We wish to acknowledge Danielle Orr Goveia, Blaine Fritz, Jennifer Summers and Fei San Lee for their contributions to this paper.

1. Vortexing and sonication
<ol>
	<li>Grow a mature P. aeruginosa ATCC 15442 biofilm grown according to ASTM Standard E25622.</li>
	<li>At the end of the 48 h growth period, prepare to treat the biofilm and sample coupons according to ASTM Standard E28718</li>
	<li>Aseptically insert autoclaved splash guards into sterile 50 mL conical tubes using flame-sterilized forceps. Repeat for all tubes that will receive treatment. Tubes for control coupons do not need a splash guard.</li>
	<li>Aseptically remove a randomly selected rod from the CDC Biofilm Reactor. Rinse coupons to remove loosely attached cells by gently dipping the rod into 30 mL of sterile buffered water.</li>
	<li>Hold the rod parallel to the bench top, over an empty, sterile 50 mL conical tube and using a flame-sterilized Allen wrench, loosen set screw to drop a biofilm coated coupon into tube. Repeat for the desired number of coupons. Remove splash guards and place in a separate container for sterilization.</li>
	<li>Using a 5 mL serological pipette, slowly pipette 4 mL of the treatment or control into the tubes so that the liquid flows down the inside of the wall of the tube.</li>
	<li>Gently tap the bottom of the tube so that any air bubbles under the coupon are displaced. Allow 30 - 60 s between each addition.</li>
	<li>At the end of the specified contact time, pipette 36 mL of neutralizer into the tubes in the same order that the treatment (or control) was applied. 
	NOTE: The final volume of combined treatment and neutralizer is important for accurately determining biofilm log density.</li>
	<li>Vortex each tube on the highest setting for 30 &#177; 5 s. Ensure that a complete vortex is achieved. 
	NOTE: Caution should be exercised when vortexing heavy coupons such as stainless steel in glass vials where breakage could occur.</li>
	<li>Determine the optimal number of tubes per bath and placement within the test tube rack prior to processing actual samples. If processing multiple samples, confirm that the water temperature in the sonicating bath is 21 &#177; 2 oC.</li>
	<li>Place tubes in tube rack suspended in degassed sonicator such that water level in bath is equal to liquid level in tubes. Sonicate at 45 kHz, 100% power and Normal function for 30 &#177; 5 s. Repeat vortex and sonication cycles and then end with a final vortex (5 cycles total). 
	NOTE: These tubes with the harvested and disaggregated biofilm are the100&#160;or 0 dilution.</li>
	<li>Serially dilute sample in buffered water. Plate on R2A agar using appropriate plating method. Incubate at 36 &#177; 2 oC for 24 h. Count colonies as appropriate to plating method used and record data.</li>
</ol>
2. Scraping and homogenization
<ol>
	<li>Grow a mature P. aeruginosa ATCC 15442 biofilm according to the ASTM Standard E264713.</li>
	<li>Set up the sampling station to include sampling board, 95% ethanol in a beaker, alcohol burner, hemostats, coupon removal tool, beakers with sterile dilution water and dilution tubes for rinsing the coupons.</li>
	<li>Turn off the pump. Remove a channel cover and use a sterile coupon removal tool and hemostats to remove the coupon, being careful not to disturb the biofilm.</li>
	<li>Rinse the coupon by gently immersing, with a fluid motion, in 45 mL of sterile dilution water (contained in a 50 mL centrifuge tube). Immediately reverse the motion to remove the coupon.</li>
