Investigations of the antibacterial properties of an anodized titanium alloy.
Titanium and its alloys are resistant to pitting, crevice corrosion and general corrosion and are suitable for use in cooling water systems in a wide range of clean and polluted waters. This superior corrosion resistance of titanium is due to the spontaneous formation of a highly tenacious oxide film, when exposed to mere atmospheric moisture . This oxide film is self healing and exceptionally stable over a wide range of redox potentials . Titanium alloys are also fully resistant to chemical species associated with microorganisms like ammoniam, sulfides, nitrites, ferrous compounds and organo-sulphur compounds and acidic conditions associated with aerobic activity of microbes. Ti6A14V alloys of titanium are being used as material for medical implants.
However, titanium and its alloys are not biotoxic and permit adhesion and growth of micro and macrofoulants. These microbes can form biofilms on the material surfaces and macro organisms can block the coolant's efficiency by reducing flow and heat transfer. Generally the biofouling of titanium in cooling water systems are combated by mechanical cleaning and chlorination, but this requires round-the clock chlorination and frequent mechanical cleaning for effective control. In the respect, surf act modification of titanium with a view on imparting it antibacterial properties assumes significance.
Anodic oxidation is a commonly used surface treatment, especially on aluminium alloys for structural applications to improve the corrosion or ear resistance . The application of anodix oxidation to the surfaces of titanium alloys is more recent. Among the benefits sited for anodizing, iron free surface, increased corrosion resistance and increased resistance to hydriding are note worthy.
Among the semiconductors known so far, titanium dioxide has been known to be the best catalyst. The anatase type of crystal, which is formed during anodisation  is reported to possess a strong oxidizing force under light irradiation below 380nm. The property exhibited is called photocatalytic activity. This property of anatase is now being effectively utilized to remove and decompose malodorants by using Ti[O.sub.2]. Significant applications with respect to this property include, the photochemical treatment of simulated dye-house effluents by novel Ti[O.sub.2] photocatalysts , inactivation of Coliforms and viruses in secondary waste water effluent  and degradation of ethylene diamine tetra acetic acid, an industrial and domestic water contaminant extensively used in detergents, fertilizers, herbicicdes etc.
Recently, development of a simple instrument for total organic carbon (TOC) analysis has been reported . The instrument utilizes photocatalytic oxidation technology to oxidize organic compounds in the aqueous sample to carbon dioxide. The carbon dioxide generated is estimated and correlated to the TOC in the aqueous sample.
MATERIALS AND METHODS
An alloy of titanium (Ti6A14V) was used in the study. Specimens in the form of discs were surface treated using acid pickling and anodisation techniques. The specimens were ultrasonically cleansed using ethanol. These specimens were pickled in an acid bath (HN03 400 g/l + HF 40 g/I) for 10 minutes to remove the oxide layer completely and make the surface free of any contaminants. One set of the acid pickled coupons were retained as such to study adhesion of the bacteria on the acid pickled surface whereas the other set of acid pickled coupons were anodized at 25[degrees]C in orthophosphoric acid (30 g/I), at 50 Volts for 10 minutes. The anodized specimens were rinsed using distilled water and then ultrasonically cleaned using detergent solution to remove all traces of acid on the surface.
Exposure studies were conducted in a pure culture of Pseudomonas sp, isolated from a freshwater reservoir in Kalpakkam, using Pseudomonas isolation medium. Biochemical and physiological tests have shown that the bacterial species used is a gram negative, oxidase positive, nitrate reducing, catalase positive, cetrimide positive, manganese oxidizing, slime forming, rod shaped bacterium, to the genus Pseudomonas sp. This is the major culprit involved in Microbiologically Influenced Corrosion (MIC) and a major component formed on titanium when exposed in natural freshwater habitats. The adhesion studies were carried out by exposing the titanium specimens in 0.1% nutrient culture of the Pseudomonas sp. The dilute culture was prepared by inoculating autoclave-sterilized 0.1% nutrient broth with 24 h actively dividing cultures of the bacteria in 100% nutrient medium. After inoculation, the medium was homogenized for 30 minutes using an orbital shaker.
The anodized and acid picked coupons were exposed to the bacterial culture in cylindrical glass containers under near-UV light illumination as well as dark conditions. The coupons were suspended from glass pags radiating from a glass rod held in position by a Teflon lid. Illumination was done using six Black light Blue (BLB) fluorescent lamps (Philips make, 4 Watts) arranged in a hexagonal configuration around the glass vessel. These tubes emit radiation of wavelength between 350 and 375nm. Equal numbers of anodized and acid pickled coupons were used to conduct the study under either condition. The exposure studies were carried out for durations of 4 days and 11 days.
