Name: biofilm removal using carbon dioxide aerosols without nitrogen purge
Uploaded: 2016-11-06T15:00:00.000+00:00
Duration: 7 min 11 s
Description: Watch this Scientific Journal Video about Biofilm Removal Using Carbon Dioxide Aerosols without Nitrogen Purge at JoVE.com

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).

Biyofilm Oluşumunun için Yüzeyin hazırlanması 1. <ol><li> Mekanik kesici ile çipleri içine 304 1 mm kalınlığında paslanmaz çelik levhalar (10 × 10 mm 2) kesin. </li><li> 10 dakika her biri için aseton, metanol ve deiyonize (Dİ) su ardışık olarak cips ultrasonik temizleme gerçekleştirin. Organik kirlenmeyi kaldırmak için, cam gibi maddelerden yapılmış bir çözücü dayanıklı kap kullanın. </li><li> 3-5 saniye DI akan su ile cips durulayın. </li><li> 3-5 saniye N2 gaz akışını kullanarak cips kurutun. </li></ol> Bakteriyel kültür 2. Hazırlık <ol><li> Bir P al -80 ° C derin dondurucuda saklanan stokunun (nazikçe Prof. Sung Kuk Lee, unist, Güney Kore tarafından sağlanan) putida KT2440. </li><li> 1 dakika sonra oda sıcaklığında erimeye ve dondurulmuş stok çözeltisi üst tabaka çamur döner. stok solüsyonu erimiş tabaka haline bir döngü daldırın. </li><li> Bir Luria üzerine çizgi bakteri bu döngü kullanmak% 1.5 agar içeren -Bertani (LB) plaka. </li><li> Bakteri kolonileri büyümesi 30 ° C&#39;de gece boyunca inkübasyona bırakılır. </li><li> taze döngüsünü kullanarak plaka tek bir koloni seçin. </li><li> tek bir bakteriyel koloniden ihtiva eden halka ile 50 ml konik bir tüp içinde LB suyu 10 ml inoküle. </li><li> 30 ° C&#39;de 16 saat ve 160 rpm&#39;de sallanan bir kuluçka makinesi içinde suyu inkübe edin. </li></ol> Yüzey 3. Biyofilm Oluşumu <ol><li> Her çip yüzeyi sterilize etmek için, 1-2 saniye her 5 kez cımbız ile hazırlanan cips her Pick up ve% 70 etanol bunları batırın. cips daldırma sırasında cımbız ile yapılan emin olun. </li><li> Geri kalan etanolü çıkarmak için otoklavlanmış DI su ve LB suyu sırayla her bir çip, 1-2 sn her biri için 5 kez, batırın. </li><li> 2 yongaları ve oyuk başına 5 mi LB suyu ile 6 oyuklu kültür plakaları, bu fiş yerleştirin. </li><li> LB suyu ulaştığı konsantrasyon kadar bakteri kültürü sulandırınız8 x 10 8 hücre / ml (600 nm dalga boyunda optik yoğunluk: ~ 0.8). </li><li> Seyreltilmiş bir bakteri kültürü 50 ul her bir inoküle. </li><li> biyofilmlerin oluşturulması için 24 saat boyunca çalkalamadan 30 ° C&#39;de inkübe edin. </li></ol> 4. CO 2 Aerosol Temizleme <ol><li> 10 mM amonyum asetat tamponunda biyofilm oluşan fiş Dip (uçucu) 5 kat gevşek bağlı planktonik bakterilerin çıkması için. </li><li> Hava hafif akan bir biyo-güvenlik kabini, bu yongaları kurutun. </li><li> Hemen kurutulduktan sonra, jet ekseni boyunca CO 2 memeden 20 mm yükleme yerinde, bir çip koyun. 40 ° açı jet ekseni yatırın. </li><li> Gaz basınç regülatörleri kullanılarak, sırasıyla 5.3 MPa ve 0,7 MPa, CO 2 ve N 2 gazı durgunluk basınçları ayarlayın. </li><li> çip orta kesiminde üzerine aerosol jet uygulayın. 2 olmalıdır katı CO dahil beyaz aerosollergörülebilir. 5 saniye CO 2 için solenoid valf &quot;konulu&quot; çevirin ve ardından 3 saniye &quot;kapalı&quot; açın: periyodik elle kontrol anahtarı kullanarak (döngü 8 sn). Azot temizleme kullanılması gerekiyorsa, N2 sürekli bir kaynağı için selenoid vana açın. </li><li> 16, 40 CO 2 aerosoller cips davranın ve ile ve azot tasfiyeler olmadan 88 saniye. Negatif kontrol olarak tedavi olmadan fiş saklayın. </li></ol> Kaldırma Verimliliği için 5. Analiz <ol><li> Kontrol ve aerosol tedavi edilen yongaları üzerinde bakteri hücreleri boyamak için DI su: 1 iM yeşil floresan nükleik asit leke (480/500 nm uyarma / emisyon dalga boyu) hazırlayın. </li><li> boyama çözeltisi içinde cips yerleştirin. </li><li> 30 dakika boyunca 37 ° C&#39;de ışıksız bir kuluçka makinesi içinde fiş inkübe edin. </li><li> inkübasyonu takiben, hafifçe aşırı floresan boya kaldırmak için distile su akan fiş ile durulayın. </li><li> cips wi kurutuninci N2 gaz akışı. </li><li> Bir Epifloresans mikroskop, 40X objektif lens ve CCD kamera kullanarak her çip için görüş 5 rastgele alanlara (321 × 240 mikron 2) mikroskopik görüntüleri floresan atın. </li><li> Böyle ImageJ gibi bir görüntü işleme yazılımı kullanarak her resim için floresan yoğunluğu elde edin. ImageJ, &quot;Süreç&quot; menüsünden &quot;Çıkart Arkaplan&quot; işlevini kullanın ve &quot;Analiz&quot; menüsünden &quot;Set Ölçümleri&quot; penceresinde &quot;Entegre yoğunluğu&quot; seçeneğini seçin. floresan yoğunluğu elde etmek için &quot;Analiz&quot; menüsünden &quot;Ölçüm&quot; yapın. </li><li> Aşağıdaki formüle göre biyofilm çıkarma verimliliğini hesaplama:% 100 x (kontrol fişleri I - muamele edilmiş fiş I) / I hesaplanan floresan yoğunluğu (kontrol fişleri I). </li><li> giderim verimleri ve standart sapmalar ortalama edinin. kullanmakHer iki durum için en az 4 cips. </li></ol>

