RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA, BANGALORE.

ANNEXURE- II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. / Name of the candidate and address ( in block letters ) / DR. SHYAM PRASAD .T .R
S/O T. L. RANGAIAH SETTY
NO. 52, VENKATAGIRI KOTE
DEVANAHALLI TALUK,
BANGALORE RURAL -562110.
2. / Name of the institution / THE OXFORD DENTAL COLLEGE,
HOSPITAL AND RESEARCH CENTER,
BANGALORE- 560068.
3. / Course of the study and subject / MASTER OF DENTAL SURGERY,
CONSERVATIVE DENTISTRY
AND ENDODONTICS.
4. / Date of admission to course / 1ST JUNE 2007
5. / Title of the topic:
AN VITRO STUDY ON ANTIMICROBIAL EFFICACY OF CALCIUM HYDROXIDE AT FIVE DIFFERENT CONCENTRATIONS WITH THREE DIFFERENT VEHICLES – BY AGAR DIFFUSION METHOD
6
7.
8. / Brief Resume Of Intended Work
6.1 NEED FOR THE STUDY:
Root canal morphology is a complex structure. It has been shown that biomechanical instrumentation when combined with placement of an antimicrobial dressing for an appropriate length of time will eliminate bacteria more effectively.
Calcium hydroxide is an established antimicrobial medicament in dentistry for past 40 years.
It has also been currently acknowledge being one of the most effective antimicrobial dressings used during endodontic therapy.
The antimicrobial action of calcium hydroxide is related to the release of hydroxyl ions in an aqueous environment. Therefore this action depends on the availability of hydroxyl ions in the solution and also the ability of these ions to diffuse through dentin and pulpal tissue remnants to reach sequestered bacteria. Differences in the velocity of hydroxyl ion dissociation are seen when various vehicles are employed along with calcium hydroxide. Calcium hydroxide shows greater antimicrobial efficacy at higher concentration.
The present study is undertaken to determine whether if varying the percent (by weight) of aqueous calcium hydroxide or using different vehicles has an effect on its antibacterial efficacy against common endodontic pathogens in a pour plate/traditional streak agar diffusion model.
6.2 REVIEW OF LITERATURE:
An vitro study was done using an agar diffusion method to determining if varying the percent (by weight) of aqueous calcium hydroxide or using different vehicles had an effect on its antibacterial efficacy against common endodontic pathogens. Each medicament was placed in one of five wells, in each of 10 agar plates (n=10) for cultures of each bacterial species. Zones of inhibition were measured after 48 hours of incubation. Vitapex had significantly smaller zones of inhibition than all other medicaments and Ultracal XS with methylcellulose was nearly as effective as 50% Ca (OH)2 in inhibitory activity1.
The study aimed to analyse two methods for determining antimicrobial effectiveness of calcium hydroxide (i) Calcium hydroxide plus saline, (ii) Calcium hydroxide plus polyethylene glycol, and (iii) Calcium hydroxide plus campohorated paramono chlorophenol. Four microorganisms (Staphylococus aureus, Enterococcus faecalis and Bacillus subtilis and one yeast candida albicans).They concluded that direct exposure test and agar diffusion test are useful to establish the antimicrobial spectrum of calcium hydroxide, thus improving infection control protocols. The direct exposure method is independent of other variables is a practical procedure and that complete antimicrobial effectiveness was observed after 48 hours for six microorganisms, by both tests, irrespective of the vehicle associated with the calcium hydroxide pastes2.
An evidence-based analysis of the antibacterial effectiveness of intracanal medicaments reinforcing the evidence that canals cannot be reliably rendered free of bacteria in 100% of cases was done. However, it is essential to reduce the microbial flora to as low a level as possible to ensure a successful outcome. Calcium hydroxide currently remains the best medicament available. Therefore to maximize reduction of bacteria in the root canal before root canal filling, Calcium hydroxide should be used as an interappointment dressing for a minimum of 7 days3.
The purpose of the study was to evaluate the influence of mixing vehicle on dissociation of calcium hydroxide in solution. In this study they concluded the use of non aqueous mixing vehicles may impede the effectiveness of Calcium hydroxide as a root canal dressing4.
