COMPARATIVE EVALUATION OF ANTIMICROBIAL EFFICACY OF ZINC OXIDE AND MAGNESIUM OXIDE
NANOPARTICLES WITH TWO DIFFERENT MORPHOLOGY-NANOROD AND NANOSPHERE AGAINST
ENTEROCOCCUS FAECALIS
– AN IN VITRO STUDY
A Dissertation submitted in partial fulfillment of the requirements
for the degree of
MASTER OF DENTAL SURGERY BRANCH – IV
CONSERVATIVE DENTISTRY AND ENDODONTICS
THE TAMILNADU DR. MGR MEDICAL UNIVERSITY CHENNAI – 600 032
2015 – 2018
DECLARATION BY THE CANDIDATE
I hereby declare that this dissertation titled “COMPARATIVE EVALUATION
OF ANTIMICROBIAL EFFICACY OF ZINC OXIDE AND MAGNESIUM
OXIDE NANOPARTICLES WITH TWO DIFFERENT MORPHOLOGY-
NANOROD AND NANOSPHERE AGAINST ENTEROCOCCUS FAECALIS -
AN IN VITRO STUDY” is a bonafide and genuine research work carried out by me
under the guidance of Dr. B. Ramaprabha MDS, Professor, Department Of
Conservative Dentistry and Endodontics, Tamil Nadu Government Dental College
and Hospital, Chennai – 600 003.
Dr. J. SRILEKHA
CERTIFICATE BY GUIDE
This is to certify that Dr. J. SRILEKHA, Post Graduate student
(2015-2018) in the Department of Conservative Dentistry and
Endodontics, TamilNadu Government Dental College and Hospital,
Chennai- 600003 has done this dissertation titled “COMPARATIVE
EVALUATION OF ANTIMICROBIAL EFFICACY OF ZINC OXIDE AND
MAGNESIUM OXIDE NANOPARTICLES WITH TWO DIFFERENT
MORPHOLOGY-NANOROD AND NANOSPHERE AGAINST
ENTEROCOCCUS FAECALIS - AN IN VITRO STUDY” under my direct
guidance and supervision in partial fulfillment of the regulations laid
down by the Tamil Nadu Dr.M.G.R Medical University Chennai -
600032, for M.D.S., Conservative Dentistry and Endodontics (Branch IV)
Degree Examination .
Dr. B. RAMAPRABHA, M.D.S. Professor & Guide
Department of Conservative Dentistry and Endodontics. Tamil Nadu Government Dental College and Hospital
Chennai- 600003
ENDORSEMENT BY HEAD OF THE DEPARTMENT
HEAD OF THE INSTITUTION
This is to certify that the dissertation “COMPARATIVE
EVALUATION OF ANTIMICROBIAL EFFICACY OF ZINC OXIDE AND
MAGNESIUM OXIDE NANOPARTICLES WITH TWO DIFFERENT
MORPHOLOGY-NANOROD AND NANOSPHERE AGAINST
ENTEROCOCCUS FAECALIS - AN IN VITRO STUDY” is a bonafide
research work done by Dr. J. SRILEKHA, Post Graduate student
(2015-2018) in the Department of Conservative Dentistry & Endodontics
under the guidance of Dr. B. RAMAPRABHA, M.D.S, Professor and
Guide, Department Of Conservative Dentistry & Endodontics, Tamil
Nadu Government Dental College and Hospital, Chennai-600003.
Dr. M. KAVITHA, M.D.S., Professor & HOD, Dept of Conservative Dentistry & Endodontics
Dr.B.SARAVANAN, M.D.S. Ph.D Principal
Tamil Nadu Government Dental College and Hospital.
Chennai- 600003
TRIPARTITE AGREEMENT
This agreement herein after the “Agreement” is entered into on this day January 2018 between the Tamil Nadu Government Dental College and Hospital represented by its Principal having address at Tamil Nadu Government Dental College and Hospital, Chennai - 600 003, (hereafter referred to as, ‘the college‘)
And Mrs. Dr. B. Ramaprabha aged 48 years working as Professor in Department
of Conservative Dentistry & Endodontics at the college, having residence address at 191/5, Green Fields Apts. R-30A, Ambattur-Thirumangalam High Road, Mugappair, Chennai-3 (herein referred to as the Principal Investigator)
And Ms. Dr. J.SRILEKHA aged 27 years currently studying as Post Graduate
student in Department of Conservative Dentistry & Endodontics, Tamil Nadu Government Dental College and Hospital, Chennai 3 (herein referred to as the PG student and coinvestigator‘).
Whereas the PG student as part of her curriculum undertakes to research on “COMPARATIVE EVALUATION OF ANTIMICROBIAL EFFICACY OF ZINC OXIDE AND MAGNESIUM OXIDE NANOPARTICLES WITH TWO DIFFERENT MORPHOLOGY-NANOROD AND NANOSPHERE AGAINST ENTEROCOCCUS FAECALIS - AN IN VITRO STUDY” for which purpose the Principal Investigator shall act as principal investigator and the college shall provide the requisite infrastructure based on availability and also provide facility to the PG student as to the extent possible as a Co-investigator.
