Name: Biofilm Removal Using Carbon Dioxide Aerosols without Nitrogen Purge (Video) | JoVE Journal
Uploaded: 2016-11-06T15:00:00.000+00:00
Duration: 7 min 11 s
Description: Learn biofilm removal using carbon dioxide aerosols without nitrogen purge for surface cleaning. This rapid, reliable method utilizes momentum transfer and surfactant action on Pseudomonas putida biofilms.

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5 minutes

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning (# 2015R1A2A2A01006446).

1. Preparation of Surface for Biofilm Formation
<ol>
	<li>Cut 1-mm-thick 304 stainless steel plates into chips (10 &#215; 10 mm2) with a mechanical cutter.</li>
	<li>Perform ultrasonic cleaning of the chips in acetone, methanol, and deionized (DI) water sequentially for 10 min each. Use a solvent-resistant container, made of substances such as glass, to remove organic contamination.</li>
	<li>Rinse the chips with flowing DI water for 3-5 sec.</li>
	<li>Dry the chips using N2 gas flow for 3-5 sec.</li>
</ol>
2. Preparation of Bacterial Culture
<ol>
	<li>Take a P. putida KT2440 (kindly provided by Prof. Sung Kuk Lee, UNIST, South Korea) stock stored in a -80 &#176;C deep-freezer.</li>
	<li>Thaw at room temperature after 1 min, and the top layer of the frozen stock solution turns to slush. Immerse a loop into the melted layer of the stock solution.</li>
	<li>Use this loop to streak the bacteria onto a Luria-Bertani (LB) plate containing 1.5% agar.</li>
	<li>Incubate the plate overnight at 30 &#176;C to grow the bacterial colonies.</li>
	<li>Pick a single colony from the plate using a fresh loop.</li>
	<li>Inoculate 10 ml of LB broth in a 50 ml conical tube with the loop containing the single bacterial colony.</li>
	<li>Incubate the broth in a shaking incubator for 16 hr at 30 &#176;C and 160 rpm.</li>
</ol>
3. Biofilm Formation on the Surface
<ol>
	<li>Pick up each of the prepared chips with tweezers and dip them in 70% ethanol 5 times for 1-2 sec each, to sterilize the surface of each chip. Ensure that the chips are held with tweezers during the dipping.</li>
	<li>Dip each chip in autoclaved DI water and in LB broth sequentially, 5 times for 1-2 sec each, to remove remaining ethanol.</li>
	<li>Place these chips in 6-well culture plates with 2 chips and 5 ml LB broth per well.</li>
	<li>Dilute the bacterial culture until the concentration in the LB broth reaches 8 &#215; 108 cells/ml (optical density at 600 nm wavelength: ~0.8).</li>
	<li>Inoculate each well with 50 &#181;l of the diluted bacterial culture.</li>
	<li>Incubate the plates at 30 &#176;C without shaking for 24 hr for formation of biofilms.</li>
</ol>
4. CO2 Aerosol Cleaning
<ol>
	<li>Dip the biofilm-formed chips in 10 mM ammonium acetate buffer (volatile) 5 times to remove loosely attached and planktonic bacteria.</li>
	<li>Dry these chips in a biosafety cabinet, where air flows mildly.</li>
	<li>Immediately after drying, place a chip on the loading place, which is 20 mm from the CO2 nozzle along the axis of the jet. Tilt the jet axis to a 40&#176; angle.</li>
	<li>Set the stagnation pressures of CO2 and N2 gas to 5.3 MPa and 0.7 MPa, respectively, using gas-pressure regulators.</li>
	<li>Apply the aerosol jet onto the central part of the chip. White aerosols including the solid CO2 should be visible. Turn &#34;on&#34; the solenoid valve for CO2 for 5 sec, and then turn it &#34;off&#34; for 3 sec (cycle: 8 sec) periodically using a manually controlled switch. If a nitrogen purge needs to be used, turn on the solenoid valve for a continuous supply of N2.</li>
	<li>Treat the chips with CO2 aerosols for 16, 40, and 88 sec with and without nitrogen purges. Retain chips without treatment as negative controls.</li>
</ol>
5. Analysis for Removal Efficiency
<ol>
	<li>Prepare 1 &#181;M green fluorescence nucleic acid stain (excitation/emission wavelength: 480/500 nm) in DI water to stain bacterial cells on the control and aerosol-treated chips.</li>
	<li>Place the chips in the staining solution.</li>
	<li>Incubate the chips in an incubator without light at 37 &#176;C for 30 min.</li>
	<li>Following the incubation, gently rinse the chips with flowing DI water to remove excessive fluorescent dye.</li>
	<li>Dry the chips with N2 gas flow.</li>
	<li>Take fluorescence microscopic images of 5 random fields of view (321 &#215; 240 &#181;m2) for each chip using an epifluorescence microscope, a 40X objective lens, and a CCD camera.</li>
	<li>Obtain the fluorescence intensity for each image using an image processing software such as ImageJ. In ImageJ, use the &#34;Subtract Background&#34; function in the &#34;Process&#34; menu, and select &#34;Integrated density&#34; in the &#34;Set Measurements&#34; window in the &#34;Analyze&#34; menu. Do the &#34;Measurement&#34; in the &#34;Analyze&#34; menu to obtain the fluorescence intensity.</li>
	<li>Calculate the biofilm removal efficiency according to the following formula: 100% &#215; (I of control chips - I of treated chips)/(I of control chips), where I is the calculated fluorescence intensity.</li>
	<li>Obtain the average removal efficiencies and standard deviations. Use at least 4 chips for each case.</li>
</ol>

