ASTM E 2180-01
The American Society for Testing and Materials (ASTM) E2180-07 Standard test method was used to determine the efficiency of incorporated antimicrobial agent in hydrophobic or polymeric materials. Days before the experiment, all required materials and equipment used were prepared and sterilized using an AMSCO 2041 Autoclave. All related procedures and methods dealing with the inactivation of E. coli were carried out in a SterilGARD Biological Safety Cabinet (Baker Company Inc). This was considered a class II cabinet because it used re-circulated HEPA filtered vertical laminar air flow within. The glass sash was kept open 20-25 cm during the experiment. Part of the air flow exhausted through the HEPA filters was re-circulated inside the room. The detailed antimicrobial efficacy testing procedures and all relevant materials used are described below:
1). A loop full of bacteria was added from the LB agar stock culture plates to a 10 mL test tube containing sterile LB medium broth. The test tube was covered with a sterile foam stopper and was then inoculated for 16 hours in the Stuart Orbital Incubator at 37°C. This step was done the day before every experiment was scheduled and methods were adjusted to obtain the required amount of cells/mL of broth for each inoculation (107 cells/mL). The no of cells was confirmed through the growth curve of that organism.
2). Augar slurry was made from 0.85 g NaCl and 0.3 g granulated dry agar (agar-agar) were dissolved in 100 mL of deionized water, and this was called the agar slurry. After sterilization for 15 minutes, the agar slurry was kept on a hot plate at 40 +/-2°C.
3). Specimens of the coated surfaces measuring 3 cm x 3cm were placed into the sterile petri dishes. Three coated surfaces per petri dish and six petri dishes in total 18 surfaces per sample. A sterile cotton swab was then dipped into sterile 0.85% saline solution to pre wet each test sample (powder coated chip). This aided in dispersing the agar slurry evenly on the coated surface. A set of control coated surfaces were also run with every experiment.
4). 1.0 ml of the inoculated culture, from step 1, was placed into the 99 mL agar slurry, and equilibrated at 40 +/- 2°C.
5). 1.0 ml of inoculated agar slurry was pipetted evenly onto each chip sample.
6). The petri dishes were then covered with their lid and edges closed with strands of pre- cut parafilm pieces. All samples except for one petri dish - “0 hour” (3 coated surfaces), were placed into the incubator at 37°C. Open reservoirs of salt water were placed inside the incubator during the experiment to avoid the drying out of the agar slurry due to low humidity.
7). The samples were removed from the incubator at the specific pre-determined contact times, and aseptically transferred from the petri dishes into the sterile 120 ml specimen cups (1 chip per cup). Each cup contained sufficient volume of Dey/Engley (D/E) neutralizing broth to form an initial 1:10 dilution of the original inoculums slurry from each coated surface. The D/E neutralization broth was used because it had the ability to neutralize antimicrobial chemicals.
8). Specimen cups containing the recovered test samples were then placed into a sonic bath and sonicated for 1 minute. Sonication was followed by 1 minute of vigorous mechanical vortexing. This step facilitated the complete release of the agar slurry from the sample.
9). Serial dilutions were made from each specimen cup with a sterile solution of 0.85% NaCl. Vortexing was done to homogenously disperse the cells before each dilution.
10). 0.1 ml was pipetted from each dilution and spread onto the agar plates.
11). The agar plates were then covered with their lids, edges wrapped with parafilm strips and placed into the incubator at 37°C for 24 hours.
12). Colony forming units on the plates were studied, counted and recorded on the following day.
References
1. Li Z, Flytzani-Stephanopoulos M, 1997, Selective catalytic reduction of nitric oxide by methane over cerium and silver ion-exchanged ZSM-5 zeolites. Applied Catalysis A: General,v ol.165,no1-2, pages15-34.
2. Niira R, Niira Y, Yamamato T, Uchida M, 1990, Antibiotic Resin Composition, USPTO Full text and Image Database. United States Patent 4938955
3. Pal A, Min X, Yu LE, Pehkonen SO, Ray MB, 2005, Photocatalytic inactivation of bioaerosols by TiO2 coated membranes, International Journal of Chemical reactor Engineering, vol.3, noA45, pages1-11
CHAPTER 4
Pre-treatment and Conditioning of Chabazites Followed by Functionalization for Making Suitable Additives used in Antimicrobial Ultra-fine Powder Coated Surface
4.1 Abstract
Silver-copper functionalized chabazites were used to develop the antimicrobial powder coated surface. Two chabazites (named as LBC and LBN) were modified to enhance their sodium content as well as ion exchange properties by conditioning with sodium nitrate. The cation exchange capacity (CEC) of the sodium-enriched LBC and LBN was improved from 3.31meq/g and 3.46meq/g for the parent zeolites to 3.77 meq/g and 4.09 meq/g, respectively. These sodium-form zeolites were used to produce silver-copper modified zeolites. The process was optimized using different combinations of the Ag and Cu ions by varying reaction time in order to achieve durable, and efficient antimicrobial additives. These additives were used in combination with a polyester resin system to produce ultrafine powder coated surfaces. Silver was utilized as an antimicrobial agent, whereas copper was used to slowdown the reduction rate of silver ion during curing process. All materials including the raw, modified and functionalized zeolites were characterized using XRD and TGA to observe the effect of treatment. ICP-OES was used for elemental analysis at every experimental stage. Color analysis proved that copper prevents silver from reduction during the curing process and maintains the color of the coated surface, which is comparable to the control surface. These coated surfaces have shown homogeneous antibacterial properties with excellent durability against E.coli for
extended periods. Cell toxicity was evident from the LDH analysis of the fresh and used antimicrobial surfaces. The controlled silver release capability of the formulated surface with high concentration of silver is promising for use in different food industry and in medical facilities.
Keywords: Zeolite, chabazite, silver, ultrafine powder coating, ion-exchange, antimicrobial, durability