		<tr>
			<td>Soil DNA isolation Kit</td>
			<td>Xcelgen</td>
			<td>NA</td>
			<td>For extraction of whole metagenomic DNA from the filtered water sample&#160;</td>
		</tr>
		<tr>
			<td>Staphylococcus aureus subsp. aureus ATCC 29213</td>
			<td>HiMedia</td>
			<td>0365P</td>
			<td>Used as a control while performing AST</td>
		</tr>
		<tr>
			<td>Taxonomical Classification</td>
			<td>Kaiju ioinformatics tool</td>
			<td>NA</td>
			<td>For classification of reads into different taxonomic groups from phylum to genus level&#160;</td>
		</tr>
		<tr>
			<td>The Comprehensive Antibiotic Resistance Database (CARD)</td>
			<td>NA</td>
			<td>NA</td>
			<td>For functional annotation of ARGs</td>
		</tr>
		<tr>
			<td>Tigecycline antibiotic disc</td>
			<td>HiMedia</td>
			<td>SD278</td>
			<td>For performing AST</td>
		</tr>
		<tr>
			<td>Trimethoprim antibiotic disc</td>
			<td>HiMedia</td>
			<td>SD039-5x50DS</td>
			<td>For performing AST</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>HiMedia</td>
			<td>TC072-500G</td>
			<td>For preparation of Gel running buffer for Agarose Gel Electrophoresis (AGE)</td>
		</tr>
		<tr>
			<td>Vancomycin antibiotic powder</td>
			<td>HiMedia</td>
			<td>CMS217</td>
			<td>For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria</td>
		</tr>
		<tr>
			<td>Weighing Balance</td>
			<td>Mettler Toledo</td>
			<td>ME204 Mettler Toledo</td>
			<td>Used for weighing media powders, reagent powders etc.</td>
		</tr>
		<tr>
			<td>NA - Not Applicable</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and identification of waterborne antibiotic-resistant bacteria and molecular characterization of their antibiotic resistance genes

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the present study, freshwater samples were collected and analyzed with respect to the total bacterial diversity and AR genes (ARGs) using both conventional culture-based techniques and a high-throughput culture-independent metagenomic approach. This paper presents a systematic protocol for the enumeration of the total and antibiotic-resistant culturable bacteria from freshwater samples and the determination of phenotypic and genotypic resistance in the culturable isolates. Further, we report the use of whole metagenomic analysis of the total metagenomic DNA extracted from the freshwater sample for the identification of the overall bacterial diversity, including non-culturable bacteria, and the identification of the total pool of different ARGs (resistome) in the water body. Following these detailed protocols, we observed a high antibiotic-resistant bacteria load in the range of 9.6 &#215; 105-1.2 &#215; 109 CFU/mL. Most isolates were resistant to the multiple tested antibiotics, including cefotaxime, ampicillin, levofloxacin, chloramphenicol, ceftriaxone, gentamicin, neomycin, trimethoprim, and ciprofloxacin, with multiple antibiotic resistance (MAR) indexes of &#8805;0.2, indicating high levels of resistance in the isolates. The 16S rRNA sequencing identified potential human pathogens, such as Klebsiella pneumoniae,&#160;and opportunistic bacteria, such as Comamonas spp., Micrococcus spp., Arthrobacter spp., and Aeromonas spp. The molecular characterization of the isolates showed the presence of various ARGs, such as blaTEM, blaCTX-M (&#946;-lactams), aadA, aac (6&#39;)-Ib (aminoglycosides), and dfr1 (trimethoprims), which was also confirmed by the whole metagenomic DNA analysis. A high prevalence of other ARGs encoding for antibiotic efflux pumps-mtrA, macB, mdtA, acrD, &#946;-lactamases-SMB-1, VIM-20, ccrA, ampC, blaZ, the&#160;chloramphenicol acetyltransferase gene catB10, and&#160;the rifampicin resistance gene rphB-was also detected in the metagenomic DNA. With the help of the protocols discussed in this study, we confirmed the presence of waterborne MAR bacteria with diverse AR phenotypic and genotypic traits. Thus, whole metagenomic DNA analysis can be used as a complementary technique to conventional culture-based techniques to determine the overall AR status of a water body.

Total bacterial load and antibiotic-resistant (AR) bacteria count 
The enumeration of the total bacterial load was carried out by spreading 10&#8722;4 to 10&#8722;6 fold dilutions of the water samples on R2A Agar, Modified medium. For the enumeration of the AR bacteria count, 10&#8722;3 to 10&#8722;6 fold dilutions were spread on media plates containing antibiotics (Figure 3). The total and AR bacteria counts were calculated a...