Whereas the parties, by this agreement have mutually agreed to the various issues including in particular the copyright and confidentiality issues that arise in this regard. Now this agreement witnesseth as follows 1. The parties agree that all the Research material and ownership therein shall become the vested right of the college, including in particular all the copyright in the literature including the study, research and all other related papers. 2. To the extent that the college has legal right to do so, shall grant to license or assign the copyright so vested with it for medical and/or commercial usage of interested persons/entities subject to a reasonable terms/conditions including royalty as deemed by the college. 3. The royalty so received by the college shall be shared equally by all the three parties.
4. The PG student and Principal Investigator shall under no circumstances deal with the copyright, Confidential information and know – how - generated during the course of research/study in any manner whatsoever, which shall sole vest with the college. 5. The PG student and Principal Investigator undertake not to divulge (or) cause to be divulged any of the confidential information or, know-how to anyone in any manner whatsoever and for any purpose without the express written consent of the college. 6. All expenses pertaining to the research shall be decided upon by the Principal Investigator/ Coinvestigator or borne solely by the PG student. (co-investigator)
7. The college shall provide all infrastructure and access facilities within and in other institutes to the extent possible. This includes patient interactions, introductory letters, recommendation letters and such other acts required in this regard.
8. The Principal Investigator shall suitably guide the Student Research right from selection of the Research Topic and Area till its completion. However the selection and conduct of research, topic and area of research by the student researcher under guidance from the Principal Investigator shall be subject to the prior approval, recommendations and comments of the Ethical Committee of the College constituted for this purpose.
9. It is agreed that as regards other aspects not covered under this agreement, but which pertain to the research undertaken by the PG student, under guidance from thePrincipal Investigator, the decision of the college shall be binding and final.
10. If any dispute arises as to the matters related or connected to this agreement herein, it shall be referred to arbitration in accordance with the provisions of the Arbitration and Conciliation Act 1996.
In witness where of the parties herein above mentioned have on this day, month and year herein above mentioned set their hands to this agreement in the presence of the following two witnesses.
College represented by its Principal PG Student
Witnesses Student Guide
1.
2.
DECLARATION
I hereby declare that no part of dissertation will be utilized for gaining financial assistance or any promotion without obtaining prior permission of the Principal, Tamil Nadu Government Dental College & Hospital, Chennai – 3. In addition I declare that no part of this work will be published either in print or in electronic media without the guide who has been actively involved in dissertation. The author has the right to preserve for publish of the work solely with the prior permission of Principal, Tamil Nadu Government Dental College & Hospital, Chennai – 3.
HOD GUIDE SIGNATURE OF THE CANDIDATE
TITLE OF DISSERTATION “COMPARATIVE EVALUATION OF ANTIMICROBIAL EFFICACY
OF ZINC OXIDE AND MAGNESIUM OXIDE
NANOPARTICLES WITH TWO DIFFERENT MORPHOLOGY-
NANOROD AND NANOSPHERE AGAINST ENTEROCOCCUS
FAECALIS - AN IN VITRO STUDY”
PLACE OF THE STUDY
Tamil Nadu Government Dental College & Hospital, Chennai- 3.
DURATION OF THE COURSE 3 YEARS
NAME OF THE GUIDE DR. B.RAMA PRABHA
HEAD OF THE DEPARTMENT DR. M. KAVITHA
ACKNOWLEDGEMENT
First and foremost I would like to extend my sincere gratitude to my
guide and Prof. Dr. B. Ramaprabha. M.D.S, who has supported me in every aspect
of this dissertation. I wish to acknowledge her reminders, constant motivation and
guidance in my PG curriculum. I am indeed grateful for all her support in this
endeavour.
I wish to place on record my deep sense of gratitude to my mentor
Dr.M.Kavitha MDS for her keen interest, inspiration, immense help and expert
guidance throughout the course of this study as Professor and Head of the Dept. of
Conservative Dentistry and Endodontics, Tamil Nadu Govt. Dental college and
Hospital, Chennai.
I take this opportunity to convey my sincere thanks and gratitude to
Dr.B.Saravanan MDS Ph.D, Principal, TamilNadu Govt. Dental College for
permitting me to utilize the available facilities in this institution.
I am extremely grateful to thank Dr.K.Amudhalakshmi MDS,
Associate Professor for constant support, motivation, guidance and encouragement
throughout my post graduate course.
I am extremely grateful to thank Dr.D.Arunaraj MDS,
Dr.A.Nandhini MDS, Dr.P.Shakunthala MDS, Associate Professors, for their
encouragement, motivation and support throughout my post graduate course.