Post exposure evaluation
The bacterial density on the coupons after exposure was estimated using plate count technique. The experiments were conducted using three replicates and each replicate was analyzed for bacterial density by Total viable count (TVC) estimation  and direct epifluorescence microscopic observation. For TVC analysis the coupons were removed from the medium and gently washed to remove the loosely adhering cells. The biofilm on the surface was ultrasonically removed and serially diluted and 0.1 ml of the dilution was plated using pour plate technique in nutrient agar. The plates were incubated for 24-48 h at 30-32[degrees]C after which the numbers of colonies were counted. Mean TVC values were calculated for each coupon.
Direct microscopic observation of the anodized and acid pickled coupons exposed the either conditions was conducted using a Nikon Eclipse E600 epifluorescence microscope (excitation filter BP 490; barrier filter O 515). The coupons were gently washed with sterile water and air-dried in the laminar air flow under aseptic conditions. The coupons were placed in a Petriplate and flooded with acridine orange (0.1% in distilled water), and after 10 min the excess stain was washed off and the coupons air dried and observed under the above said eqifluorescence microscope. It is seen that the acridine orange (AO) is a metachromatic dye which differentially stains double stranded (ds) and single stranded (ss) nucleic acids. When AO intercalates into dsDNA, it emits green fluorescence upon excitation at 480-490nm and when AO complexes with single stranded, nucleic acid i.e. RNA  orange-red fluorescence is obtained. Microscopic counting was done by randomly selecting ten different fields and counting the total number of fluorescing cells. Results were expressed as number of cells [cm.sup.2]. Statistical analysis of the data was done using MYSTAT Software, a Tukey-Kramer Multiple Comparison test was performed to assess significance between the bacterial counts on the different surfaces exposed to light and dark conditions.
Total viable counts and direct acridine orange of the bacteria present of different types of coupons are presented in Tables 1 & 2. Significant difference in bacterial attachment was observed between the coupons exposed under dark and light conditions. Of the blacklight illuminated coupons the anodized coupons showed very less attachment when compared to the acid pickled coupons. Within the dark exposed coupons, anodized coupons showed remarkably lesser attachment when compared to the acid pickled coupons. The illuminated acid pickled and anodized coupons showed significant reduction in attachment when compared to the dark acid pickled and dark anodized ones. (Table 1)
A similar trend was seen in the acridine orange counts also. The anodized coupon under illumination showed the least attachment followed by the anodized coupon, exposed under dark conditions, closely followed by the light acid pickled coupon. Figure 1 shows epifluorescence micrographs of titanium coupons exposed to the bacterial culture for 4 days under illumination. The number of cells which are attached and also fluorescing were seen to be very high in the case to the acid pickled coupons exposed under light and dark conditions. The anodized surface of the coupons exposed under illuminated conditions had reduced attachment, of which the number of fluorescing cells were very les. Most of the cells were green, indicating that they were metabolically inactive.
[FIGURE 1 OMITTED]
The total viable count of the 11 day old biofilm showed remarkable decrease in colony count in case of the anodized coupons under illumnation as compared to the acid pickled coupon exposed to similar conditions (Table 2). Among the coupons exposed in dark the anodized coupons recorded least attachment.
Direct Acridine Orange Counts further confirmed the significant reduction in attachment seen in case of the illuminated anodized coupons when compared to the dark anodized and light and dark exposed acid pickled coupons. Epifluorescence micrographs of 11-day old biofilm on titanium coupons exposed under illumination are given in Figure 2. There was a remarkable decrease in the overall attachment, here too the anodized surface exposed under illumination, were seen to have the least attachment.
[FIGURE 2 OMITTED]
There was significant reduction in the bacterial density of the liquid medium in which anodized specimens were exposed under illumination for 4 days (6.6 x 10 cfu / [cm.sup.2]). The experiments conducted without introducing the coupons into the inoculated medium showed that illumination had considerably less significant effect on the suspended cells in the absence of the anodized coupons (1.4 x [10.sup.5] cfu / [cm.sup.2]).
The results were statistically confirmed by the Tukey-Kramer Multiple comparison test which showed that the variation in adhesion on the illuminated anodized surfaces was indeed highly significant. The presence of anatase type of Ti[O.sub.2] was confirmed by Glancing Angle X-ray Diffraction studies, which showed a peak at the 2[theta] value of 25.3. The photocatalytic activity of the anodized specimens was evaluated by immersing the specimens in a OA M KI for 2 h under illumination by BLB lamps. The solution turned blue in the presence of starch indicator in the case of anodized surfaces indicating photocatalytic decomposition of KI to iodine. Similar experiments conducted with acid pickled surfaces showed no coloration indicating the absence of phototcataytic activity on those surfaces.