	<li>Place the coupon into a beaker containing 45 mL of sterile dilution water. Scrape the biofilm-covered coupon surface in a downward direction for approximately 15 s, using a sterile spatula or scraper. Rinse the spatula or scraper by stirring it in the beaker. Repeat the scraping and rinsing process 3-4 times, ensuring full coverage of the coupon surface.</li>
	<li>Rinse the coupon by holding it at a 60&#176; angle over the sterile beaker and pipetting 1 mL of sterile dilution water over the surface of the coupon. Repeat for a total of 5 rinses. The final volume in the beaker is 50 mL. 
	NOTE: The final volume of combined treatment and neutralizer is important for accurately determining biofilm log density.</li>
	<li>Replace each channel cover as the coupons are removed.</li>
	<li>Working in the biosafety cabinet, homogenize the scraped biofilm sample. Attach a sterile homogenizer probe to the homogenizer, place the probe tip in the liquid, turn the homogenizer on and ramp up to 20,500 rpm.</li>
	<li>Homogenize the sample for 30 s. Turn down the RPMs and switch the homogenizer off.</li>
	<li>Sanitize the probe between biofilm samples by homogenizing a 9 mL of sterile dilution blank at 20,500 rpm for 30 s as described above. Homogenize a 9 mL tube of 70% ethanol for 30 s, detach the probe and let stand in the ethanol tube for 1 min. Homogenize two additional dilution blanks. 
	NOTE: A disposable homogenizer probe may be used for each sample.</li>
	<li>Serially dilute the samples in buffered water. Plate on R2A agar using the appropriate plating method. Incubate plates at 36 &#177; 2oC for 24 h, count colonies as appropriate to plating method used and record data.</li>
</ol>
3. Scraping, vortexing and sonication
<ol>
	<li>Grow a mature Escherichia coli ATCC 53498 biofilm in silicone catheter tubing10.</li>
	<li>Prepare sampling materials: rinse tubes, sterile centrifuge tube, empty sterile Petri dish, flame sterilized stainless steel hemostat and scissors, timer, and ruler.</li>
	<li>With the pump paused, use 70% ethanol to clean the outside of the tubing. Measure 2 cm from the end, avoiding the area attached to the connector, and mark the tubing to determine cutting locations.</li>
	<li>With flame sterilized scissors, cut the tubing on the 2 cm mark and place the segment in empty sterile Petri dish. Wipe the tubing with 70% ethanol and reconnect the distal end to waste tubing.</li>
	<li>Rinse tubing segment to remove planktonic cells. With flame sterilized forceps, gently immerse tubing segment into 20 mL of sterile dilution water and then immediately remove. Place the segment into 10 mL of neutralizer.</li>
	<li>With flame sterilized forceps, hold the tubing segment and scrape with sterile wooden applicator stick until all inner areas of the tubing have been scraped. Occasionally rinse the stick in the 10 mL of neutralizer and place the segment back into the sample tube. The scraped tubing segment is the 100 or 0 dilution. 
	NOTE: The final volume of combined treatment and neutralizer is important for accurately determining biofilm log density.</li>
	<li>Vortex each tube on the highest setting for 30 &#177; 5 s. Place the tube in tube rack suspended in sonicator such that water level in bath is equal to liquid level in tubes. Sonicate at 45 kHz, 100% power and Normal function for 30 &#177; 5 seconds. Repeat vortex and sonication cycles then end with a final vortex. 
	NOTE: This tube with the harvested and disaggregated biofilm is the 100 dilution.</li>
	<li>Serially dilute the samples in buffered water. Plate on Tryptic Soy Agar using the appropriate plating method.</li>
	<li>Incubate plates at 36 &#177; 2 oC for 24 h. Count colonies as appropriate to plating method used, record data and calculate the arithmetic mean.</li>
</ol>