In this study antimicrobial activity of several calcium hydroxide preparations in infected rootcanal with Enterococcus faecalis (E faecalis) was done. The authors concluded that the calcium hydroxide can decrease the numbers of E. faecalis at all depths of dentinal tubules within 24 hours and that thin preparations of calcium hydroxide may be more effective in the elimination of E. faecalis from dentinal tubules than thick preparations5
The purpose of this study was evaluation of three antibacterial effects of chlorhexidine, metronidazole and calcium hydroxide associated with three vehicles. This study revealed that calcium hydroxide/CPMC paste was effective against all bacterial strains tested. Chlorhexidine was also inhibitory to all strains. It was about as effective as calcium hydroxide/CPMC paste against most of the strains. Metronidazole also caused inhibition of growth of all obligate anaerobes tested and was more effective than calcium hydroxide/CPMC against two strains. Calcium hydroxide mixed with distilled water or glycerin failed to show zones of bacterial inhibition, probably because of limitations of the agar diffusion test6.
This study investigates that intracanal medicaments, eliminates candida albicans from the infection of the radicular dentin when exposed to four different medicaments:
(a) Calcium hydroxide/glycerin; (b)Calcium hydroxide/0.12% chlorhexidine digluconate;(c)Calciumhydroxide/camphorated paramonochlorophenol/glycerin and (d) Calcium- hydroxide/0.12% chlorhexidine digluconate/zinc oxide. They concluded that the calciumhydroxide CMCP/glycerin paste and chlorhexidine digluconate mixed with zinc oxide were the most effective in eliminating C. albicans cells from dentinal specimens7.
6.3 OBJECTIVES OF THE STUDY:
1. To test whether the antimicrobial efficacy of calcium hydroxide is concentration dependent.
2.To test whether the use of different vehicles has its effect on antimicrobial activity
3.To test whether it is effective against the selected microbes
MATERIALS AND METHODS:
7.1 SOURCE OF DATA:
Bacterial strain will be obtained from microbial type culture collection, chandigarh www.mtcc.imtech.res.in and national chemical laboratory, Pune.
7.2 Method of collection of data:
Experimental Calcium hydroxide material
1.  CalciumHydroxide mixied with sterile saline (Vehicle) for 40 % by weight ( n =10 )
2.  CalciumHydroxide mixed with sterile saline (Vehicle) for 50 % by weight ( n =10 )
3.  Calciumhydroxide mixed with sterile saline (Vehicle) for 60% by weight ( n =10 )
4.  Vitapex{neo-dent int}Consist of 30% CA(0H)2 and 40.4% iodoform in a 22.4% silicon base ( vehicle) ( n =10 )
5.  Ultracal XS ( ultradent prducts inc ) consist of 35% calciumhydroxide in methylcellulose base (vehicle) ( n =10 )
Experimental micro-organism
1.  Staphylococus aureus (ATCC 6538)
2.  Enterococcus faecalis (ATCC 29212)
3.  Candida albicans (ICB/USP-562)
Methodology
The selection of test endopathogens for this study was arbitrary and not a random sampling from necrotic teeth.
A Combination of the pour plate and cotton swab streaking methods will be to ensure a complete and even distribution of the bacterial cultures.
Methods of Microbial Culture
1.  Bacterial Culture Method
2.  Fungal Culture Method
1.  Bacterial Culture Method:
Isolates of Staph .aureus, and E. faecalis will be inoculated on brain heart infusion broth ( BHI ) at 37° C for 48 hrs.
The purity of staph .aureus and E. Faecalis in the inoculums will be confirmed at the end of three days and five days by taking sample cultivating on agar plates and incubating at 37° C for 48 hrs
The density of inoculums consisting of isolates of Staphylococus aureus and E. Faecalis about 0.5 Mc Farland ( consist of 1.5 X 108 bacteria /ml) will be seeded on Muellar Hinton Agar culture plate separately with pour plate method and incubated at 37o C for 48hrs.
A combination of pour plate and cotton swab streaming method was used to ensure a complete and even distribution of bacterial culture.