I sincerely thank Dr.M.S.Sharmila MDS, Dr.M.Sudharshana
Ranjani MDS, Dr.N.Smitha MDS, Dr.S.Venkatesh MDS, Dr.S.Jyotilatha MDS,
Dr.S.Dhanalakshmi MDS, Dr.Bhakthavatchalam MDS, Assistant Professors for
their guidance and encouragement throughout the past three years.
I am extremely grateful to Prof. S. Balakumar, Director and Mr. Ajay
Rakesh Ph.D Scholar, Department of Nanoscience and Nanotechnology, Madras
University, Guindy campus, Chennai for their meticulous help and guidance in
synthesis of nanoparticles in my study.
I am extremely grateful to Mr.R.Selvarajan teaching faculty, Department
of Nanoscience and Nanotechnology, Anna University, Chennai, for his meticulous
help in synthesis and characterization of nanoparticles.
I extremely grateful to Dr. S. Ramesh, Professor and Head of the
Department, Department of Centralized instrumentation lab, Tamilnadu Veterinary
and Animal sciences University (TANUVAS), Chennai, for his meticulous help in
TEM analysis of nanoparticles
I express my heartfelt gratitude to Dr. K. Padmavathy, Associate
professor, Department of Microbiology, Balaji Dental College for her suggestions and
timely help throughout this study.
I specially thank Biostatistician, Dr. Junaid Mohammed MDS,
Assistant Professor, Meenakshi Ammal Dental College, Chennai for all his statistical
guidance and help.
None of my aims in life would have been fulfilled without constant
support and encouragement of my loving mother Mrs. Malar vizhi, my dear
brother J. Jaganathan, my Father N. Jayakumar and my well wisher Mr.
Radhakrishnan who stood by me in all the good and bad times of my life.
I also thank my dear co-pgs, seniors and juniors for their timely help and
friendship.
Above all I pray and thank THE ALMIGHTY GOD for His continuous
grace and blessings in my every endeavour.
In His time He makes all things beautiful!
PLAGAIRISM REPORT
CERTIFICATE - II This is to certify that this dissertation work titled Comparative evaluation of
antimicrobial efficacy of Zinc oxide and Magnesium oxide nanoparticles with
two different morphology- Nanorod and Nanosphere against Enterococcus
faecalis – An vitro study of the candidate Dr. J. Srilekha with registration Number
24151 7006 for the award of M.D.S - Conservative Dentistry and Endodontics in
the branch of IV. I personally verified the urkund.com website for the purpose of
plagiarism Check. I found that the uploaded thesis file contains from introduction to
conclusion pages and result shows 1% percentage of plagiarism in the dissertation.
Guide & Supervisor sign with Seal.
ABSTRACT
AIM: To evaluate the antibacterial effectiveness of zinc oxide and magnesium oxide nano-particles with two different morphology- nanorod and nanosphere against Enterococcus faecalis. MATERIALS AND METHODS: Zinc oxide nanorod was prepared by Hydro-thermal method and Zinc oxide nanosphere prepared by Sol-gel method. While Magnesium oxide nanorod and Magnesium nanosphere was prepared by Co-precipitation and Sol-gel method. Synthesized nanoparticles were characterised by UV-spectrophotometer, FTIR, TEM. Nanoparticles were grouped as follows: Group I-10%DMSO (negative control), Group II-ZnO-R, Group III- ZnO-S, Group IV- MgO-R, Group V- MgO-S, Group VI-3% NaOCl (positive control). The antimicrobial efficacy of nanoparticles was determined against ATCC 29212 and ORAL ISOLATE E.faecalis by Agar well diffusion assay (at five different volumes - 50,75,100,125,150μl). Broth microdilution method was chosen for determining MIC/ MBC and Time kill assay to evaluate the time needed for the nanoparticles to destroy the bacteria. The values were tabulated and subjected to statistical analysis. RESULT: Zinc oxide nanorod was effective against both strains at 75μl. Zinc oxide nanosphere was effective against ATCC E.faecalis at 100 μl but was effective against oral isolate only at 150 μl. Magnesium oxide nano-rod was effective against ATCC at 100 μl but against oral isolate only at 125 μl. Magnesium nanosphere showed activity against ATCC at 125 μl but against oral isolate showed activity at 100 μl. 3% NaOCl was effective against both strains at 50 μl. Zinc oxide nanorod activity was on par with 3% NaOCl. Considerable antibacterial activity was present in all nanoparticles at different volumes. In Time Kill assay all the nanoparticles were effective within 15 mins against both strains except Magnesium oxide nanorod which showed bacterial growth at 15 mins against ATCC but killed the bacteria within 30 mins. CONCLUSION: Zinc oxide nanorods exhibited good activity against both strains of E.faecalis. Considerably, all the nanoparticles were effective at different volumes and concentrations against E.faecalis. So the zinc oxide and magnesium oxide nanoparticles (nanorod and nanosphere) can provide a new horizon in the disinfection stratergy in the domain of Endodontics. KEYWORDS: zinc oxide, magnesium oxide, nanosphere, nanorods, polar facets, ROS, alkaline effect.