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	<li>Jain, A., Gupta, Y., Agrawal, R., Khare, P., Jain, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms+-+A+microbial+life+perspective:+A+critical+review.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofouling+control+with+ultrasound.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approaches+to+prevention,+removal+and+killing+of+biofilms.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+electric+currents+on+bacterial+detachment+and+inactivation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+laser+ablation+of+laboratory+developed+biofilms+using+an+Nd:YAG+laser+of+532+nm+wavelength.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+cleaning+techniques+used+in+the+food+industry+in+terms+of+the+removal+of+bacterial+biofilms.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+biofilms+using+carbon+dioxide+aerosols.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas-phase+removal+of+biofilms+from+various+surfaces+using+carbon+dioxide+aerosols.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+application+of+bacterial+predation+and+carbon+dioxide+aerosols+to+effectively+remove+biofilms.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+different-age+biofilms+using+carbon+dioxide+aerosols.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Carbon+Dioxide+Aerosols+on+the+Viability+of+Escherichia+coli+during+Biofilm+Dispersal.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+Dioxide+Snow+Cleaning.">Carbon Dioxide Snow Cleaning.</a> Particul. Sci.Technol. 25 (1), 37-57 (2007).</li></ol>

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 uniform focal length and reduced light scattering. The bacterial species P. putida is also commonly used in the field of biofilm research. Fluorescence intensity was employed for the quantification of the amount of biofilm attached to the surface. Two-dimensional fluorescent images were used to quantify the amount of biofilm on the surface. This method may be sufficient for 24-hr-grown thin biofilms; however, confocal 3D images should be used for thick biofilms. This method is simple and enables biological recognition of bacterial cells. Since the stain penetrates the bacterial membrane and stains nucleic acids irrespective of cell viability, the green fluorescence shows all live and dead bacterial populations. In addition, fluorescence intensity was higher if the bacterial cells were overlapping (Figure 2), which could not have been taken into account if the methods were solely based on the coverage area of the cells 5, 7, 8, 10. 
It should be noted that the stagnation pressure of the CO2 gas is a critical factor for removal efficiency. The white CO2 aerosol jet was not visible, and the removal efficiency was dramatically decreased, when the gas pressure was below 4 MPa7. This decrease is attributed to the lower proportion of solid CO2 generated. The measured biofilm removal efficiencies with and without nitrogen purges were not significantly different (t-test, p &gt; 0.55) (Figure 3), even though dry nitrogen gas is commonly used to increase the removal efficiency. In fact, for the removal of submicron particles, dry nitrogen gas has often been used to reduce the moisture condensation caused by cryogenic CO2 aerosols12. In this study, however, the moisture might positively affect the removal of the biofilm.
Although CO2 aerosols can significantly inactivate bacteria11, the dispersal of viable bacteria during the cleaning process can result in secondary contamination and infection through aerosol inhalation. Therefore, this removal method needs to be utilized in conjunction with other disinfection methods such as ultraviolet irradiation, especially if the bacteria are pathogenic. In addition to providing damage-free and residue-free cleaning12, this technique can potentially be applied to the removal of biofilms from surfaces with complicated topographies or narrow gaps and protrusions.