<ol><li>Jain, A., Gupta, Y., Agrawal, R., Khare, P., Jain, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biofilms+-+A+microbial+life+perspective%3A+A+critical+review%5BTitle%5D)+AND+%22Crit.+Rev.+Ther.+Drug%22%5BJournal%5D)">Biofilms - A microbial life perspective: A critical review.</a> Crit. Rev. Ther. Drug. 24 (5), 393-443 (2007).</li>
<li>Bott, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biofouling+control+with+ultrasound%5BTitle%5D)+AND+%22Heat+Transfer+Eng%22%5BJournal%5D)">Biofouling control with ultrasound.</a> Heat Transfer Eng. 21 (3), 43-49 (2000).</li>
<li>Meyer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Approaches+to+prevention%2C+removal+and+killing+of+biofilms%5BTitle%5D)+AND+%22Int.+Biodeterior.+Biodegradation%22%5BJournal%5D)">Approaches to prevention, removal and killing of biofilms.</a> Int. Biodeterior. Biodegradation. 51 (4), 249-253 (2003).</li>
<li>Hong, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+electric+currents+on+bacterial+detachment+and+inactivation%5BTitle%5D)+AND+%22Biotechnol.+Bioeng%22%5BJournal%5D)">Effect of electric currents on bacterial detachment and inactivation.</a> Biotechnol. Bioeng. 100 (2), 379-386 (2008).</li>
<li>Nandakumar, K., Obika, H., Utsumi, A., Ooie, T., Yano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+laser+ablation+of+laboratory+developed+biofilms+using+an+Nd%3AYAG+laser+of+532+nm+wavelength%5BTitle%5D)+AND+%22Biotechnol.+Bioeng%22%5BJournal%5D)">In vitro laser ablation of laboratory developed biofilms using an Nd:YAG laser of 532 nm wavelength.</a> Biotechnol. Bioeng. 86 (7), 729-736 (2004).</li>
<li>Gibson, H., Taylor, J. H., Hall, K. E., Holah, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effectiveness+of+cleaning+techniques+used+in+the+food+industry+in+terms+of+the+removal+of+bacterial+biofilms%5BTitle%5D)+AND+%22J.+Appl.+Microbiol%22%5BJournal%5D)">Effectiveness of cleaning techniques used in the food industry in terms of the removal of bacterial biofilms.</a> J. Appl. Microbiol. 87 (1), 41-48 (1999).</li>
<li>Kang, M. Y., Jeong, H. W., Kim, J., Lee, J. W., Jang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Removal+of+biofilms+using+carbon+dioxide+aerosols%5BTitle%5D)+AND+%22J.+Aerosol+Sci%22%5BJournal%5D)">Removal of biofilms using carbon dioxide aerosols.</a> J. Aerosol Sci. 41 (11), 1044-1051 (2010).</li>
<li>Cha, M., Hong, S., Kang, M. Y., Lee, J. W., Jang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gas-phase+removal+of+biofilms+from+various+surfaces+using+carbon+dioxide+aerosols%5BTitle%5D)+AND+%22Biofouling%22%5BJournal%5D)">Gas-phase removal of biofilms from various surfaces using carbon dioxide aerosols.</a> Biofouling. 28 (7), 681-686 (2012).</li>
<li>Dwidar, M., Hong, S., Cha, M., Jang, J., Mitchell, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Combined+application+of+bacterial+predation+and+carbon+dioxide+aerosols+to+effectively+remove+biofilms%5BTitle%5D)+AND+%22Biofouling%22%5BJournal%5D)">Combined application of bacterial predation and carbon dioxide aerosols to effectively remove biofilms.</a> Biofouling. 28 (7), 671-680 (2012).</li>
<li>Cha, M., Hong, S., Lee, S. Y., Jang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Removal+of+different-age+biofilms+using+carbon+dioxide+aerosols%5BTitle%5D)+AND+%22Biotechnol.+Bioprocess+Eng%22%5BJournal%5D)">Removal of different-age biofilms using carbon dioxide aerosols.</a> Biotechnol. Bioprocess Eng. 19 (3), 503-509 (2014).</li>
<li>Singh, R., Monnappa, A. K., Hong, S., Mitchell, R. J., Jang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+Carbon+Dioxide+Aerosols+on+the+Viability+of+Escherichia+coli+during+Biofilm+Dispersal%5BTitle%5D)+AND+%22Sci.+Rep%22%5BJournal%5D)">Effects of Carbon Dioxide Aerosols on the Viability of Escherichia coli during Biofilm Dispersal.</a> Sci. Rep. 5, 13766(2015).</li>
<li>Sherman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Carbon+Dioxide+Snow+Cleaning%5BTitle%5D)+AND+%22Particul.+Sci.Technol%22%5BJournal%5D)">Carbon Dioxide Snow Cleaning.</a> Particul. Sci.Technol. 25 (1), 37-57 (2007).</li>
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The authors declare that they have no competing financial interests.