Watch this Scientific Journal Video about Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes at JoVE.com

Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes

Here, we present a detailed protocol for the isolation and identification of antibiotic-resistant bacteria from water and the molecular characterization of their antibiotic resistance genes (ARGs). The use of culture-based and non-culture-based (metagenomic analysis) techniques provides complete information about the total bacterial diversity and the total pool of different ARGs present in freshwaters from Mumbai, India.

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the ...

isolation-identification-waterborne-antibiotic-resistant-bacteria

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6.1K Views.  University of Mumbai. Here, we present a detailed protocol for the isolation and identification of antibiotic-resistant bacteria from water and the molecular characterization of their antibiotic resistance genes (ARGs). The use of culture-based and non-culture-based (metagenomic analysis) techniques provides complete information about the total bacterial diversity and the total pool of different ARGs present in freshwaters from Mumbai, India.

Video: Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes

Helminth Collection and Identification from Wildlife

Wild animals are commonly parasitized by a wide range of helminths. The four major types of helminths are &#34;roundworms&#34; (nematodes), &#34;thorny-headed worms&#34; (acanthocephalans), &#34;flukes&#34; (trematodes), and &#34;tapeworms&#34; (cestodes). The optimum method for collecting helminths is to examine a host that has been dead less than 4-6 hr since most helminths will still be alive. A thorough necropsy should be conducted and all major organs examined. Organs are washed over a 106 &#956;m sieve under running water and contents examined under a stereo microscope. All helminths are counted and a representative number are fixed (either in 70% ethanol, 10% buffered formalin, or alcohol-formalin-acetic acid). For species identification, helminths are either cleared in lactophenol (nematodes and small acanthocephalans) or stained (trematodes, cestodes, and large acanthocephalans) using Harris&#39; hematoxylin or Semichon&#39;s carmine. Helminths are keyed to species by examining different structures (e.g. male spicules in nematodes or the rostellum in cestodes). The protocols outlined here can be applied to any vertebrate animal. They require some expertise on recognizing the different organs and being able to differentiate helminths from other tissue debris or gut contents. Collection, preservation, and staining are straightforward techniques that require minimal equipment and reagents. Taxonomic identification, especially to species, can be very time consuming and might require the submission of specimens to an expert or DNA analysis.

Wild animals are commonly parasitized by a wide range of helminths.&#160; The four major types of helminths are &#8220;roundworms&#8221; (nematodes), &#8220;thorny-headed worms&#8221; (acanthocephalans), &#8220;flukes&#8221; (trematodes), and &#8220;tapeworms&#8221; (cestodes).&#160; Here we describe how helminths are collected from a vertebrate animal and how they are preserved and taxonomically identified.&#160;

Wild animals are commonly parasitized by a wide range of helminths. The four major types of helminths are &#34;roundworms&#34; (nematodes), ...

Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae, Heterorhabditidae)

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to two families: Steinernematidae and Heterorhabditidae. Until now, more than 70 species have been described in the Steinernematidae and there are about 20 species in the Heterorhabditidae. The nematodes have a mutualistic partnership with Enterobacteriaceae bacteria and together they act as a potent insecticidal complex that kills a wide range of insect species.
Herein, we focus on the most common techniques considered for collecting EPN from soil. The second part of this presentation focuses on the insect-baiting technique, a widely used approach for the isolation of EPN from soil samples, and the modified White trap technique which is used for the recovery of these nematodes from infected insects. These methods and techniques are key steps for the successful establishment of EPN cultures in the laboratory and also form the basis for other bioassays that consider these nematodes as model organisms for research in other biological disciplines. The techniques shown in this presentation correspond to those performed and/or designed by members of S. P. Stock laboratory as well as those described by various authors.

Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae

Entomopathogenic nematodes are soil-inhabiting roundworms that parasitize a wide range of insects. We demonstrate sampling methods used for the isolation of these nematodes from the soil using two techniques: the insect baiting and the modified White trap, for the recovery of nematodes from soil samples and infected insect cadavers, respectively.

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to ...

Protocols for Robust Herbicide Resistance Testing in Different Weed Species

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented protocols, based on whole-plant bioassays performed in a greenhouse, can be readily adapted to a wide range of weed species and herbicides through appropriate variants. Seed samples from plants that survived a field herbicide treatment are collected and stored dry at low temperature until used. Germination methods differ according to weed species and seed dormancy type. Seedlings at similar growth stage are transplanted and maintained in the greenhouse under appropriate conditions until plants have reached the right growth stage for herbicide treatment. Accuracy is required to prepare the herbicide solution to avoid unverifiable mistakes. Other critical steps such as the application volume and spray speed are also evaluated. The advantages of this protocol, compared to others based on whole plant bioassays using one herbicide dose, are related to the higher reliability and the possibility of inferring the resistance level. Quicker and less expensive in vivo or in vitro diagnostic screening tests have been proposed (Petri dish bioassays, spectrophotometric tests), but they provide only qualitative information and their widespread use is hindered by the laborious set-up that some species may require. For routine resistance testing, the proposed whole plant bioassay can be applied at only one herbicide dose, so reducing the costs.