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

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 aerosoller P çıkarmak için kullanıldı putida SUS304 yüzeyler (Şekil 1) den Biyofilmlererde. yüzeylerin çoğu büyüme 24 saat sonra biyofilm ile kaplanmıştır. Biyofilm çoğu CO 2 aerosoller (Şekil 2) kullanılarak çıkarıldı. Beklendiği gibi, Şekil 3 artmış CO2 aerosol tedavi süresi olarak biyofilm giderme veriminde bir artış gösterir. 88 saniye tedavi süresi için, çıkarma verimi 97.74 ve% ile, sırasıyla, bir nitrojen akışı olmayan 98.29% kadar yüksek olduğu belirlenmiştir. hava sıcaklığı 23-24 ° C idi ve temizleme deneyi yapılmıştır nispi nem,% 26-50 idi. <img alt="Şekil 1" src="/files/ftp_upload/54827/54827fig1.jpg" /> CO 2 aerosoller kullanarak bir biyofilm kaldırılmasını gösteren Şekil 1. şematik. Aerosoller oluştuğunda<html> yüksek basınçlı CO2 gazı adyabatik bir venturi meme (0.4 mm) ile genişletildi ve gaz sıcaklığı hızla düştüğünde. Bu yüksek hızlı CO2 aerosoller bir biyofilm ile kaplanmış bir yüzeye uygulanır. Azot temizleme kullanıldığında, N2 gaz merkezi CO2 meme çevreleyen 8 memeleri yoluyla sağlanır. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54827/54827fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="şekil 2" src="/files/ftp_upload/54827/54827fig2.jpg" /> 24-hr-yetiştirilen P. Şekil 2. Floresan mikroskobik görüntüleri 304 paslanmaz çelik çipleri putida biyofilm. fiş temizlenmiş azot olmadan CO2 farklı tedavi kez aerosoller (0, 16, 40 ve 88 sn) ile muamele edildi. Tek renkli görüntüler yeşil renkli edildi ve beyaz ölçek çubuklar 50 mikron göstermektedir.ps: //www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54827/54827fig2large.jpg &quot;target =&quot; _ blank &quot;&gt; bu rakamın daha büyük bir versiyonunu görmek için lütfen buraya tıklayınız. <img alt="Şekil 3," src="/files/ftp_upload/54827/54827fig3.jpg" /> P. Şekil 3. giderim verimleri ve azot tasfiyeler olmadan farklı CO 2 aerosol tedavi süreleri, 304 paslanmaz çelik yüzeyler putida biyofilm. giderme verimleri biyofilm floresan yoğunlukları temel alınarak hesaplanmıştır. Hata çubukları standart sapmaları temsil etmektedir. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54827/54827fig3large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

Watch this Scientific Journal Video about Biofilm Removal Using Carbon Dioxide Aerosols without Nitrogen Purge at JoVE.com

Biofilm Removal Using Carbon Dioxide Aerosols without Nitrogen Purge

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

Biyofilm Kaldırma azotla temizleme karbon dioksiti aerosollerle

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

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6.3K Görüntüleme.  Ulsan National Institute of Science and Technology (UNIST). Biofilms on surfaces can be effectively and rapidly removed by using a periodic jet of carbon dioxide aerosols without a nitrogen purge.

