Name: Quantitative Screening of Nanoparticle-Treated Fabric: The Log Reduction Method
Uploaded: 2023-04-07T14:35:00
Description: Watch this Scientific Journal Video about Quantitative Screening of Nanoparticle-Treated Fabric: The Log Reduction Method at JoVE.com

The video describes a protocol for testing the antimicrobial efficacy of nanoparticles-treated fabrics. It outlines preparing microbial cultures, spiking fabric swatches, conducting control experiments, and analyzing colony counts to calculate microbial log reduction. The results highlight the effectiveness of thymol-encapsulated chitosan nanoparticles against specific pathogens and their decreased efficacy after wash cycles.

Quantitative Screening of Nanoparticle-Treated Fabric: The Log Reduction Method

quantitative screening of nanoparticle-treated fabric: the log reduction method

Antimikrobielle Wirksamkeit von nano-kräuterbeschichteten Geweben

The protective white coat is a mandatory personal protective equipment (PPE) item in microbiology laboratories and healthcare facilities, and it protects from direct exposure to pathogens, spills, and burns. These cotton coats promote microbial growth due to many factors-the woven fabric provides attachment sites and aeration, cotton and starch used in the manufacturing process along with exfoliated epithelial cells from the user supply nutrients, and the proximity to the user gives warmth and moisture. The accumulation of microbes on textiles can also cause health problems such as allergies and nosocomial infection, unpleasant odors, and fabric deterioration1.
Unlike regular clothing, protective coats are infrequently washed or disinfected, as found in many surveys2,3. Many studies show evidence of lab coats acting as a vector of microbial transmission and the risk of nosocomial infections in the healthcare setting2,4, particularly resistant strains3 such as methicillin-resistant Staphylococcus aureus (MRSA); thus, they raise health concerns of PPE, which is meant to protect from microbial contamination. There are not enough cross-sectional studies on lab coat-associated infections in the context of Biosafety Level 2 (BSL-2) facilities or microbiology teaching labs, but many regulatory authorities restrict the use of lab coats within the containment level. However, many academic institutions in North America struggle to meet the requirements due to practical constraints, such as laundering and storing inside the facility, the incidents of wearing lab coats in public areas such as cafeterias and libraries are common. One practical solution to these issues is the application of antimicrobial agents in textile finishing.
Antimicrobial fabrics are gaining increasing popularity in sportswear, activewear, and socks, mainly intended to reduce body odor. However, the use of these fabrics is not common in PPE development, except for some silver-coated cotton masks and healthcare garments5. We report the development of an antimicrobial fabric for lab coats, which inhibits common pathogens found in BSL-2 labs and renders effective protection from the cross-contamination of common pathogens.
Currently, a variety of antimicrobial fabrics and finishings are available in the market, but most of these use heavy metal colloidal particles (e.g., silver, copper, zinc), organometallics, or synthetic chemicals such as triclosan and quaternary ammonium compounds, which are not environmentally friendly1 and may lead to health issues such as skin irritation and allergies6. Some synthetic formulations pose concerns due to non-target microbes, such as normal flora or inducing antimicrobial resistance (AMR). The US Food and Drug Administration (FDA) regulates commercial antimicrobial fabrics, which must be non-toxic to the user and free from eco-toxicity. Therefore, antimicrobial fabrics based on natural biocides that inhibit a broad spectrum of microbes are preferable. Essential oils (EOs) are used widely as antimicrobial and therapeutic agents, but their use in antimicrobial finishing is limited due to their durability6,7,8. Based on our knowledge and market research on nano-herbal finishing8, no herbal-based antimicrobial fabric is commercially available. This is because synthetic coatings are easy to manufacture and have long durability. A few nano-herbal-coated textiles reported only for research purposes include neem7, moringa9, and curry leaves9.
The present study uses two bioactive components extracted from oregano EOs, carvacrol and thymol, which are effective against a wide range of bacterial pathogens and viruses but are generally recognized as safe for humans10. However, these bioactive components are volatile, and therefore their antimicrobial potential is short-lived if applied directly to the fabric. Nano-herbal encapsulation is a process in which bioactive components or drugs are loaded inside a polymeric shell that protects the core from environmental degradation, and thus enhances the shelf life. In addition, the small size of the polymeric particles, which generally range from 10 nm to 100 nm, enhances the efficacy of the application and slows the release of the bioactive compounds onto the fabric. These bioactive compounds are used for various purposes, such as food preservation10, but not for textile coating.
Among many polymeric encapsulants, chitosan is an attractive candidate due to many of its attributes, such as nontoxicity, biodegradability, mucoadhesivity, and biocompatibility11. It is a natural polysaccharide, obtained by the deacetylation process from chitin, which is found in seashells and fungal cell walls. It is used in biochemical and food preservation applications such as drug or protein delivery11,12,13, controlled release14, and antimicrobial films10. Chitosan is not readily soluble in water but forms a colloidal suspension in acidic media. Bioactive molecules are loaded into chitosan nanoparticles (NPs) by a simple two-step ionic gelation method14,15,16. In this process, hydrophobic bioactive compounds such as carvacrol and thymol form an oil-in-water emulsion, which is aided by a surfactant, Tween 80. Subsequently, a polyanionic compound, pentasodium tripolyphosphate (TPP), is used to form the cross-linkages between the amino groups along the polycationic polymer molecules and phosphate groups of TPP molecules to stabilize the complex. This complexation process solidifies the bioactive compounds within the matrix of chitosan, which is subsequently purified and coated onto cotton swatches to produce antimicrobial fabric.
The nano-formulations must be tested first for antimicrobial effectiveness in emulsion form before being applied to the fabric. This can be conveniently evaluated by a qualitative method, such as Kirby-Bauer disk diffusion, well diffusion, and the cylinder plate assay. However, the cylinder plate assay17 provides the flexibility to load varying volumes of the formulation and compare the zone of clearance. In this method, the antimicrobial formulations are loaded in stainless steel cylinders and placed on a soft agar layer, which is inoculated with the test microorganism or pathogen. The diameter of the zone of clearance produced against the test organism is proportional to the inhibitory potential of the antimicrobial formulation, and therefore can be used as an alternative to broth dilution methods. However, the size of the clear zones is only a comparative or qualitative measure within a specific plate unless specific standards are maintained. Antimicrobial agents act against the pathogens either by inhibiting their growth (biostatic) or killing the cells (biocidal), which can be quantified by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), respectively. However, the efficacy and behavior of the bioactive chemicals are different in their formulations (liquid state) and when coated on a substrate such as a fabric18. This is because multiple factors play a role in the efficacy, such as the stability of the adherence of the antimicrobial agents to the fabric, moisture content, substrate type, and adherence of the microbes. If the intended purpose is only bacteriostatic activity, a qualitative assay such as the &#34;Parallel Streak Method&#34;19 can provide a relatively quick and easy evaluation of diffusible antimicrobial formulation. However, if the bactericidal effects are to be determined, &#34;Assessment of Antibacterial Finishes on Textile Materials&#34;20 can be employed, which provides the log reduction of the spiked pathogen.