<ol>
	<li>Donlan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms:+microbial+life+on+surfaces.">Biofilms: microbial life on surfaces.</a> Emerging Infectious Diseases. 8 (9), 881-890 (2002).</li><li>ASTM International. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ASTM+Standard+E2562,+2017,+Standard+Test+Method+for+Quantification+of+Pseudomonas+aeruginosa+Biofilm+Grown+with+High+Shear+and+Continuous+Flow+using+CDC+Biofilm+Reactor.">ASTM Standard E2562, 2017, Standard Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown with High Shear and Continuous Flow using CDC Biofilm Reactor.</a> ASTM International. , (2017).</li><li>Azeredo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+review+on+biofilm+methods.">Critical review on biofilm methods.</a> Critical Reviews In Microbiology. 43 (3), 313-351 (2017).</li><li>Gomes, I. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+reactors+for+the+study+of+medical+biofilms:+a+review+of+the+principles+and+latest+modifications.">Standardized reactors for the study of medical biofilms: a review of the principles and latest modifications.</a> Critical Reviews in Biotechnology. 38 (5), 657-670 (2018).</li><li>Goeres, D. M., et al., Simoes, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+fabrication+of+biofilm+reactors.">Design and fabrication of biofilm reactors.</a> Recent Trends in Biofilm Sciences and Technology. , 71-88 (2020).</li><li>Goeres, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+growing+a+biofilm+under+low+shear+at+the+air-liquid+interface+using+the+drip+flow+biofilm+reactor.">A method for growing a biofilm under low shear at the air-liquid interface using the drip flow biofilm reactor.</a> Nature Protocols. 4 (5), 783-788 (2009).</li><li>ASTM International. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ASTM+Standard+E2871,+Standard+Test+Method+for+Determining+Disinfectant+Efficacy+Against+Biofilm+Grown+in+the+CDC+Biofilm+Reactor+Using+the+Single+Tube+Method.">ASTM Standard E2871, Standard Test Method for Determining Disinfectant Efficacy Against Biofilm Grown in the CDC Biofilm Reactor Using the Single Tube Method.</a> ASTM International. , (2019).</li><li>Goeres, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+a+Biofilm+Efficacy+Test:+The+Single+Tube+Method.">Validation of a Biofilm Efficacy Test: The Single Tube Method.</a> Journal of Microbiological Methods. , (2019).</li><li>The development and validation of a standard in vitro method to evaluate the efficacy of surface modified urinary catheters. Theses and Dissertations at Montana State University Available from: <a target="_blank" href="https://scholarworks.montana.edu/xmlui/handle/1/15149">https://scholarworks.montana.edu/xmlui/handle/1/15149</a> (2019)</li><li>Hamilton, M. A., Buckingham-Meyer, K., Goeres, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Checking+the+Validity+of+the+harvesting+and+Disaggregating+Steps+in+Laboratory+Tests+of+Surface+Disinfectants.">Checking the Validity of the harvesting and Disaggregating Steps in Laboratory Tests of Surface Disinfectants.</a> Journal of AOAC International. 92 (6), 1755-1762 (2009).</li><li>Conlon, B. P., Rowe, S. E., Lewis, K., Donelli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persister+Cells+in+Biofilm+Associated+Infections.">Persister Cells in Biofilm Associated Infections.</a> Biofilm-based Healthcare-associated Infections. , (2015).</li><li>ASTM International. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ASTM+Standard+E2647,+Standard+Test+Method+for+Quantification+of+Pseudomonas+aeruginosa+Biofilm+Grown+Using+Drip+Flow+Biofilm+Reactor+with+Low+Shear+and+Continuous+Flow.">ASTM Standard E2647, Standard Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown Using Drip Flow Biofilm Reactor with Low Shear and Continuous Flow.</a> ASTM International. , (2020).</li><li>Rosenberg, M., Azevedo, N., Ivask, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propidium+iodide+staining+underestimates+viability+of+adherent+bacterial+cells.">Propidium iodide staining underestimates viability of adherent bacterial cells.</a> Scientific Reports. , (2019).</li><li>Stiefel, P., Schmidt-Emrich, S., Manuira-Weber, K., Ren, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+aspects+of+using+bacterial+cell+viability+assays+with+the+fluorophores+SYTO9+and+propidium+iodide.">Critical aspects of using bacterial cell viability assays with the fluorophores SYTO9 and propidium iodide.</a> BMC Microbiology. , (2015).</li><li>Kobayashi, N., Bauer, T. W., Tuohy, M. J., Fujishiro, T., Procop, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+Ultrasonication+Improves+Detection+of+Biofilm-formative+Bacteria+Around+a+Metal+Implant.">Brief Ultrasonication Improves Detection of Biofilm-formative Bacteria Around a Metal Implant.</a> Clinical Orthopaedics and Related Research. 457, 210-213 (2007).</li><li>Lourenco, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimum+information+about+a+biofilm+experiment+(MIABiE),+standards+for+reporting+experiments+and+data+on+sessile+microbial+communities+living+at+interfaces.">Minimum information about a biofilm experiment (MIABiE), standards for reporting experiments and data on sessile microbial communities living at interfaces.</a> Pathogens and Disease. , 1-7 (2014).</li><li>Nascentes, C. C., Korn, M., Sousa, C. S., Arruda, M. A. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Ultrasonic+Baths+for+Analytical+Applications:+A+New+Approach+for+Optimisation+Conditions.">Use of Ultrasonic Baths for Analytical Applications: A New Approach for Optimisation Conditions.</a> Journal of Brazilian Chemical Society. 12 (1), 57-63 (2001).</li><li>Elma GmbH &amp; Co. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KG+TI-H+Ultrasonic+Cleaning+Units:+Operating+Instructions.">KG TI-H Ultrasonic Cleaning Units: Operating Instructions.</a> Elma GmbH &amp; Co. , (2009).</li><li>Stamper, D. M., Holm, E. R., Brizzolara, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+times+and+energy+densities+for+ultrasonic+disinfection+of+Escherichia+coli,+Pseudomonas+aeruginosa,+Enterococcus+avium+and+sewage.">Exposure times and energy densities for ultrasonic disinfection of Escherichia coli, Pseudomonas aeruginosa, Enterococcus avium and sewage.</a> Journal of Environmental Engineering and Science. 7 (2), 139-146 (2008).</li><li>Suslick, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonochemistry.">Sonochemistry.</a> Science. 247 (4949), (1990).</li></ol>