CONTENTS
SL.NO TITLE PAGE NO
1 INTRODUCTION 1-4
2 AIM AND OBJECTIVES 5
3 REVIEW OF LITERATURE 6-17
4 MATERIALS AND METHODS 18-34
5 RESULTS 35-54
6 DISCUSSION 55-72
7 SUMMARY 73-74
8 CONCLUSION 75
9 BIBLIOGRAPHY i-xiii
FIG NO. COLOUR PLATES
1 ZINC ACETATE
2 SODIUM HYDROXIDE
3 ZINC CHLORIDE
4 MAGNESIUM NITRATE
5 POLYETHYLENE GLYCOL
6 LIQUID AMMONIA
7 METHANOL
8 ETHANOL
9 BEAKERS
10a-f SYNTHESIS OF ZINC OXIDE NANOROD
11a-f SYNTHESIS OF ZINC OXIDE NANOSPHERE
12a-f SYNTHESIS OF MAGNESIUM OXIDE NANOROD
13a-f SYNTHESIS OF MAGNESIUM OXIDE NANOSPHERE
14 UV-VISIBLE SPECTROPHOTOMETER
15 TRANSMISSION ELECTRON MICROSCOPE (TEM)
16a-b TEM IMAGES OF ZINC OXIDE NANOROD
17a-b TEM IMAGES OF ZINC OXIDE NANOSPHERE
18a-b TEM IMAGES OF MAGNESIUM OXIDE NANOROD
19a-b TEM IMAGES OF MAGNESIUM OXIDE NANOSPHERE
20 MACCONKEY AGAR, MUELLER HITON BROTH AND AGAR
21 E.FAECALIS ATCC 29212 STRAIN
22 CORK BORER
23 INCUBATOR
24 DIGITAL COLONY COUNTER
25 MICROPIPETTES
26 VORTEX MIXER
27 STERILE LOOP
28 STERILE COTTON SWAB
29 LAMINAR AIR FLOW CHAMBER
30 ANTIBIOTIC ZONE SCALE C
31 ELECTRONIC BALANCE
32 AGAR PLATES
33 DMSO –DIMETHYL SULPHOXIDE
34 3 % SODIUM HYPOCHLORITE
35 MUELLER HINTON AGAR PLATE
36 MAC CONKEY AGAR PLATE
37 ATCC E.FAECALIS REVIVED ON MACCONKEY AGAR
38 ORAL ISOLATE E.FAECALIS REVIVED ON MACCONKEY AGAR
39 BROTH CULTURE OF ATCC AND ORAL ISOLATE E.FAECALIS
40 DIFFERENT CONCENTRATION OF NANOPARTICLES
41 LAWN CULTURE WITH WELLS PUNCHED
42 BROTH MICRODILUTION FOR THE TEST SOLUTION
43a-e AGAR DIFFUSION ASSAY (ZONE OF INHIBITION) AND BROTH MICRODILUTION OF ZINC OXIDE NANOROD AND NANOSPHERE
44a-e AGAR DIFFUSION ASSAY (ZONE OF INHIBITION) AND BROTH MICRODILUTION OF MAGNESIUM OXIDE NANOROD AND NANOSPHERE
45a-b MBC OF ZINC OXIDE NANOROD
46a-b MBC OF ZINC OXIDE NANOSPHERE
47 MBC OF MAGNESIUM OXIDE NANOROD
48a-b MBC OF MAGNESIUM OXIDE NANOSPHERE
49a-d AGAR DIFFUSION ASSAY, BROTH MICRODILUTION AND MBC OF 3% NaOCl AND 10% DMSO
50a-b TIME KILL ASSAY FOR ZINC OXIDE NANOROD
51a-b TIME KILL ASSAY FOR ZINC OXIDE SPHERE
52a-b TIME KILL ASSAY FOR MAGNESIUM OXIDE NANOROD
53a-b TIME KILL ASSAY FOR MAGNESIUM OXIDE NANOSPHERE
NPs NANOPARTICLES
ZnO- S ZINC OXIDE NANOSPHERE
ZnO-R ZINC OXIDE NANOROD
MgO-S MAGNESIUM OXIDE NANOSPHERE
MgO-R MAGNESIUM OXIDE NANOROD
TEM TRANSMISSION ELECTRON MICROSCOPE
NaOCl SODIUM HYPOCHLORITE
ABBREVIATIONS USED
LIST OF TABLES
TABLE
NO TITLE PAGE
NO
1 FTIR ANALYSIS – NARROW AND BROAD RANGE PEAK VALUES
21
2 ZONE OF INHIBITION IN MILLIMETERS FOR ALL GROUPS
35
3 TIME KILL ASSAY – CFU/ml 38
4 MINIMUM INHIBITORY CONCENTRATION / MINIMUM BACTERICIDAL CONCENTRATION
40
5 ATCC E.FAECALIS – ONE WAY ANOVA ANALYSIS - Descriptive analysis of all groups
42
6 ATCC E.FAECALIS - ANOVA ANALYSIS for Zone
of Inhibition of all groups
43
7 TUKEY POST HOC TEST- Multiple comparison of all the groups for ATCC E.faecalis
44
8 ORAL ISOLATE E.FAECALIS – ONE WAY ANOVA ANALYSIS - Descriptive analysis of all groups
48
9 ORAL ISOLATE E.FAECALIS - ANOVA ANALYSIS for Zone Of Inhibition of all the Groups
49
10 TUKEY POST HOC TEST –Multiple Comparison of all the Groups for oral isolate E.faecalis
50
LIST OF GRAPHS
SL. NO
GRAPH
PAGE
NO 1
UV-VISIBLE SPECTROPHOTOMETER ANALYSIS-
ZINC OXIDE NANORODS
22
2
UV-VISIBLE SPECTROPHOTOMETER ANALYSIS-
ZINC OXIDE NANOSPHERE
22
3
UV-VISIBLE SPECTROPHOTOMETER ANALYSIS-
MAGNESIUM OXIDE NANORODS
23
4
UV-VISIBLE SPECTROPHOTOMETER ANALYSIS-
MAGNESIUM OXIDE NANOSPHERE
23
5
FTIR ANALYSIS – ZINC OXIDE NANOROD
24
6
FTIR ANALYSIS – ZINC OXIDE NANOSPHERE
24
7
FTIR ANALYSIS – MAGNESIUM OXIDE
NANOROD
25
8
FTIR ANALYSIS – MAGNESIUM OXIDE
NANOSPHERE
25
9
HISTOGRAM REPRESENTATION OF THE ZONE OF
INHIBITION OF ALL GROUPS AGAINST ATCC STRAIN AT DIFFERENT VOLUMES
36
10
HISTOGRAM REPRESENTATION OF THE ZONE OF
INHIBITION OF ALL GROUPS AGAINST ORAL ISOLATE STRAIN AT DIFFERENT VOLUMES
37
11
HISTOGRAM REPRESENTATION OF TIME KILL ASSAY OF ALL THE GROUPS FOR ATCC AND ORAL
ISOLATE E.FAECALIS
39
12
HISTOGRAM REPRESENTATION OF MIC/MBC OF
ALL THE GROUPS FOR BOTH STRAINS
41
INTRODUCTION
1
The success of endodontic treatment depends mainly on the eradication of
micro-organisms from the root canal system and prevention of re-infection.23 A
