An Antimicrobial Fabric Using Nano-Herbal Encapsulation of Essential Oils

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 neutralizes 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 utilized within a short time. The application of the technique in the manufacturing of protective coat 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 coat in biosafety level 2 labs.

Our future research questions would focus on the effectiveness of preventing microbial contamination in the long run in continuous usage. To begin, prepare chitosan solution by dissolving 0.6 grams of chitosan flakes and 50 milliliters of 1%acetic acid. Agitate overnight at room temperature to get a homogenous emulsion.

Add 0.5 grams of Tween 80 and stir at 60 degrees Celsius for two hours at 1000 RPM to get a homogenous solution. After bringing the solution to room temperature, add 0.75 grams of bioactive compound carvacrol or thymol dropwise or gradually while stirring the mixture for 20 minutes. Add 50 milliliters of pentasodium tripolyphosphate dropwise to the mixture while stirring at room temperature.

Continue stirring for 30 minutes to get a homogenous emulsion. For purification, centrifuge the emulsion at 10, 000g for 30 minutes at four degrees Celsius. Decant the supernatant and collect the form nanoparticles.

Wash the particles using aqueous Tween 80 with double the volume of pellets formed to remove the unbound or free bioactive compounds. Then wash the particles with deionized water twice to get rid of impurities. Reconstitute the nanoparticles by resuspending the pellet in 30 milliliters of deionized water.

Tick a nanoparticles treated fabric and cut it into 50 millimeters by 25 millimeter swatches. Inoculate the microbial cultures from fresh purity plates to Trypticase Soy Broth at 35 degrees Celsius overnight with moderate shaking. Dilute the overnight cultures to 150 million CFU per milliliter, or corresponding to the turbidity of 0.5 McFarland standard.

To inoculate the diluted culture with a four millimeter sterile loop, load a loop full of broth culture and transfer it to the surface of the Mueller-Hinton agar or MHA agar plate by making five parallel streaks of six centimeters in length and one centimeter apart from each other. Gently press the fabric swatch using a sterile spatula across the five streaks so that the fabric is in the center and touches all five streak lines. Incubate at 35 degrees Celsius for 24 hours.

For the qualitative evaluation of antimicrobial efficacy, examine the incubated plate for the interruption of growth along the streaks beyond the edges of the fabric. Calculate the average width of a zone of inhibition along the streak line W on either side of the fabric swatch using the equation shown on the screen. The results of the parallel streak method are presented in this figure.

The untreated fabric and the thymol nanoparticles coated fabric against S-aureus and the untreated fabric and the thymol nanoparticles coated fabric against C-albicans are shown here. These results show clear zones for the treated fabric swatches. Tick a nanoparticles treated fabric and cut it into 50 millimeters by 50 millimeters square swatches.

Prepare the overnight cultures of microbes of interest by inoculating the isolated colonies from the purity plates to sterile Trypticase Soy Broth. Incubate at 35 degrees Celsius for 18 to 24 hours. Dilute the overnight cultures to 150 million CFU per milliliter or corresponding to the turbidity of 0.5 McFarland standard.

Next, to determine the appropriate volume of cultures for spiking, add a series of volumes of the diluted broth culture onto the fabric swatches and choose the volume such that the fabric swatch absorbs the water fully and leaves no residual or free liquid. Spike the volume of each culture onto the swatches placed in sterile petri plates. Similarly, perform the negative control with the same type of untreated fabric swatches for each corresponding test.

To recover microbes by viable plate counts, air dry the inoculated swatches inside the petri plates at room temperature for the required contact periods to be tested. Transfer the swatches aseptically to separate sterile centrifuge tubes and screw the caps tightly. Add Letheen Broth or any relevant neutralizing buffers to make a 1 to 100 dilution.

After closing centrifuge tubes, vortex for one minute at a medium speed. Serially dilute the suspension with the sterile PBS and subsequent 1 to 10 dilution such that the colony counts of the untreated group becomes too low to count. Plate the dilution on appropriate media plates, which support the growth of the microorganism or any media that optimize the growth and provide good contrast to make the colony counting accurate.

Incubate the bacterial plates at 35 degrees Celsius for two days and the fungal plates at room temperature for five days. Count the viable colony forming units directly using a colony counter or imaging software. Calculate the microbial log reduction R due to the antimicrobial fabric using the equation shown on the screen.

Here, A is the log value of the number of colony forming units recovered from untreated fabric, and B is the same for treated fabric. The log reduction results of antimicrobial fabric coated with thymol encapsulated chitosan nanoparticles tested against S-aureus on blood agar, E.coli on purple lactose agar, P-aeruginosa on cetrimide agar, and C-albicans on Sabouraud dextrose agar are shown here. The pathogens were spiked on untreated and treated swatches for 30 minutes and recovered upon neutralization and dilution plating.

The dilution ratios are shown between the treated and untreated series. The log reduction of three bacteria in one fungus due to the contact of antimicrobial fabrics impregnated with two bioactive compounds are shown here. The antimicrobial efficacy is weakened after washed cycles for both carvacrol and thymol-coated fabrics.