A robust and flexible approach to confirm herbicide resistance in weed populations is presented. This protocol allows the herbicide resistance levels to be inferred and applied to a wide range of weed species and herbicides with minor adaptations.

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented ...

Spotting Cheetahs: Identifying Individuals by Their Footprints

The cheetah (Acinonyx jubatus) is Africa's most endangered large felid and listed as Vulnerable with a declining population trend by the IUCN1. It ranges widely over sub-Saharan Africa and in parts of the Middle East. Cheetah conservationists face two major challenges, conflict with landowners over the killing of domestic livestock, and concern over range contraction. Understanding of the latter remains particularly poor2. Namibia is believed to support the largest number of cheetahs of any range country, around 30%, but estimates range from 2,9053 to 13,5204. The disparity is likely a result of the different techniques used in monitoring.
Current techniques, including invasive tagging with VHF or satellite/GPS collars, can be costly and unreliable. The footprint identification technique5 is a new tool accessible to both field scientists and also citizens with smartphones, who could potentially augment data collection. The footprint identification technique analyzes digital images of footprints captured according to a standardized protocol. Images are optimized and measured in data visualization software. Measurements of distances, angles, and areas of the footprint images are analyzed using a robust cross-validated pairwise discriminant analysis based on a customized model. The final output is in the form of a Ward's cluster dendrogram. A user-friendly graphic user interface (GUI) allows the user immediate access and clear interpretation of classification results.
The footprint identification technique algorithms are species specific because each species has a unique anatomy. The technique runs in a data visualization software, using its own scripting language (jsl) that can be customized for the footprint anatomy of any species. An initial classification algorithm is built from a training database of footprints from that species, collected from individuals of known identity. An algorithm derived from a cheetah of known identity is then able to classify free-ranging cheetahs of unknown identity. The footprint identification technique predicts individual cheetah identity with an accuracy of &gt;90%.

The cheetah (Acinonyx jubatus) is an iconic, endangered species, but conservation efforts are challenged by habitat shrinkage and conflict with commercial farmers. The footprint identification technique, a robust, accurate and cost-effective image classification system, is a new approach to monitoring cheetahs.

The cheetah (Acinonyx jubatus) is Africa's most endangered large felid and listed as Vulnerable with a declining population trend by the IUCN1. It ranges ...

Characterization and Application of Passive Samplers for Monitoring of Pesticides in Water

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study provides a protocol for the passive sampler preparation, calibration, extraction method and instrumental analysis. Sampling rates (RS) and passive sampler-water partition coefficients (KPW) were calculated for silicone rubber, polar organic chemical integrative sampler POCIS-A, POCIS-B, SDB-RPS and C18 disk. The uptake of the selected compounds depended on their physicochemical properties, i.e., silicone rubber showed a better uptake for more hydrophobic compounds (log octanol-water partition coefficient (KOW) &#62; 5.3), whereas POCIS-A, POCIS-B and SDB-RPS disk were more suitable for hydrophilic compounds (log KOW &#60; 0.70).

A protocol about the characterization and application of five different passive sampling devices is presented.

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study ...

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with &#39;end-point&#39; PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. The methodology includes the co-extraction of DNA and RNA; preparation of DNA-free RNA; and 16S rRNA gene and transcript quantification via RT-q-PCR, including standard curve construction.

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of ...

Extraction and Characterization of Surfactants from Atmospheric Aerosols

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in the Earth&#39;s atmosphere, a central process in meteorology, hydrology, and for the climate system. But because specific extraction and characterization of these compounds have been lacking for decades, very little is known on their identity, properties, mode of action and origins, thus preventing the full understanding of cloud formation and its potential links with the Earth&#39;s ecosystems.
In this paper we present recently developed methods for 1) the targeted extraction of all the surfactants from atmospheric aerosol samples and for the determination of 2) their absolute concentrations in the aerosol phase and 3) their static surface tension curves in water, including their Critical Micelle Concentration (CMC). These methods have been validated with 9 references surfactants, including anionic, cationic and non-ionic ones. Examples of results are presented for surfactants found in fine aerosol particles (diameter &#60;1 &#956;m) collected at a coastal site in Croatia and suggestions for future improvements and other characterizations than those presented are discussed.