Six wells of 5mm deep and 5mm diameter were punched inoculated agar plates with sterile copper tube. Five wells will be completely filled with each of five experimental calcium hydraoxide mixture and remaining center well be left empty for negative control to ensure confluent growth across the agar plate.
Each of two bacterial species and candida albicans were challenged on 150 X 15mm plates 10 times with each medicament (n =10) for total of 150 samples.
The diameter of zone of bacterial inhibition around each medicament was measured to the nearest 0.01mm using a digital caliper.
To obtain average zone of inhibition on one side of well, 5mm well width is subtracted from the measurement, then which were then divided by factor 2
Average zone of Inhibition = X – 5mm well width
______
2
X = Total diameter of inhibition
Inhibition Zone around Each well recorded in milli meter and submitted to ANOVA and TUKEY test (alpha=0.005)
Positive control will be conducted for both Staphylococus aureus and E. Faecalis with doxycycline mixed with liquid agar 1mg /mL placed in centre well.
2. Fungal Culture
Initially the candida albicans inoculated on BHI at 37oC for 7 days.
The inoclumn is seeded on sabouraud agar plate in place of Mueller Hinton Agar.
The above procedure is repeated to know the zone of inhibition.
Positive control will be conducted for C. albicans with flucanazole mixed with liquid agar 1mg /mL placed in centre well.
The order of antimicrobial efficacy of calcium hydroxide at different concentration is recorded on bar diagram and tabular column.
7.3 Does the study require any investigation or intervention to be
conducted on patients or other humans or animals?
NOT APPLICABLE
7.4 Has ethical clearance been obtained from your institution?
NOT APPLICABLE
LIST OF REFERENCES:
1.  Michael L. Blanscet, DDS, MS, Patricia A. Tordik, DMD, and Gary G. Goodell, DDS, MS, MA; An Agar Diffusion Comparison of the Antimicrobial Effect of Calcium hydroxide at Five Different Concentrations with Three Different Vehicles. J Endod: 2008 – Volume 34: 1246-48
2.  Carlos Estrela, DDS, MSc, PhD, Cyntia Rodrigues de Araujo Estrela, DDS, MSc, Lili Luschke Bammann, DDS, MSc, PhD, and Jesus Djalma Pecora, DDS, MSc, PhD. Two Methods to Evaluate the Antimicrobial Action of Calcium Hydroxide Paste. J Endod 2001:27:722-723.
3.  Amanda Law, BDS (Hons), FRACDS, and Harold Messer, MDSc, PhD; AnEvidence-Based Analysis of the AntiBacterial Effectiveness of Intracanal Medicaments. J Endod 2004:30:689-94
4.  Kamran SAfavi, DMD, Med, and Tomoko A. Nakayama, PhD. Influence of Mixing Vehicle on Dissociation of Calcium Hydroxide in solution. J Endod 2000:26:649-51.
5.  Michel J. Behnon, DDS, Lesley A. West, DDS, MS, Frederick R. Liewehr, DDS. Antimicrobial activity of Several Calcium hydroxide preparations in root canal dentin J Endod 2001:27:765-767.
6.  Siqueira JF Jr. de Uzeda M. Intracanal medicaments: evaluation of the antibacterial effects of chlorhexidine, metronidazole and calcium hydroxide associated with three vehicles. J Endod 1997;23:167-9.
7.  Jose F. Siqueira, Jr., DDS, MSc, PhD, Isabela N. Rocas DDS. Elimination of candida albicans infection of the radicular dentin by intracanal medications J Endod 2003:29:501-503.
9. / Signature of the candidate
10. / Remarks of the guide
11. / Name and designation of
( in block letters)
11.1  Guide
11.2  Signature
11.3  Co- guide
11.4  Signature
11.5  Head of the department
11.6 Signature / DR.SRIREKHA
PROFESSOR
DEPT. OF CONSERVATIVE DENTISTRY & ENDODONTICS
DR. LEKHA
ASSOCIATE PROFESSOR
DEPT. OF CONSERVATIVE DENTISTRY & ENDODONTICS
DR. GURURAJ D. NADIG
PROFESSOR & H.O.D
DEPT. OF CONSERVATIVE DENTISTRY & ENDODONTICS
12. / 12.1  Remarks of the chairman and principal
12.2  Signature