bacteria free root canal system is difficult to achieve due to the anatomical
complexities of root canals, organic residues and unreachable bacteria located deep
inside the dentinal tubules.47,63 Bacteria in the root canal are present either as free-
floating planktonic single cells or attached to each other or to the root canal walls to
form a biofilm.31,16 Sundqvist and Fidgor reported that “root canal infection is not a
random event”. Species that establish a persistent endodontic infection are selected
by the phenotypic traits that they share and that are suited to the modified
environment. Some of these shared characteristics include the capacity to penetrate
and invade dentin, a growth pattern of chains or cohesive filaments, resistance to
antimicrobials used in endodontic treatment, as well as an ability to grow in mono-
infections, to survive periods of starvation and to evade the host response.78
E. faecalis has proved a potentially important microorganism to colonize in
endodontic infections and being the dominant microorganism in post-treatment
apical periodontitis, has often been isolated from the root canal in pure culture.77 In
mixed infections, E. faecalis is typically the dominant isolate. Enterococci survive
very harsh environments including extreme alkaline pH (9.6).62 E. faecalis
overcomes the challenges of survival within the root canal system in several ways. It
has been shown to exhibit widespread genetic polymorphisms.30 It possesses serine
protease, gelatinase, and collagen-binding protein, which help it bind to
dentin.26 It is small enough to proficiently invade and live within dentinal tubules. It
has the capacity to endure prolonged periods of starvation until an adequate
INTRODUCTION
2
nutritional supply becomes available. E. Faecalis has a proton pump that provides a
means of maintaining pH homeostasis. This is accomplished by “pumping” protons
into the cell to lower the internal pH and hence resist high ph.42,44
Endodontic infections are currently treated by mechanical debridement followed by
chemical disinfection. Irrigants are used during the endodontic treatment to flush out
and remove loose debris, in lubrication of the dentinal walls, to dissolve organic
matter in the canal, and to provide antimicrobial action.73 However, clinical studies
have shown that even after meticulous chemomechanical disinfection and
obturation of the root canals, bacteria may still persist in the un-instrumented
portions and anatomical complexities of the root canal.49 Therefore, it is vital to
understand that the current limitations in endodontic disinfection strategies are not
only due to the biofilm mode of bacterial growth within the root canals, but also
collectively due to the anatomical complexities of the root canal system.55
Considering the nature of the challenges presented by the root canal environment and
endodontic microbes, a reliable therapeutic requirement of endodontic disinfection
should eliminate the biofilm structure and destroy the resident bacteria completely,
even in locations untouched by root canal instrumentation procedures. Because of the
shortcomings of current antibiofilm strategies in root canal treatment, advanced
disinfection strategies are being developed and tested, the recent approach being the
use of nanoparticles in endodontic disinfection.36 In the recent times, the advances in
the field of Nanosciences and Nanotechnology has brought to fore the nanosized
inorganic and organic particles which are finding increasing applications as
INTRODUCTION
3
amendments in industrial use, medicine, dentistry and therapeutics. 22, 67
Nanoparticles are microscopic particles with dimensions in the range of 1–100
nm. Nanoparticles are recognized to have properties that are very unique from their
bulk or powder counterparts. Antibacterial nanoparticles have been found to have
a broad spectrum of antimicrobial activity and lesser incidence of microbial
resistance development than antibiotics. The nanoparticles possess unique
physico-chemical, optical and biological properties which can be manipulated