Autor im Rampenlicht: Ein antimikrobielles Gewebe unter Verwendung der Nano-Kräuterverkapselung von ätherischen Ölen  

Ein antimikrobielles Gewebe mit nanopflanzlicher Verkapselung ätherischer Öle

This research outlines the protocol to develop a safe and eco-friendly antimicrobial fabric which can be used to design lab coats. The study answers many key questions in the field of antimicrobial fabric development. For example, how effectively the treated fabric neutralizers pathogens.The key challenge in antimicrobial fabric development is to choose the right ratios of bioactive compounds without compromising antimicrobial efficacy while maintaining skin friendly finishes. Our antimicrobial fabric provides effective protection against four human pathogens with up to four log reduction, or simply 99.99%effectiveness. It also resists up to 10 wash cycles while retaining an efficacy of up to 90%The major advantage of this method over existing standard techniques is that it provides a simplified and realistic approach to accurately evaluate the efficacy of antimicrobial fabrics.It better represents the typical scenarios of lab coat usage, such as any accidental microbial spills that must be neutralized within a short time. The application of the technique in the manufacturing of protective coats will reduce the biohazard risk in biosafety labs and nosocomial infections in healthcare setting. We are in the process of testing full length antimicrobial lab coats in Biosafety Level 2 labs.Our future research questions would focus on the effectiveness of preventing microbial contamination in the long run and continuous usage.