The authors have no disclosures.

Minimum Information for Harvesting and Disaggregation Methods 
To create reproducible biofilm data across the scientific community, it is imperative that authors include as much detail as possible regarding each of the growth, treatment, sampling and analysis steps of a biofilm method. The standardization of biofilm methods has aided in this endeavor as it allows the researcher to reference a specific method and any relevant modifications. However, many papers include only a sentence or two to descr...

The definition of biofilm has evolved over the last few decades and encompasses microbial association with a variety of biological and/or non-biological surfaces, inclusion of noncellular components1 that display differing growth and genetic expression2 within a matrix. Biofilm provides protection from environmental stresses such as drying and may render the action of chemical disinfectants less effective resulting in the survival of microbes. The survivors within a biofilm can potentially provide a source of pathogenic microorganisms that are a public health concern3.
Biofilm methods are comprised of four steps, growth, treatment, sampling (harvesting and disaggregation), and analysis. Growth, the first step, where the user determines the organism growth conditions, temperature, media, etc., is the most considered and reported upon in the biofilm literature4,5,6,7. The treatment step evaluates antimicrobials (e.g., disinfectants) to determine their efficacy either against a mature biofilm3,8,9 or the antimicrobial may be incorporated into the surface to determine the ability of the product to prevent or reduce biofilm growth10. The third step, sampling, includes steps to harvest the biofilm from the surface on which it was growing and to disaggregate the removed clumps3,8,11. The fourth step, analysis, may include viable cell counts, microscopy, fluorescence measurements, molecular outcomes, and/or a matrix component assessment8,9. Assessment of data provides information about the outcome of an experiment. Of the four, sampling is often the most overlooked step because it presumes that the chosen biofilm harvesting and/or disaggregation technique is 100% effective, often without verification11.
Planktonic suspensions of bacteria, often considered to be homogenous, require simple vortexing prior to analysis. Biofilms, however, are complex communities composed of microorganisms (prokaryotic and/or eukaryotic), exopolysaccharides, proteins, lipids, extracellular DNA and host cells12. Steps beyond traditional planktonic microbiological culture methods are needed in order to adequately harvest biofilm from a surface and then disaggregate it into a homogenous single cell suspension. An extensive literature review (information not included in this publication) demonstrated that the choice of the removal and disaggregation technique is dependent on a number of factors, including species present in the biofilm, surface that the biofilm is attached to (non-porous or porous), accessibility to growth surfaces (easily removable coupon or physical destruction of apparatus in which the biofilm is growing), surface geometry (area and shape), density of biofilm on growth surfaces, and available laboratory equipment.
When biofilm is harvested from a surface, the resulting cell suspension is heterogenous. If this nonuniform suspension is to be accurately enumerated, it must be disaggregated into individual cells. Viable plate counts assume that a colony forming unit originates from one bacterium. If aggregates of biofilm are placed on the growth medium, it is impossible to distinguish individual cells which could lead to inaccurate estimates. For example, during disinfectant efficacy testing, if a treatment removes biofilm very effectively from a surface compared to the control, the log reduction could appear artificially large compared to the control. On the other hand, a chemical disinfectant that fixes biofilm onto a surface compared to the control will appear to have a lower log reduction11. This type of scenario could lead to biased interpretation of experimental data.