Video: Biofilm Removal Using Carbon Dioxide Aerosols without Nitrogen Purge

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Co-kültür Modelleri Pseudomonas aeruginosa</emCanlı İnsan Havayolu Hücreler Yetiştirilen> Biyofilmler

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

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Preparation of Hydrophobic Metal-Organic Frameworks via Plasma Enhanced Chemical Vapor Deposition of Perfluoroalkanes for the Removal of Ammonia

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high surface area microporous materials, such as metal-organic frameworks (MOFs), unique challenges present themselves for PECVD treatments. Herein the protocol for development of a MOF that was previously unstable to humid conditions is presented. The protocol describes the synthesis of Cu-BTC (also known as HKUST-1), the treatment of Cu-BTC with PECVD of perfluoroalkanes, the aging of materials under humid conditions, and the subsequent ammonia microbreakthrough experiments on milligram quantities of microporous materials. Cu-BTC has an extremely high surface area (~1,800 m2/g) when compared to most materials or surfaces that have been previously treated by PECVD methods. Parameters such as chamber pressure and treatment time are extremely important to ensure the perfluoroalkane plasma penetrates to and reacts with the inner MOF surfaces. Furthermore, the protocol for ammonia microbreakthrough experiments set forth here can be utilized for a variety of test gases and microporous materials.

Herein the procedures for plasma enhanced chemical vapor deposition of perfluoroalkanes on microporous materials such as metal-organic frameworks to enhance their stability and hydrophobicity are described. Furthermore, breakthrough testing of milligram quantities of samples is described in detail.

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Kimya

Preparation of Biopolymer Aerogels Using Green Solvents

Although the first reports on aerogels made by Kistler1 in the 1930s dealt with aerogels from both inorganic oxides (silica and others) and biopolymers (gelatin, agar, cellulose), only recently have biomasses been recognized as an abundant source of chemically diverse macromolecules for functional aerogel materials. Biopolymer aerogels (pectin, alginate, chitosan, cellulose, etc.) exhibit both specific inheritable functions of starting biopolymers and distinctive features of aerogels (80-99% porosity and specific surface up to 800 m2/g). This synergy of properties makes biopolymer aerogels promising candidates for a wide gamut of applications such as thermal insulation, tissue engineering and regenerative medicine, drug delivery systems, functional foods, catalysts, adsorbents and sensors. This work demonstrates the use of pressurized carbon dioxide (5 MPa) for the ionic cross linking of amidated pectin into hydrogels. Initially a biopolymer/salt dispersion is prepared in water. Under pressurized CO2 conditions, the pH of the biopolymer solution is lowered to 3 which releases the crosslinking cations from the salt to bind with the biopolymer yielding hydrogels. Solvent exchange to ethanol and further supercritical CO2 drying (10 - 12 MPa) yield aerogels. Obtained aerogels are ultra-porous with low density (as low as 0.02 g/cm3), high specific surface area (350 - 500 m2/g) and pore volume (3 - 7 cm3/g for pore sizes less than 150 nm).

A new way for the production of biopolymer-based aerogels by carbon dioxide (CO2) induced gelation is shown. The technique utilizes pressurized carbon dioxide (5 MPa) for the production of biopolymer hydrogels and supercritical CO2 (12 MPa) to convert gels into aerogels. The only solvents needed besides CO2 are water and ethanol.

Yeşil Solventler kullanma Biyopolimer Aerogels Hazırlanması

Although the first reports on aerogels made by Kistler1 in the 1930s dealt with aerogels from both inorganic oxides (silica and others) and biopolymers ...

Non-equilibrium Microwave Plasma for Efficient High Temperature Chemistry

A flowing microwave plasma based methodology for converting electric energy into internal and/or translational modes of stable molecules with the purpose of efficiently driving non-equilibrium chemistry is discussed. The advantage of a flowing plasma reactor is that continuous chemical processes can be driven with the flexibility of startup times in the seconds timescale. The plasma approach is generically suitable for conversion/activation of stable molecules such as CO2, N2 and CH4. Here the reduction of CO2 to CO is used as a model system: the complementary diagnostics illustrate how a baseline thermodynamic equilibrium conversion can be exceeded by the intrinsic non-equilibrium from high vibrational excitation. Laser (Rayleigh) scattering is used to measure the reactor temperature and Fourier Transform Infrared Spectroscopy (FTIR) to characterize in situ internal (vibrational) excitation as well as the effluent composition to monitor conversion and selectivity.

This article describes a flowing microwave reactor that is used to drive efficient non-equilibrium chemistry for the application of conversion/activation of stable molecules such as CO2, N2 and CH4. The goal of the procedure described here is to measure the in situ gas temperature and gas conversion.

