Take 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 1:100 dilution. After closing centrifuge tubes, vortex for one minute at a medium speed. Serially dilute the suspension with the sterile PBS in subsequent 1:10 dilutions, such that the colony counts of the untreated group becomes too low to count.
Plant the dilutions 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 and one fungus due to the contact of antimicrobial fabrics impregnated with two bioactive compounds are shown here.
The antimicrobial efficacy is weakened after wash cycles for both carvacrol and thymol-coated fabrics.