In preparation for the&#160;publication, a review of the literature determined that common approaches to harvesting and disaggregating biofilm include scraping, swabbing, sonication, vortexing or a combination of these. Scraping is defined as physical removal of biofilm from surfaces with a sterile stick, spatula or other tool. Swabbing refers to removal of biofilm from surfaces with a cotton tipped stick or other fixed absorbent material. Sonication refers to disruption of biofilm from surfaces via ultrasonic waves distributed through water. Vortexing refers to the use of a mixer to achieve a liquid vortex of the sample inside a tube. Homogenization uses rotating blades to shear harvested biofilm clumps into a single cell suspension. In this paper, we present three harvesting and disaggregation methods for two different surface types, hard/non-porous and porous.
A list of recommended minimum information that researchers should include in the methods sections of publications is provided. We hope that inclusion of this information enables other researchers to reproduce their work. There is no perfect harvesting and disaggregation method, therefore, recommendations for how to check the technique are also provided.
Three common methods to harvest and disaggregate biofilm from common growth surfaces are demonstrated in this article. This information will enable researchers to better understand the overall precision and bias of a biofilm test method. Methods described are as follows: (1) A Pseudomonas aeruginosa biofilm grown on polycarbonate coupons (hard non-porous surface) under high fluid shear in the CDC Biofilm Reactor is harvested and disaggregated following a five step combination of vortexing and sonication to achieve biofilm harvest and disaggregation (2) A P. aeruginosa biofilm grown on borosilicate glass coupons (hard non-porous surface) in the drip flow reactor under low fluid shear is harvested and disaggregated using scraping and homogenization (3) An Escherichia coli biofilm grown in silicone tubing (porous surface) is harvested and disaggregated using scraping, followed by sonication and vortexing.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>50 mL conical vials</td>
			<td>Thermo Scientific</td>
			<td>339652</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL glass beakers</td>
			<td>Fisher Scientific</td>
			<td>FB102100</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL serological pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-678-12D</td>
			<td>For adding treatment to vials containing coupons.</td>
		</tr>
		<tr>
			<td>50 mL serological pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-678-14C</td>
			<td>For adding neutralizer to vials at the end of treatment contact time.</td>
		</tr>
		<tr>
			<td>Applicator sticks</td>
			<td>Puritan</td>
			<td>807</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostats</td>
			<td>Fisher Scientific</td>
			<td>16-100-115</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal spatula</td>
			<td>Fisher Scientific</td>
			<td>14-373</td>
			<td></td>
		</tr>
		<tr>
			<td>PTFE policemen</td>
			<td>Saint-Gobain</td>
			<td>06369-04</td>
			<td></td>
		</tr>
		<tr>
			<td>S 10 N - 10 G - ST Dispersing tool</td>
			<td>IKA</td>
			<td>4446700</td>
			<td>For homogenization of biofilm samples.</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fisher Scientific</td>
			<td>08-951-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone Foley catheter, size 16 French</td>
			<td>Medline Industries</td>
			<td>DYND11502</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone tubing, size 16</td>
			<td>Cole-Parmer</td>
			<td>EW96400-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Splash Guards</td>
			<td>BioSurface Technologies, Inc.</td>
			<td>CBR 2232</td>
			<td></td>
		</tr>
		<tr>
			<td>T 10 basic ULTRA-TURRAX Disperser</td>
			<td>IKA</td>
			<td>3737001</td>
			<td>For homogenization of biofilm samples.</td>
		</tr>
		<tr>
			<td>Tubing connectors</td>
			<td>Cole-Parmer</td>
			<td>EW02023-86</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic Cleaner</td>
			<td>Elma</td>
			<td>TI-H15</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex-Genie 2</td>
			<td>Scientific Industries</td>
			<td>SI-0236</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex-Genie 2 Vertical 50 mL Tube Holder</td>
			<td>Scientific Industries</td>
			<td>SI-V506</td>
			<td></td>
		</tr>
	</tbody>
</table>