Previously, we conducted optimization studies on CO2 gas pressure, jet angle, and distance to the solid surface in CO2 aerosol cleaning7. Unlike our previous studies, in the present study, a nitrogen purge was not included in the aerosol (Figure 1). Moreover, 304 stainless steel was used in this protocol, since it is one of the most common stainless steels and is widely used in the food industry. The polished surface is beneficial for fluorescence analysis because of a un...

Biofilms are complex bacterial community structures in which bacterial cells are embedded within self-produced matrices of extracellular polymeric substances, held together, and protected from the external environment. Biofilms can present a public health risk and cause economic losses because they can form on various surfaces, including medical implant materials and devices, food processing equipment, and heat exchangers. In fact, biofilms have been found to be associated with 65% of all bacterial infectious diseases in humans, according to the Centers for Disease Control and Prevention1.
Autoclaves and disinfectants such as chlorine have generally been used for the inactivation of biofilms. However, the use of an autoclave is limited for surfaces that can neither withstand high temperature steam nor be placed into the autoclave. Disinfectants are not suitable for surfaces sensitive to chemical treatment or prone to collecting toxic oxidation products2. In addition, the biofilm should not only be inactivated, but also removed in order to prevent the attachment of new cells onto the surface, thereby forming a new biofilm3. However, it is difficult to remove biofilms using methods based on viscous fluid shear because the flow velocity near the surface is almost zero and the shear force usually cannot overcome the adhesive forces of micron- and submicron-sized substances. Moreover, the biofilm matrix is known to act as a physical and chemical barrier1.
Many physical and mechanical techniques have been developed to remove biofilms from surfaces, including ultrasonic vibration1, electric currents4, laser irradiation5, and high-pressure water sprays6. Each technique has its own pros and cons. Ultrasonic vibration and electric current can be used to control biofilm formation; however, they require a particular configuration and conductive surfaces, respectively, requiring additional shear stress1, 4. Laser irradiation can be applied to a limited area and to hard surfaces; however, some live and dead cells remain on the surface5. High-pressure water sprays effectively remove biofilms; however, their high momentum can cause damage to soft substrates6.