suitably for desired applications in the field of medicine and dentistry. The
nanoparticles are broadly grouped into organic and inorganic nanoparticles. The
latter have gained significant importance due to their ability to withstand adverse
processing conditions.53
The antimicrobial activity of the nanoparticles is because of the surface area in
contact with the microorganisms. The small size, varied morphology (sphere, rod,
flower, wires) and the high surface to volume ratio i.e., large surface area of the
nanoparticles enhances their interaction with the microbes to carry out a broad range
of antimicrobial activities. So these nanoparticles pave a new way for endodontic
disinfection.89 Various nanoparticles like Chitosan, Silver, BAG, Quaternary
ammonium polyethylenimine nanoparticles (QPEINPs), Zinc oxide nanoparticles
have been investigated for their antibacterial effectiveness in Endodontics.71 Zinc
oxide and Magnesium oxide nanoparticles of two varied morphologies sphere and
rod were choosen for this study for their antibacterial effectiveness due to the
following advantages.
INTRODUCTION
4
Zinc oxide nanoparticles: ZnO nanoparticles currently being investigated as an
antibacterial agent in both microscale and nanoscale formulations. ZnO exhibits
significant antimicrobial action when particle size is reduced from micrometer to the
nanometer range; nano-sized ZnO can interact with bacterial surface or with the
bacterial core where it enters inside the cell, and subsequently exhibits distinct
bactericidal mechanisms. The interactions between these unique materials and
bacteria are mostly toxic, which have been exploited for antimicrobial applications.69
Magnesium oxide nanoparticles: MgO is an important inorganic material with a
wide band-gap. MgO nanoparticles have shown promise for application in tumor
treatment in medicine. MgO nanoparticles are a promising antibacterial agent due to
their high resistance to harsh processing conditions. Three main antibacterial
mechanisms have been proposed, such as the formation of ROS, the interaction of
nanoparticles with bacteria, subsequently damaging the bacterial cell and an alkaline
effect.11
Antimicrobial effectiveness is evaluated using Agar Diffusion assay to gauge the
zone of inhibition and to determine Minimum inhibitory and Minimum Bactericidal
concentration by Broth microdilution assay. Time kill curve was done to ascertain
the time required for the nanoparticles to kill the bacteria.18
The purpose of this in vitro study was to evaluate the antimicrobial
effectiveness of Zinc oxide and Magnesium oxide nanoparticles with two different
morphologies - Rod and Sphere against Enterococcus faecalis.
AIM AND OBJECTIVES
5
AIM
To evaluate the antibacterial effectiveness of zinc oxide and magnesium
oxide nano-particles with two different morphology- nanorod and nanosphere
against Enterococcus faecalis.
OBJECTIVES
1. Synthesis of zinc oxide and magnesium oxide nanoparticles with two
distinct morphology – nano rod and nano sphere
2. Characterisation of nanoparticles by UV spectrophotometer, Fourier
transform infrared spectroscopy for analysing absorbance peaks for
ZnO and MgO NPs and Transmission electron microscope for size
and shape analysis.
3. Antibacterial activity against Enterococcus faecalis – ATCC 29212
and oral isolate
REVIEW OF LITERATURE
6
ZINC OXIDE NANOPARTICLE
Padmavathy and Vijayaraghavan (2008) compared ZnO-NPs of three different
sizes (45, 12 nm, and 2 nm) to determine ZnO bactericidal efficiency against E.coli.
Study found that the nanosize of 12 nm showed best efficiency compared to 45 nm
and 2 nm. The antibacterial action was attributed to ROS release.52
Jones et al, (2008) evaluated the mechanisms to explain the antibacterial activity of
ZnO NPs against S.aureus, which include the formation of ROS, lipid peroxidation,
electrostatic interactions, and alkaline effects. The strong electrostatic interaction
between the bacterial cell surface and the ZnO NPs leads to the death of the
bacteria.34
Kasemets et al (2009) in their study identified that the release of Zn ions was
responsible for ZnO NPs toxicity toward Saccharomyces cerevisiae bacteria.
