We present a protocol for the antimicrobial characterization of advanced materials. Here, the antimicrobial activity on material surfaces is measured by two methods that complement each other: one is based on the agar disk diffusion test, and the other is a standard procedure based on the ISO 22196:2007 norm.
This method can help answer key questions in the material engineering field, such as antimicrobial properties, which are very desirable for many bioengineering applications. This technique can be utilized as a consistent protocol across laboratories to help less experienced researchers who require in-depth step-by-step procedures to follow for accurate results. We first had the idea for this method when we had started looking for antimicrobial protocols, and realized there were no detailed protocols on this one in the literature.
In this study, four advanced materials with different chemical natures and a control material, will be tested for antimicrobial activity. Prepare each material by cutting it into 10 mm diameter disks. Next, sterilize each specimen by immersion in 70%ethanol for 10 minutes, followed by ultra-violet radiation for one hour per side.
Three different microorganisms will be used to test the antimicrobial activity of the advanced materials. The gram positive bacteria, Staphylococcus aureus, the gram negative bacteria, Escherichia coli, and the yeast, Candida albicans. Using a sterile cotton swab, resuspend a few colonies of each microorganism in 25 mL of tryptic soy broth, or TSB, contained in a sterile centrifuge tube.
Vortex for one minute to achieve uniform mixing. Measure the absorbence at 540 nm for each microorganism culture with a spectrophotometer. Adjust the concentration of each microorganism to a suitable number of colony forming units, or CFU per milliliter.
Vortex the microbial suspension for five seconds to improve the microorganism dispersion, and briefly submerge a sterile cotton swab in this microbial suspension. Remove the excess liquid from the swab by pressing the swab against the tube wall containing the culture. Evenly streak each microbial broth suspension with the sterile cotton swab onto the surface of a tryptic soy agar, or TSA plate, in three planes to cover the whole surface with the microorganism.
Let the plates dry for five minutes after the inoculation. Leave the microbial broth suspensions on the bench top for use later to check microbial suspension concentration and purity. Sterilize a pair of tweezers by immersing it in a beaker with 96%ethanol, and then flaming with an alcohol burner.
Use the sterile tweezers to place the sample disks to be tested at the center of the TSA plates. Incubate the TSA plates in an inverted position at 37 degrees Celsius for 24 hours. To analyze the diffusion test results, measure the diameter of the inhibition zone and the sample disk diameter with a digital caliper, or by taking a photograph and using a suitable image processing software.
This procedure is necessary for verifying that the microbial concentrations determined earlier are correct, and ensuring the absence of microbial environmental contamination. Using a micropipette with suitable autoclaved tips and aseptic conditions, dispense sterile TSB into sterile microcentrifuge tubes. Include a sample that will be utilized as a negative control.
Perform six decimal serial dilutions with the microbial broth suspensions utilized earlier for streaking the TSA plates. Using a pre-sterilized Drigalski spatula, spread 100 l of each selected dilution on a TSA plate. Spread 100 l of TSB medium without microorganism on a TSA plate as a negative control.
Incubate the TSA plates at 37 degrees Celsius for 24 hours. On the following day, count the number of colonies to check that the CFU per milliliter is similar to that determined earlier. Check the negative control plate to confirm that there is no microbial environmental contamination.
Begin this procedure by culturing the different microorganisms to be tested in TSB, in an orbital shaker at 37 degrees Celsius overnight. On the following day, measure the OD540 of each overnight culture, and then dilute each culture in 20 mL of TSB in a 50 mL pre-sterilized centrifuge tube to a concentration of approximately ten to the sixth CFU per milliliter. Place four disks of each type of material and four control disks in separate wells of a sterile 48-well plate.
Pipette 150 l of the microbial suspension onto each disk surface. Incubate the plate in the 37 degree Celsius incubator for 24 hours. After the 48-well plate has been incubated for 24 hours, pipette 850 l of sterile PBS onto the surface of each of the four sample disks and four control disks, and mix it with the microbial suspension.
Collect each PBS microbial suspension mixture and each disk from the 48-well plate, and transfer to a 15 mL pre-sterilized tube. Vortex the PBS microbial suspension mixtures and disks for one minute. Sonicate at 50 Hz for five minutes, and vortex again for one minute to ensure that no viable microorganisms remain adhered to the material surface.
Perform decimal serial dilutions of the sonicated cultures and sterile microcentrifuge tubes containing TSB. Spread 100 l of each dilution on a TSA plate, and incubate the plates aerobically at 37 degrees Celsius for 24 hours. After 24 hours, count the number of colonies and express this number in CFU per milliliter.
The contact method results are then analyzed as described in the text protocol. The results of the agar disk diffusion tests show non antimicrobial activity for the first material, and increasing antibacterial activity against S.aureus and E.coli for the other three materials. This graph shows the normalized halo for each material disk against S.aureus and E.coli after 24 hours of incubation.
The results of the contact method also indicate non antimicrobial activity for the first material, and increasing antibacterial activity against gram positive and gram negative bacteria for the other three materials. C is the viable bacteria recovered from the control disk after 24 hours of incubation. This graph shows the percentage loss of viability for the four materials against S.aureus and E.coli on the material surfaces.
Sample M1 exhibited no antimicrobial activity. Although not shown here, none of the four materials are able to inhibit the growth of the yeast, Candida albicans, by other method. After watching this video you should have a good understanding of how to determine antimicrobial activity by these two complementary methods.