Methods are presented for the targeted extraction of surfactants present in atmospheric aerosols and the determination of their absolute concentrations and surface tension curves in water, including their Critical Micelle Concentration (CMC).

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in ...

Characterization of Aquatic Biofilms with Flow Cytometry

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond quickly to environmental changes and can be thus used as indicators of water quality. Currently, biofilm assessment is mostly based on integrative and functional endpoints, such as photosynthetic or respiratory activity, which do not provide information on the biofilm community structure. Flow cytometry and computational visualization offer an alternative, sensitive, and easy-to-use method for assessment of the community composition, particularly of the photoautotrophic part of freshwater biofilms. It requires only basic sample preparation, after which the entire sample is run through the flow cytometer. The single-cell optical and fluorescent information is used for computational visualization and biological interpretation. Its main advantages over other methods are the speed of analysis and the high-information-content nature. Flow cytometry provides information on several cellular and biofilm traits in a single measurement: particle size, density, pigment content, abiotic content in the biofilm, and coarse taxonomic information. However, it does not provide information on biofilm composition on the species level. We see high potential in the use of the method for environmental monitoring of aquatic ecosystems and as an initial biofilm evaluation step that informs downstream detailed investigations by complementary and more detailed methods.

Flow cytometry in combination with visual clustering offers an easy-to-use and fast method for studying aquatic biofilms. It can be used for biofilm characterization, detection of changes in biofilm community structure, and detection of abiotic particles embedded in the biofilm.

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond ...

Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior

Copepods of the genus Tigriopus, which are common zooplankton in rocky tide pools, show precopulatory mate-guarding behavior where a male clasps a potential mate to form a pair. While this phenomenon has attracted interest of researchers, methods for its analysis have not been well described. Here we describe procedures for: 1) individual culturing and staging of Tigriopus juveniles and adults, and 2) video-based analysis of their mate-guarding behavior. The culturing method enables experimental control of paring experience of animals as well as the ability to track their development before behavioral tests. The analysis method allows quantitative evaluation of several aspects of the mate-guarding behavior, including capturing attempts by males and swimming trajectory of mate-guarding pairs. Although these methods were originally established for ethological studies on Tigriopus, with proper modifications they can also be applied to studies of other zooplankton in different research fields, such as physiology, toxicology, and ecological genetics.

Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior

Mate-guarding behavior plays an important role in reproduction of intertidal copepods of the genus Tigriopus. However, methods for studying this behavior have not been well described. Here we describe methods for: 1) individual culture of virgin Tigriopus animals, and 2) quantitative analysis of their mate-guarding behavior.

Copepods of the genus Tigriopus, which are common zooplankton in rocky tide pools, show precopulatory mate-guarding behavior where a male clasps a ...

Sampling, Identification and Characterization of Microplastics Release from Polypropylene Baby Feeding Bottle during Daily Use

Microplastics (MPs) are becoming a global concern due to the potential risk to human health. Case studies of plastic products (i.e., plastic single-use cups and kettles) indicate that MP release during daily use can be extremely high. Precisely determining the MP release level is a crucial step to identify and quantify the exposure source and assess/control the corresponding risks stemming from this exposure. Though protocols for measuring MP levels in marine or freshwater has been well developed, the conditions experienced by household plastic products can vary widely. Many plastic products are exposed to frequent high temperatures (up to 100 &#176;C) and are cooled back to room temperature during daily use. It is therefore crucial to develop a sampling protocol that mimics the actual daily-use scenario for each particular product. This study focused on widely used polypropylene-based baby feeding bottles to develop a cost-effective protocol for MP release studies of many plastic products. The protocol developed here enables: 1) prevention of the potential contamination during sampling and detection; 2) realistic implementation of daily-use scenarios and accurate collection of the MPs released from baby feeding bottles based on WHO guidelines; and 3) cost-effective chemical determination and physical topography mapping of MPs released from baby feeding bottles. Based on this protocol, the recovery percentage using standard polystyrene MP (diameter of 2 &#181;m) was 92.4-101.2% while the detected size was around 102.2% of the designed size. The protocol detailed here provides a reliable and cost-effective method for MP sample preparation and detection, which can substantially benefit future studies of MP release from plastic products.

This study detailed a reliable and cost-effective protocol for microplastics collection and detection from the daily use of plastic products.

Microplastics (MPs) are becoming a global concern due to the potential risk to human health. Case studies of plastic products (i.e., plastic single-use ...