This is a prospective in vitro study in which the efficacy of chlorhexidine, povidone iodine and cetyl pyridinium will be evaluated. Healing abutments will be collected from different centers using convenient sampling meeting the inclusion criteria.   These healing abutments will be tested for the turbidity until 96 contaminated healing abutments are obtained, which will be divided into 3 equal groups for their decontamination which will be allocated randomly by onsite computer randomization method. And a total of 32 new healing abutments serving as a control group will be taken from various manufacturers as each manufacturing company has different designs and size of healing abutments.

This study will follow a 3step experimental procedure

Procedure-

In the 1^st step- A total of 10 new unused HAs and 24 used HA will be stained using phloxine B stain and photographs will be taken.  And then divided into 3 equal groups Group 1-chlorhexidine 0.2%; Group 2-povidone iodine 1% solutions Group 3-cetyl pyridinium chloride0.05% for sterilization. The staining will be performed by isolating each HAs into a plastic bag containing 2ml of phloxine B and the plastic bag will be placed in an ultra-sonic bath for 15 minutes. After which the HAs will be washed in de ionized water and then air dried. These dried HAs will be observed in oblique light and photographs (Canon 1500D, lens-100mm macro lens) zoomed in their main body, thread and screw holes will be taken. The photographs will be projected on to the computer with 15X magnification and the unused HAs will serve as controls.

In the 2^nd step- 22 new HAs with the remaining used HAs present in the sterilized pouches as collected from different centers will be inserted into a sterile test tube holding 10ml of BHI which is a growth media for fastidious microorganisms. An additional test tube containing 10ml of BHI will be set up to serve as a control. All the test tubes will be cultured for 10 days at 37^o C in 5% CO₂ milleu. These test tube then will be checked for turbidity to assess the presence of microbial colonies. The test tubes that will show turbidity from them inoculum will be obtained and added into the petri dishes containing BHI and PDA. PDA is a general-purpose medium used for yeasts and molds. The petri dishes with inoculum will be cultured at 30^oC for 5 days and the process will be repeated several times until the pure culture bacterial colonies are obtained. These colonies of bacteria will be stored in nutrient agar butt at -20^o C. The bacterial colonies then obtainedwill be subjected to gram stain and biochemical tests, mainly the catalase test and growth in alkaline medium (PH: 9.2) to establish the characteristics and identity of the of the cultured colonies in order to identify the bacteria rapidly further microbial analysis will be done by using VITEK 2. The fungal growth if obtained in the petri dishes will be subjected to multiple cultures for 7 days at 30^oC until the pure cultures are obtained. The identification of the fungus will be done based on the morphology, reverse appearance and coloration. These colorations of the colonies will be assessed in three medias- PDA, malt extract agar and capeks solution.

In the 3^rd step- all the contaminated abutments obtained from cultures and 24 used HAs obtained from staining procedure will be divided into three equal groups and cleaned with Group 1-chlorhexidine 0.2%; Group 2-povidone iodine 1% solutions Group 3-cetyl pyridinium chloride0.05%. This will be done by placing the healing abutments in the plastic lock seal bags containing the 10ml of each solution and incubated in the ultrasonic bath for 15mins and subsequent steam autoclaving. The abutments will be extracted and incubated for 10days again in the petri dishes using same procedure as mentioned above. These cultures will be assessed for turbidity, bacterial colonies and molds using microbial analysis. Then the SEM examination will be done to observe the body, threads and screw holes of used HAs after the treatment with respective solutions.

Data Analysis plan and Method-

Data collected will be compiled on to a MS Office excel worksheet & will be subjected to statistical analysis using an appropriate package like SPSS software. Descriptive statistics like frequency (n) & percentage (%) ofcategorical data, mean & Standard deviation of numerical data in each group / subgroup will be depicted.

Frequency (n) & percentage (%) of various categories in each group / subgroup will be compared using chi square test.

Normality of numerical data will be checked using Shapiro – Wilk test or Kolmogorov-Smirnov test. Depending on the normality of data, statistical tests will be determined.  

For a numerical continuous data following a normal distribution, inter group comparison (>2 groups) will be done using one way ANOVA test, else a non-parametric substitute like Kruskal Wallis ANOVA test will be used.Intra group comparisons for a numerical continuous data following a normal distribution will be done using paired t test (for 2 observations) or repeated measures ANOVA for >2 observations, else a non-parametric substitute like Wilcoxon signed rank test (for 2 observations) or Friedman’s test for >2 observations will be used. Frequency (n) & percentage (%) of various responses in each time interval will be compared using chi square test / Mc Nemar’s test.

Keeping alpha error at 5% and Beta error at 20%, power at 80%, p<0.05 will be considered statistically significant.