harvesting and disaggregation: an overlooked step in biofilm methods research

Biofilm methods consist of four distinct steps: growing the biofilm in a relevant model, treating the mature biofilm, harvesting the biofilm from the surface and disaggregating the clumps, and analyzing the sample. Of the four steps, harvesting and disaggregation are the least studied but nonetheless critical when considering the potential for test bias. This article demonstrates commonly used harvesting and disaggregation techniques for biofilm grown on three different surfaces. The three biofilm harvesting and disaggregation techniques, gleaned from an extensive literature review, include vortexing and sonication, scraping and homogenization, and scraping, vortexing and sonication. Two surface types are considered: hard non-porous (polycarbonate and borosilicate glass) and porous (silicone). Additionally, we provide recommendations for the minimum information that should be included when reporting the harvesting technique followed and an accompanying method to check for bias.

Validation/Confirmation of a Harvesting Method 
Several studies that were conducted in our laboratory examined the ability of vortexing and sonication to effectively harvest biofilm grown in the biofilm reactor (ASTM E2562)2 using the Single Tube Method (ASTM E2871)8.
A P. aeruginosa ATCC 15442 biofilm was grown according to ASTM E25622 on borosilicate glass coupons. After 48 hours, four coup...

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Harvesting and Disaggregation: An Overlooked Step in Biofilm Methods Research

This paper details methods that demonstrate three common biofilm harvesting and disaggregation techniques on two surface types, ruggedness testing of a harvesting method and minimum information to consider when choosing and optimizing harvesting and disaggregation techniques to increase reproducibility.

Biofilm methods consist of four distinct steps: growing the biofilm in a relevant model, treating the mature biofilm, harvesting the biofilm from the ...

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3.7K Views.  Montana State University. This paper details methods that demonstrate three common biofilm harvesting and disaggregation techniques on two surface types, ruggedness testing of a harvesting method and minimum information to consider when choosing and optimizing harvesting and disaggregation techniques to increase reproducibility.

Video: Harvesting and Disaggregation: An Overlooked Step in Biofilm Methods Research

Harvesting Sperm and Artificial Insemination of Mice

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals. Peromyscus species are used in a wide range of research including toxicology, epidemiology, ecology, behavioral, and genetic studies. Here they provide a useful model for demonstrations of artificial insemination. 

Methods similar to those displayed here have previously been used in several deer mouse studies, yet no detailed protocol has been published. Here we demonstrate the basic method of artificial insemination. This method entails extracting the testes from the rodent, then isolating the sperm from the epididymis and vas deferens. The mature sperm, now in a milk mixture, are placed in the female&#x2019;s reproductive tract at the time of ovulation. Fertilization is counted as day 0 for timing of embryo development. Embryos can then be retrieved at the desired time-point and manipulated.

Artificial insemination can be used in a variety of rodent species where exact embryo timing is crucial or hard to obtain. This technique is vital for species or strains (including most Peromyscus) which may not mate immediately and/or where mating is hard to assess. In addition, artificial insemination provides exact timing for embryo development either in mapping developmental progress and/or transgenic work. Reduced numbers of animals can be used since fertilization is guaranteed. This method has been vital to furthering the Peromyscus system, and will hopefully benefit others as well.

This protocol demonstrates methods for extracting sperm from the testes of males and then inseminating female mice. This procedure is useful when precise time is needed in developmental studies as well as transgenic work.

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals.  Peromyscus species are used in a wide range of research ...

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a particularly attractive option for studying membrane proteins, especially in the context of ligand-receptor interactions. The method pioneered by Sligar and colleagues is based on the amphipathic properties of an engineered highly a-helical scaffold protein derived from the apolipoprotein A1. The hydrophobic faces of the scaffold protein interact with the fatty acyl side-chains of the lipid bilayer whereas the polar regions face the aqueous environment. Analyses of membrane proteins in nanodiscs have significant advantages over liposome because the particles are small, homogeneous and water-soluble. In addition, biochemical and biophysical methods normally reserved to soluble proteins can be applied, and from either side of the membrane. In this visual protocol, we present a step-by-step reconstitution of a well characterized bacterial ABC transporter, the MalE-MalFGK2 complex. The formation of the disc is a self-assembly process that depends on hydrophobic interactions taking place during the progressive removal of the detergent. We describe the essential steps and we highlight the importance of choosing a correct protein-to-lipid ratio in order to limit the formation of aggregates and larger polydisperse liposome-like particles. Simple quality controls such as gel filtration chromatography, native gel electrophoresis and dynamic light scattering spectroscopy ensure that the discs have been properly reconstituted.