According to their study, ZnO-NPs toxicity is attributed to the solubility of Zn ions
in the medium including the bacteria.35
Yang et al (2009) investigated the cytotoxicity, genotoxicity and oxidative effects of
zinc oxide, carbon nanotube and silicon dioxide nanoparticles on primary mouse
embryo fibroblast cells. As observed in the methyl thiazolyl tetrazolium (MTT) and
water-soluble tetrazolium (WST) assays, ZnO induced much greater cytotoxicity
than other nanoparticles. The results denoted that oxidative stress may be a main
route in inducing the cytotoxicity of nanoparticles. Compared with ZnO
nanoparticles, carbon nanotubes were moderately cytotoxic but induced more DNA
damage determined by the comet assay.93
REVIEW OF LITERATURE
7
Wahab et al. (2010) carried a non-hydrolytic solution process using zinc acetate
dihydrate to prepare ZnO-NPs. The method yielded structures of spherical surface
that showed high antibacterial activity against the tested pathogens Staphylococcus
aureus, Escherichia coli, Salmonella typhimurium, and Klebsiella pneumonia. NPs
solution inhibiting the growth of microbial strain is found to be 5 μg/ml for K.
pneumoniae, whereas for E. coli, S. aureus, and S. typhimurium, it was calculated to
be 15 μg/ml.87
Jalal et al.(2010) obtained strong antibacterial activity against E. coli at increased
concentration. As a result, an increase in hydrogen peroxide amount was produced
from ZnO surface, a lethal agent to bacteria. 29
Shrestha et al (2010). highlighted the efficacy of CS-np and ZnO-np to reduce
biofilm bacteria and disrupt biofilm structure. The antibacterial property of these
nanoparticles was retained even after aging for 90 days against E.faecalis biofilm.
Result showed that CS-np and ZnO-np possess a potential antibiofilm capability
against E.faecalis.72
Zhang et al (2011) evaluated ZnO-NPs in aqueous solution exposed to UV radiation
found to have phototoxic effect that can produce reactive oxygen species (ROS)
such as hydrogen peroxide (H2O2) and superoxide ions (O2-). Reactive oxygen
species are extremely essential for antimicrobial action.95
Narayanan et al. (2012) tested the antibacterial activity of ZnO-NPs against human
pathogens such as P. aeruginosa, E. coli, S. aureus, and E. faecalis. They emerged
with the result that ZnO-NPs have strong antibacterial activity toward these human
REVIEW OF LITERATURE
8
pathogens. The antibacterial action is by the growth inhibition as a result of cell
membrane damage through penetration of ZnO-NPs.50
Talebian et al (2013) studied that flower-shaped nanoparticles have higher biocidal
activity against S. aureus, and E. coli than the spherical and rod-shaped ZnO-NPs.
The antibacterial property of ZnO NPs were influenced by the physiological
condition of the bacterial cells, different shapes and crystal growth , particle size and
optical properties of the NPs. ZnO flower-like NPs showed significantly higher
photocatalytic inactivation than ZnO rod- and sphere-like NPs against E. coli
compared with S. aureus. It was found that the antibacterial activity of ZnO increased
with decreasing crystallite size.81
Stankovic et al (2013) have synthesized ZnO powder hydrothermally with the
addition of different stabilizing agents (polyvinyl pyrrolidone (PVP), polyvinyl
alcohol (PVA) and poly l-glutamic acid) (PGA) leading to different shapes. The
synthesized ZnO has shown nanorods of hexagonal prismatic and hexagonal pyramid
like structures, with some spherical and ellipsoid shapes. These different
morphologies displayed pronounced antibacterial effect toward the targeted bacteria
E.coli and S. aureus. 75
Vidic et al (2013) evaluated nanostructured Zn-MgO produced by combustion
technique which exhibit advantageous properties from both of its pure components;
high antibacterial activity of nano-ZnO and low cytotoxicity of nano-MgO. This
mixed metal oxide inhibited Gram-positive bacteria (B. subtils) completely and
Gram-negative bacteria (E. coli) partially upon 24 h treatment. Zn MgO
nanoparticles were shown to damage bacterial cells by causing extensive injury to
REVIEW OF LITERATURE
9
membranes that resulted in a leakage of the cell contents. Comparatively, pure ZnO
nanorods and nanotetrapods exhibited the highest but non selective activity as they
completely eradicated both bacterial strains and mammalian HeLa cells, under the
same treatment protocol. In contrast, pure MgO nanocubes only partially inhibited
bacterial growth being at the same time harmless to mammalian cells.85
Rago et al (2014) investigated the antimicrobial properties against two Gram-positive
bacteria (Staphylococcus aureus and Bacillus subtilis) of ZnO microrods (MRs) and
nanorods (NRs). They concluded that ZnO-NRs have a superior antimicrobial effect
against both S. aureus and B. subtilis at much lower doses when compared to ZnO-MRs. 59
He et al.(2014) have observed that deposition of small Au particles of 3nm diameter
onto the surface of ZnO nanoparticles significantly enhanced the photocatalytic and
antibacterial activity of ZnO. The deposition of Au onto ZnO nanoparticles resulted
in production of holes and electrons at the particle surface which dramatically
increased light-induced generation of hydroxyl radical, superoxide and singlet
oxygen. When incubated with E. coli, the ZnO/Au hybrid nanostructures showed
about three times higher antibacterial efficiency than pure ZnO nanoparticles.24
Wu et al (2015) evaluated ZnO nanowires that were synthesized in heterojunction of
silver-loaded nanowires through UV light decomposition process. Nanowires were
found to exhibit higher antibacterial activities to E. coli. It disrupted the bacterial
membrane and released lethal active species.90
Zanni et al (2016) investigated the antimicrobial and antibiofilm properties of a
novel nano-material composed of zinc oxide nanorods-decorated graphene
nanoplatelets (ZNGs). The antimicrobial activity of ZNGs was evaluated against
REVIEW OF LITERATURE
10
Streptococcus mutans, the main bacteriological agent in the etiology of dental caries.