Verimli Yüksek Sıcaklık Kimyası İçin Denge Dışı Mikrodalga Plazma

A flowing microwave plasma based methodology for converting electric energy into internal and/or translational modes of stable molecules with the purpose ...

Precision Milling of Carbon Nanotube Forests Using Low Pressure Scanning Electron Microscopy

A nanoscale fabrication technique appropriate for milling carbon nanotube (CNT) forests is described. The technique utilizes an environmental scanning electron microscope (ESEM) operating with a low pressure water vapor ambient. In this technique, a portion of the electron beam interacts with the water vapor in the vicinity of the CNT sample, dissociating the water molecules into hydroxyl radicals and other species by radiolysis. The remainder of the electron beam interacts with the CNT forest sample, making it susceptible to oxidation from the chemical products of radiolysis. This technique may be used to trim a selected region of an individual CNT, or it may be used to remove hundreds of cubic microns of material by adjusting ESEM parameters. The machining resolution is similar to the imaging resolution of the ESEM itself. The technique produces only small quantities of carbon residue along the boundaries of the cutting zone, with minimal effect on the native structural morphology of the CNT forest.

Low pressure scanning electron microscopy in a water vapor ambient is used to machine nanoscale to microscale features in carbon nanotube forests.

Alçak Basınç Taramalı Elektron Mikroskobu kullanılarak Karbon Nanotüp Ormanlar Hassas freze

A nanoscale fabrication technique appropriate for milling carbon nanotube (CNT) forests is described. The technique utilizes an environmental scanning ...

Mühendislik

Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

Applications such as sensors, batteries, and fuel cells have been improved through the use of highly porous aerogels when functional compounds are encapsulated within the aerogels. However, few reports on encapsulating proteins within sol&#8211;gels that are processed to form aerogels exist. A procedure for encapsulating cytochrome c (cyt. c) in silica (SiO2) sol-gels that are supercritically processed to form bioaerogels with gas-phase activity for nitric oxide (NO) is presented. Cyt. c is added to a mixed silica sol under controlled protein concentration and buffer strength conditions. The sol mixture is then gelled and the liquid filling the gel pores is replaced through a series of solvent exchanges with liquid carbon dioxide. The carbon dioxide is brought to its critical point and vented off to form dry aerogels with cyt. c encapsulated inside. These bioaerogels are characterized with UV-visible spectroscopy and circular dichroism spectroscopy and can be used to detect the presence of gas-phase nitric oxide. The success of this procedure depends on regulating the cyt. c concentration and the buffer concentration and does not require other components such as metal nanoparticles. It may be possible to encapsulate other proteins using a similar approach making this procedure important for potential future bioanalytical device development.

Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

This procedure describes how to encapsulate cytochrome c (cyt. c) in silica (SiO2) sol-gels, process these gels to form bioaerogels, and use these bioaerogels to rapidly recognize nitric oxide (NO) through a gas-phase reaction. This type of protocol may aid in the future development of biosensors or other bioanalytical devices.

Sitokrom Kapsüllenen<em&gt; C</em&gt; Metal Nanopartiküller olmadan Silika Aerojel Nanoarchitectures Gaz faz biyoaktivitesinin İstinat ederken

Applications such as sensors, batteries, and fuel cells have been improved through the use of highly porous aerogels when functional compounds are ...

Biyomühendislik

Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases

The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The persistence of bacterial infection is often associated with quorum-sensing-mediated biofilm formation. Thus, the disruption of this signaling circuit presents an attractive anti-virulence strategy. Quorum-quenching lactonases have been reported to be effective disrupters of quorum-sensing circuits. However, there have been very few reports of the effective use of these enzymes in disrupting bacterial biofilm formation. This protocol describes a method to disrupt biofilm formation in a clinically relevant A. baumannii S1 strain through the use of an engineered quorum-quenching lactonase. Acinetobacter baumannii is a major human pathogen implicated in serious hospital-acquired infections globally and its virulence is attributed predominantly to its biofilm's tenacity. The engineered lactonase treatment achieved significant A. baumannii S1 biofilm reduction. This study also showed the possibility of using engineered quorum-quenching enzymes in future treatment of biofilm-mediated bacterial diseases. Lastly, the method may be used to evaluate the competency of promising quorum-quenching enzymes.