Nanodiscs are small discoid particles that incorporate membrane proteins into a small patch of phospholipid bilayer. We provide a visual protocol that shows the step-by-step incorporation of the MalFGK2 transporter into a disc.

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to eradicate not only due to the emergence of antibiotic-resistant strains but also its ability to form biofilms. Recently, photodynamic therapy (PDT) has been indicated as one of the potential treatments for controlling biofilm infections. However, further studies are required to improve our knowledge of its effect on bacterial biofilms, as well as the underlying mechanisms. This manuscript describes an in vitro model of PDT with 5-aminolevulinic acid (5-ALA), a precursor of the actual photosensitizer, protoporphyrin IX (PpIX). Briefly, mature S. aureus biofilms were incubated with ALA and then exposed to light. Subsequently, the antibacterial effect of ALA-PDT on S. aureus biofilm was quantified by calculating the colony forming units (CFUs) and visualized by viability fluorescent staining via confocal laser scanning microscopy (CLSM). Representative results demonstrated a strong antibacterial effect of ALA-PDT on S. aureus biofilms. This protocol is simple and can be used to develop an in vitro model to study the treatment of S. aureus biofilms with ALA-PDT. In the future, it could also be referenced in PDT studies utilizing other photosensitizers for different bacterial strains with minimal adjustments.

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

This manuscript describes a protocol to study the antimicrobial effect of 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) on a Staphylococcus aureus biofilm. This protocol can be used to develop an in vitro model to study the treatment of bacterial biofilms with PDT in the future.

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

A Step-by-Step Guide to Mosquito Electroantennography

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when acquiring a blood-meal. To locate a host to feed on, mosquitoes rely on a wide range of sensory cues, including visual, mechanical, thermal, and olfactory. The study details a technique, electroantennography (EAG), that allows researchers to assess whether the mosquitoes can detect individual chemicals and blends of chemicals in a concentration-dependent manner. When coupled with gas-chromatography (GC-EAG), this technique allows to expose the antennae to a full headspace/complex mixture and determines which chemicals present in the sample of interest, the mosquito can detect. This is applicable to host body odors as well as plant floral bouquets or other ecologically relevant odors (e.g., oviposition sites odorants). Here, we described a protocol that permits long durations of preparation responsiveness time and is applicable to both female and male mosquitoes from multiple genera, including Aedes, Culex, Anopheles, and Toxorhynchites mosquitoes. As olfaction plays a major part in mosquito-host interactions and mosquito biology in general, EAGs and GC-EAG can reveal compounds of interest for the development of new disease vector control strategies (e.g., baits). Complemented with behavioral assays, the valence (e.g., attractant, repellent) of each chemical can be determined.

The present article details a step-by-step protocol for successful and low noise electroantennograms in several genera of mosquitoes, including both females and males.

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when ...

A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health conditions in both the laboratory and the clinic. However, pre-existing neutralizing antibodies (NAbs) against AAV capsids pose an ongoing challenge for the successful administration of gene therapies in both large animal experimental models and human populations. Preliminary screening for host immunity against AAV is necessary to ensure the efficacy of AAV-based gene therapies as both a research tool and as a clinically viable therapeutic agent. This protocol describes a colorimetric in vitro assay to detect neutralizing factors against AAV serotype 6 (AAV6). The assay utilizes the reaction between an AAV encoding an alkaline phosphatase (AP) reporter gene and its substrate NBT/BCIP, which generates an insoluble quantifiable purple stain upon combination. 
In this protocol, serum samples are combined with an AAV expressing AP and incubated to permit potential neutralizing activity to occur. Virus serum mixture is subsequently added to cells to allow for viral transduction of any AAVs that have not been neutralized. The NBT/BCIP substrate is added and undergoes a chromogenic reaction, corresponding to viral transduction and neutralizing activity. The proportion of area colored is quantitated using a free software tool to generate neutralizing titers. This assay displays a strong positive correlation between coloration and viral concentration. Assessment of serum samples from sheep before and after administration of a recombinant AAV6 led to a dramatic increase in neutralizing activity (125 to &gt;10,000-fold increase). The assay displayed adequate sensitivity to detect neutralizing activity in &gt;1:32,000 serum dilutions. This assay provides a simple, rapid, and cost-effective method to detect NAbs against AAVs.