Watch this Scientific Journal Video about Quantitative Screening of Nanoparticle-Treated Fabric: The Log Reduction Method at JoVE.com

Quantitatives Screening von nanopartikelbehandelten Geweben: Die Log-Reduktionsmethode

Nehmen Sie einen mit Nanopartikeln behandelten Stoff und schneiden Sie ihn in 50 Millimeter mal 50 Millimeter große quadratische Muster. Bereiten Sie die Overnight-Kulturen der interessierenden Mikroben vor, indem Sie die isolierten Kolonien von den Reinheitsplatten in sterile Trypticase-Sojabrühe impfen. Bei 35 Grad Celsius 18 bis 24 Stunden inkubieren.Verdünnen Sie die Kulturen über Nacht auf 150 Millionen KBE pro Milliliter oder entsprechend der Trübung von 0,5 McFarland-Standard. Um das geeignete Volumen der Kulturen für das Aufspießen zu bestimmen, geben Sie als Nächstes eine Reihe von Volumina der verdünnten Brühenkultur auf die Stoffmuster und wählen Sie das Volumen so, dass das Stoffmuster das Wasser vollständig aufnimmt und keine Reste oder freie Flüssigkeit hinterlässt. Streuen Sie das Volumen jeder Kultur auf die Proben, die in sterile Petriplatten gelegt werden.Führen Sie in ähnlicher Weise die Negativkontrolle mit der gleichen Art von unbehandelten Stoffmustern für jeden entsprechenden Test durch. Um Mikroben durch brauchbare Keimzahlen zu gewinnen, trocknen Sie die beimpften Proben in den Petriplatten bei Raumtemperatur an der Luft für die erforderlichen Kontaktzeiten, um sie zu testen. Übertragen Sie die Proben aseptisch in separate sterile Zentrifugenröhrchen und schrauben Sie die Kappen fest.Fügen Sie Letheen-Brühe oder andere relevante neutralisierende Puffer hinzu, um eine Verdünnung von 1:100 zu erreichen. Nach dem Verschließen der Zentrifugenröhrchen eine Minute lang bei mittlerer Geschwindigkeit vortexen. Die Suspension wird in nachfolgenden 1:10-Verdünnungen seriell mit dem sterilen PBS verdünnt, so dass die Koloniezahlen der unbehandelten Gruppe zu niedrig werden, um gezählt zu werden.Pflanzen Sie die Verdünnungen auf geeignete Medienplatten, die das Wachstum des Mikroorganismus unterstützen, oder auf andere Medien, die das Wachstum optimieren und einen guten Kontrast bieten, um die Koloniezählung genau zu machen. Inkubieren Sie die Bakterienplatten zwei Tage lang bei 35 Grad Celsius und die Pilzplatten fünf Tage lang bei Raumtemperatur. Zählen Sie die lebensfähigen koloniebildenden Einheiten direkt mit einem Koloniezähler oder einer Bildgebungssoftware.Berechnen Sie die mikrobielle logarithmische Reduktion R aufgrund des antimikrobiellen Gewebes anhand der auf dem Bildschirm angezeigten Gleichung. Hier ist A der logarithmische Wert der Anzahl der koloniebildenden Einheiten, die aus unbehandeltem Gewebe gewonnen werden, und B ist für behandeltes Gewebe gleich. Die logarithmischen Reduktionsergebnisse von antimikrobiellen Geweben, die mit Thymol-verkapselten Chitosan-Nanopartikeln beschichtet sind, wurden gegen S. aureus auf Blutagar, E. coli auf violettem Laktose-Agar, P. aeruginosa auf Cetrimid-Agar und C. albicans auf Sabouraud-Dextrose-Agar getestet.Die Erreger wurden 30 Minuten lang auf unbehandelte und behandelte Proben gespickt und nach Neutralisation und Verdünnungsplattierung wiederhergestellt. Die Verdünnungsverhältnisse zwischen der behandelten und der unbehandelten Reihe sind dargestellt. Die logarithmische Reduktion von drei Bakterien und einem Pilz durch den Kontakt von antimikrobiellen Geweben, die mit zwei bioaktiven Verbindungen imprägniert sind, wird hier gezeigt.Die antimikrobielle Wirksamkeit wird nach dem Waschgang sowohl bei Carvacrol- als auch bei Thymol-beschichteten Stoffen geschwächt.

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