Biofilm removal using CO2 aerosols has been previously proposed. It has shown promising results, with high removal efficiencies within a short time7-11. CO2 aerosols are generated by adiabatic expansion of a high-pressure CO2 gas through a nozzle, and they are applied to the surfaces contaminated with a biofilm. This cleaning technique utilizes the momentum transfer of solid CO2 particles and the solvent action of the melted CO2 liquids, followed by the aerodynamic shear force of the CO2 gas12. Compared with high-pressure N2 gas jets, CO2 aerosol jets at the same stagnation pressure are much more effective in removing E. coli biofilms7. Moreover, although the momentum of the solid CO2 that is delivered to the bacteria is considerably high, the momentum of the total aerosol jet applied to the solid surface is substantially lower than that of water jets. Therefore, damage-free cleaning is possible using this CO2 aerosol technique.
In this protocol, periodic jets of CO2 aerosols without nitrogen purges were used to remove Pseudomonas putida biofilms from a polished stainless steel surface. In fact, nitrogen purges have been used in many CO2 aerosol studies to increase the removal efficiency by reducing the moisture condensation generated during the cleaning, even though heating with a hot plate or infrared lamps and employing dry boxes have also been adopted12. The surface biofilm formation and the optimized cleaning procedures are described below. The removal efficiency was evaluated by measuring the fluorescence intensity of the biofilm on the surface.

<table><tbody><tr><td>304 stainless steel</td><td>Steelni&lt;br/&gt; (South Korea)</td><td></td><td>Polished and diced ones</td></tr><tr><td>Ultrasonic cleaner</td><td>Branson&lt;br/&gt; (USA)</td><td>5510E-DTH</td><td></td></tr><tr><td>Luria-Bertani (LB)</td><td>Becton, Dickinson and Company&lt;br/&gt; (USA)</td><td>244620</td><td>500 g</td></tr><tr><td>Agar</td><td>Becton, Dickinson and Company&lt;br/&gt; (USA)</td><td>214010</td><td>500 g</td></tr><tr><td>6-well culture plate</td><td>SPL Life Sciences&lt;br/&gt; (South Korea)</td><td>32006</td><td></td></tr><tr><td>Ammonium acetate buffer</td><td>Sigma-Aldrich&lt;br/&gt; (USA)</td><td>667404</td><td>10 mM</td></tr><tr><td>Dual gas unit</td><td>Applied Surface Technologies&lt;br/&gt; (USA)</td><td>K6-10DG</td><td>One nozzle for CO&lt;sub&gt;2&lt;/sub&gt; gas&lt;br/&gt; &amp;amp; 8 nozzles for N&lt;sub&gt;2&lt;/sub&gt; gas</td></tr><tr><td>SYTO9</td><td>Thermo Fissher Scientific&lt;br/&gt; (USA)</td><td>Invitrogen</td><td>Excitaion: 480 nm&lt;br/&gt; Emission: 500 nm</td></tr><tr><td>Epifluorescence microscope&amp;nbsp;</td><td>Nikon (Japan)</td><td>Eclipse 80i</td><td></td></tr><tr><td>40X objective lens</td><td>Nikon&lt;br/&gt; (Japan)</td><td>Plan Fluor</td><td>NA: 0.75</td></tr><tr><td>CCD camera&amp;nbsp;</td><td>Photometrics&lt;br/&gt; (USA)</td><td>Cool SNAP HQ2</td><td>Monochrome</td></tr></tbody></table>

biofilm removal using carbon dioxide aerosols without nitrogen purge

Biofilm Removal Using Carbon Dioxide Aerosols without Nitrogen Purge

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

CO2 aerosols were used to remove the P. putida biofilms from SUS304 surfaces (Figure 1). Most of the surfaces were covered with the biofilm after 24 hr of growth. Most of the biofilm was removed using the CO2 aerosols (Figure 2). As expected, Figure 3 shows an increase in biofilm removal efficiency as the CO2 aerosol treatment time increased. For the treatment time of 88 sec, the removal efficiency was determined to be as high as...