Quorum-quenching enzymes are anti-virulent and anti-bacterial options that can mitigate pathogenesis without risk of incurring resistance, by preventing the expression of virulence factors and genes associated with antibiotic resistance and biofilm formation. In this study, we report a method that demonstrates the efficacy of quorum-quenching enzymes in bacterial biofilm disruption.

Engineered Nisabı giderici Lactonases kullanarak Patojen Biyofilmler Anti-öldürücü bozulması

The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

Araştırma yöntemleri<em&gt; B. subtilis</emKüçük Molekül Biyofilm İnhibitörleri Karakterizasyonu için Bir Model Olarak&gt; Biyofilmler

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural integrity. The nosocomial pathogen, Pseudomonas aeruginosa, is known to adopt a biofilm mode of growth, causing chronic pulmonary infection in patients with cystic fibrosis (CF). The computer program, COMSTAT, is a useful tool for quantifying antimicrobial-induced changes in P. aeruginosa biofilm architecture by extracting data from three-dimensional confocal images. However, standardized operation of the software is less commonly addressed, which is important for optimal reporting of biofilm behavior and cross-center comparison. Thus, the aim of this protocol is to provide a simple and reproducible framework for quantifying in vitro biofilm structures under varying antimicrobial conditions via COMSTAT. The technique is modeled using a CF P. aeruginosa isolate, grown in the form of biofilm replicates, and exposed to tobramycin and the anti-Psl monoclonal antibody, Psl0096. The step-by-step approach aims to reduce user ambiguity and minimize the chance of overlooking crucial image-processing steps. Specifically, the protocol emphasizes the elimination of subjective variations associated with the manual operation of COMSTAT, including image segmentation and the selection of appropriate quantitative analysis functions. Although this method requires users to spend additional time processing confocal images prior to running COMSTAT, it helps minimize misrepresented biofilm heterogenicity in automated outputs.

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Antimicrobial-induced changes to Pseudomonas aeruginosa biofilm architecture differ among clinical isolates cultured from patients with cystic fibrosis and chronic pulmonary infection. Following confocal microscopy, COMSTAT software can be utilized to quantify variations in biofilm architecture (e.g., surface area, thickness, biomass) for individual isolates to assess the efficacy of anti-infective agents.

Antimikrobiyallerin İn vitro Biyofilm Mimarisi Üzerindeki Etkilerinin COMSTAT Yazılımı Kullanılarak Ölçülmesi

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural ...

<meta charset="utf8"></head><body>Antimikrobiyal Test ve Mikrobiyal Dinamik Analizi için Uyarlanabilir CF Modeli

Development of a Polymicrobial Colony Biofilm Model to Test Antimicrobials in Cystic Fibrosis

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as colony-forming units or metabolic activity. Furthermore, many biofilm models do not reflect the biological and physiochemical properties of the human host environment. This is an important issue in many conditions, but most noticeably in cystic fibrosis (CF). A large proportion of people with CF suffer from both chronic and intermittent infections, and in vitro, antibiotic susceptibility testing poorly correlates with patient treatment outcomes. Some biofilm models incorporate CF lung-relevant media, including synthetic sputum mimics, but do not consider the polymicrobial nature of the environment, which alters biofilm architecture, physiology, and the way microbes respond to treatment. The solid-air interface colony biofilm model described here is highly adaptable and incorporates both CF-relevant media and a polymicrobial context. This model can also be used for mid-throughput screening of antimicrobials and to study their effect on polymicrobial dynamics. Output measurements from the model can be colony-forming units, metabolic activity, and confocal microscopy analysis. The model can easily be adapted to different microorganisms, media, temperatures, and variable oxygen conditions and can be used to test a wide range of chemical, biological, and physical treatments.

<meta charset="utf8"></head><body>Kistik Fibrozisde Antimikrobiyalleri Test Etmek İçin Polimikrobiyal Koloni Biyofilm Modelinin Geliştirilmesi

This methodology allows for the establishment of a polymicrobial biofilm model in cystic fibrosis for antimicrobial sensitivity testing in research and clinical laboratories. This model provides accurate and reliable results over a range of outputs.

Kistik Fibrozisde Antimikrobiyalleri Test Etmek İçin Polimikrobiyal Koloni Biyofilm Modelinin Geliştirilmesi

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as ...

Biyofilm Kaldırma azotla temizleme karbon dioksiti aerosollerle

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Co-kültür Modelleri Pseudomonas aeruginosa Biyofilmler