The results showed that there was a reduction in attachment on the anodized surfaces exposed under illumination. A three-order of magnitude decrease in attachment was observed between the acid pickled and anodized surfaces. Epifluorescence direct counts also showed a two order decrease in attachment on the anodized surfaces. The experiments conducted by the anodized coupons, the number of suspended cells in the medium was unaffected, suggesting the possible role of anodized surfaces in reducing attachment. Earlier studies in our laboratory, on adhesion of Pseudomonas sps on stainless steel surfaces had shown that surface topography and surface chemistry could play an important role in bacterial attachment and subsequent growth on material surfaces. Anatase type of Ti[O.sub.2] possesses photocatalytic activity and produces electron-hole pairs when illuminated with near-UV photons. This has a very strong oxidizing force and produce highly reactive free redicals like OH and [O.sub.2]. These free radicals all capable of sterilizing the anodized surfaces by direct or indirect oxidation of the bacterial cells, these explaining the reduced attachment on the anodized surfaces.
According to Ogawa et al (1996) , the photocatalytic activity is more dominant in anatase than rutile. Hence, the reason for the difference in attachment seen between the illuminated anodized coupons and dark exposed coupons is directly correlated with the presence of the anatase type of Ti[O.sub.2], on the anodized surface.
The number of cells fluorescing orange were more on the acid pickled coupon, when denoted that they are metabolically active. The anodized surfaces had more of green fluorescing cells and less of orange fluorescing cells, when indicated that most of the cells on the anodized surface have been rendered metabolically inactive by the photocatalytic activity of the oxide film.
SUMMARY & CONCLUSIONS
1. The effect of illumination by 'black light blue' fluorescent lamps on the attachment of Pseudonmonas sp. onto acid pickled and anodized Ti6Al4V alloy was carried out.
2. The bacterial medium used was 0.1 nutrient culture of Pseudnomonas sp. isolated from a freshwater reservoir at Kalpakkam.
3. The exposure studies were done for 4 days and 11 days under dark as well as illuminated conditions.
4. Total viable counts of bacteria, determined using plate count technique showed three-order of magnitude reduction in TVC values on anodized coupons illumination.
5. Direct acridine ornge counts using epifluorescence microscopy also showed a reduction in the number of cells on anodized alloy.
6. There was a marginal reduction in the number of bacterial cells in the liquid medium in which the anodized coupons where exposed for 4 days. However, no significant reduction in bacterial density was observed when the bacterial culture was exposed without introducing the anodized coupons.
7. The study confirms the antibacterial properties exhibited by the anodized surfaces under illumination.
The authors are very grateful to the Dr. V. S. Raghunathan, Associate Director, Materials Characterization Group and Dr. Baldev Raj, Director, Materials, Chemistry and Reprocessing Groups, Indira Gandhi Centre for Atomic Research for their encouragement.
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Judy Gopal, P. Muraleedharan, and P. George and H.S. Khatak
Corrosion Science & Technology Division
Indira Gandhi Center for Atomic Research, Kalpakkam 603 102
Table 1: Bacterial density in 4 day-old biofilm S.No Specimen Total Viable Count (Mean) cfu / [cm.sup.2] 1. Anodized (Dark) 2.8 x [10.sup.4] (8200*) 2. Anodized (illuminated) 2.6 x 10 (15) 3. Acid pickled (Dark) 4.6 x [10.sup.4] (4800) 4. Acid pickled Illuminated 2.9 x [10.sup.3] (840) S.No Direct acridine orange counts (Mean) cells/[cm.sup.2] 1. 6.1 x [10.sup.4] (11000) 2. 1.5 x [10.sup.2] (167) 3. 9.8 x [10.sup.4] (24000) 4. 5.5 x [10.sup.4] (15000) (* Standard deviation for the three specimens is given in parenthesis) Table 2: Bacterial density of 11 day-old-biofilm S.No Specimen Total Viable Count (Mean) cfu / [cm.sup.2] 1. Anodized (Dark) 4.1 x [10.sup.3] (770) 2. Anodized (illuminated) 6 (6.3) 3. Acid pickled (Dark) 4.5 x [10.sup.3] (1400) 4. Acid pickled (Illuminated) 1.4 x [10.sup.3] (10) Direct acridine orange counts (Mean) cells/[cm.sup.2] 1. 9.9 x [10.sup.3] * (2700) 2. 2 x [10.sup.3] (800) 3. 3,9 x [10.sup.4] (8000) 4. 3.7 x [10.sup.3] (2500) (* Standard deviation for the three specimens is given in parenthesis)
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|Author:||Gopal, Judy; Muraleedharan, P.; George, P.; Khatak, H.S.|
|Publication:||Trends in Biomaterials and Artificial Organs|
|Date:||Jul 1, 2003|
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