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

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

The overall goal of this experiment is to evaluate the effectiveness of biofilm removal using carbon dioxide aerosols without a nitrogen purge. Carbon dioxide aerosols can be used to remove biofilms utilizing momentum transfer and surfactant action over the aerosols. This is an effective and reliable method to remove biofilms in a short period of time, and is also a biofilm gas-phase cleaning technique.To begin the experiment, use mechanically cut 1-millimeter thick three zero four stainless steel chips, 10 x 10 square millimeters inside. Next, ultrasonically clean the chips in acetone, methanol, and deionized or DI water sequentially. Rinse the chips with flowing DI water for four seconds, then dry the chips using nitrogen gas flow for four seconds.Retrieve a Pseudomonas putida KT2440 stock stored at 80 degrees Celsius. Thaw the culture at room temperature for one minute, when the top layer of the frozen stock solution turns to slush. Submerge a loop into the melted layer of the stock solution, and streak the bacteria onto a Luria Broth, or LB plate containing 1.5%agar.Incubate the plate overnight at 30 degrees Celsius. The next day, add 10 milliliters of LB in a 50 mL conical tube. Use a fresh loop to pick a single colony from the plate and inoculate the LB.Incubate the broth in a shaking incubator.Pick up each of the prepared chips with tweezers and dip them in 70%ethanol five times for 1-2 seconds each to sterilize the surface of each chip. Next, rinse each chip in autoclaved deionized or DI water. Then, in LB sequentially to remove the remaining ethanol.Place the chips into six well culture plates with two chips in five millileters of LB broth per well. Then, dilute the bacterial culture until the concentration in the LB broth reaches 8 x 10 to the eighth cells per milliliter. Inoculate each well with 50 microliters of the diluted bacterial culture.Then, incubate the plates. Dip the biofilmed form chips in 10 millimolar ammonium acetate buffer five times to remove loosely attached and planktonic bacteria. Then, dry the chips in a biosafety cabinet where air flows mildly.Immediately after drying, place a chip on the loading place, 20 millileters from the carbon dioxide nozzle along the axis of the jet. Tilt the jet axis to a 40 degree angle. Next, set the stagnation pressures of carbon dioxide and nitrogen gas using gas pressure regulators.Apply the aerosol jet onto the central part of the chip. Turn on the solenoid valve for carbon dioxide for five seconds, and then turn it off for three seconds periodically using a manually controlled switch. If a nitrogen purge needs to be used, turn on the solenoid valve for a continuous supply of nitrogen.Once the setup is complete, treat the chips with carbon dioxide aerosols with and without nitrogen purges and compare chips with different aerosol treatment times. Retain the chips without treatment as negative controls. Prepare one micromolar of green fluorescence nucleic acid stain in DI water to stain bacterial cells on the control and aerosol treated chips.Put the staining solution into the wells with the chips, and incubate them. Following the incubation, gently rinse the chips with flowing DI water to remove excessive fluorescent dye. Then, dry the chips with nitrogen gas flow.Take fluorescence microscopic images of five random fields of view for each chip using an epifluorescence microscope. Finally, move on to calculations. Fluorescence microscopic images for 24 hour grown Pseudomonas putida biofilms were treated with carbon dioxide aerosols for zero, 16, 40, and 88 seconds without nitrogen purges.Removal efficiencies calculated based on the fluorescence intensities of the biofilms without nitrogen purges were higher than removal efficiencies with nitrogen purges. As you have seen in this video, the tri-phase cleaning technique effectively removes biofilm in a short period of time. It can be applied to a wide variety of bio-contaminated surfaces.

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Learn biofilm removal using carbon dioxide aerosols without nitrogen purge for surface cleaning. This rapid, reliable method utilizes momentum transfer and surfactant action on Pseudomonas putida biofilms.

Biofilm Removal Using Carbon Dioxide Aerosols without Nitrogen Purge

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