A comprehensive laboratory protocol and analysis workflow are described for a rapid, cost-effective, and straightforward colorimetric cell-based assay to detect neutralizing elements against AAV6.

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health ...

Assessment of Acinetobacter Biofilm Formation and Characterization of Biofilms

Quantification, Viability Assessment, and Visualization Strategies for Acinetobacter Biofilms

Acinetobacter causes nosocomial infections and its biofilm formation can contribute to the survival on dry surfaces such as hospital environments. Thus, biofilm quantification and visualization are important methods to assess the potential of Acinetobacter strains to cause nosocomial infections. The biofilms forming on the surface of the microplate can be quantified in terms of volume and cell numbers. Biofilm volumes can be quantified by staining using crystal violet, washing, destaining using ethanol, then measuring the solubilized dye using a microplate reader. To quantify the number of cells embedded in the biofilms, the biofilms are scrapped off using cell scrapers, harvested in the saline, vigorously agitated in the presence of glass beads, and spread on Acinetobacter agar. Then, the plates are incubated at 30 &#176;C for 24-42 h. After incubation, the red colonies are enumerated to estimate the number of cells in biofilms. This viable count method can also be useful for counting Acinetobacter cells in mixed-species biofilms. Acinetobacter biofilms can be visualized using fluorescent dyes. A commercially available microplate designed for microscopic analysis is employed to form biofilms. Then, the bottom-surface attached biofilms are stained with SYTO9 and propidium iodide dyes, washed, then visualized with confocal laser scanning microscopy.

Author Spotlight: A Comprehensive Protocol for Acinetobacter Biofilm Quantification, Assessment, and Visualization 

This protocol describes the preparation of the inoculum, the biofilm quantification on microtiter plates using crystal violet dye, the viable count in biofilms, and the visualization of biofilms of Acinetobacter.

Acinetobacter causes nosocomial infections and its biofilm formation can contribute to the survival on dry surfaces such as hospital environments. Thus, ...

Standardized Dissection and Sampling Protocol for Adult Xenopus Organs

Techniques for Rapidly Sampling Six Crucial Organs in Adult Xenopus

Xenopus has been a powerful model organism for understanding vertebrate development and disease for over a hundred years. While experimental analysis and dissection techniques of the embryo have been well documented, descriptions of adult Xenopus structures and organs, together with techniques for working with adults, have not been updated to take into consideration the requirements of such modern approaches as quantitative proteomics and single-cell transcriptomics. The cell-type and gene-centric perspectives require contrasting observations in embryonic stages to those in adult tissues. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location all along the larval to adult transition, most notably during massive metamorphosis remodeling. Establishing robust standards for organ identification and dissection is crucial to ensure datasets resulting from studies performed at different laboratories can be consistent. The present protocol identifies six of the organs in the adult Xenopus, demonstrating methods for dissection and sampling of the heart ventricle, liver, fat body, pancreas, paired kidney, and skin of the adult Xenopus. Depending on the preservation methods, the dissected organs can be used for quantitative proteomics, single cell/nuclei transcriptomics, in situ hybridization, immunohistochemistry, histology, etc. This protocol aims to standardize tissue sampling and facilitate multi-lab investigations of the adult organ systems.

Author Spotlight: Standardizing Tissue Sampling in Proteomics and Immunochemistry Research

This article presents a guide for sampling six significant and diverse organs in adult Xenopus that can be rapidly and easily accessed: the heart ventricle, liver lobe, pancreas, fat bodies, paired kidneys, and skin.

Xenopus has been a powerful model organism for understanding vertebrate development and disease for over a hundred years. While experimental analysis and ...