Cell viability assay demonstrated that ZNGs exerted a high bactericidal effect on S.
mutans cells in a dose-dependent manner.94
MAGNESIUM OXIDE NANOPARTICLE
Huang et al. (2005) reported that antibacterial activity was increased with the
decrease of the particle size of MgO. A relationship between the bactericidal efficacy
against B. subtilis ATCC 9372 and the particle size of nano-MgO was demonstrated.
For particles in the size range ~ 45-70 nm, the bactericidal efficacy of nano-MgO
increased slowly with decreasing particle size. Below ~ 45 nm however, the
bactericidal efficacy showed a much stronger dependence on particle size.25
Makhluf et al. (2005) demonstrated that small MgO nanoparticles had an efficient
antibacterial activity towards Escherichia coli (E. coli) and Staphylococcus aureus
(S. aureus). Small, electron-dense black dots could be observed in the cytoplasm of
MgO-nanoparticle-treated bacteria.43
Avanzato et al. (2009) investigated the antibacterial activity of magnesium oxide-
germanium oxide composite powder. The prepared nano-composite powder showed
good bactericidal activity toward both gram-negative (E. coli) as well as gram-
positive bacteria (S. aureus).7
Yamamoto et al (2010) has reported that the increase of the surface area of MgO
particles leads to an increase of the O2 − concentration in solution and thus results in
a more effective destruction of the cell wall of the bacteria.92
REVIEW OF LITERATURE
11
Jin and He (2011) found that higher MgO nanoparticle concentrations resulted in
greater bacterial in-activation. An approximate seven log unit reduction in E. coli O
157: H7 was achieved by an 8 mg/mL MgO nanoparticle treatment at 24 h. At 7 h,
the anti E. coli O157: H7 activity of MgO nanoparticles was dependent on its
concentration, as in the case of low inoculum levels. The treatment with 3 mg/mL or
higher MgO nanoparticles significantly reduced cell concentrations to undetectable
levels after 24 h at room temperature, results indicating 3 mg/mL MgO nanoparticles
would be enough to kill all cells.33
Sundrarajan et al. (2012) investigated the effect of MgO nanoparticles size on the
antibacterial activity. Their results indicated that small-sized MgO nanoparticles had
better antibacterial activities towards both gram positive (S. aureus) and gram
negative (E. coli) bacteria. Furthermore, MgO nanoparticles had more activity
towards gram positive bacteria compared to gram negative bacteria.79
Krishnamoorthy et al. (2012) prepared MgO nanoparticles using magnesium nitrate
and sodium hydroxide as precursors and cellulose as a stabilizing agent. The size of
the prepared MgO nanoparticles was in the range of 10 to 30 nm. Calcination
temperature could significantly affect the morphology and size of MgO
nanoparticles.38
Rao et al. (2013) have shown that doping MgO with different metal ions may give
opposite effects on nanoparticles’ antibacterial properties. Li-doped MgO was more
efficient than pure MgO, while Zn- and Ti-doped nano-MgO displayed poorer
antibacterial activity than MgO. The authors concluded that doping with Li+
REVIEW OF LITERATURE
12
promoted the generation of oxygen vacancies and increased the basicity of the oxide,
which favoured generation and stabilization of superoxide anion, O-2. In contrast,
Ti2+ and Zn2+, having higher valence than Li+, less efficiently favored these two
phenomena although Ti-doped MgO was somehow more efficient than Zn-doped
MgO in eliminating E. coli which was ascribed to smaller sizes of Ti-doped MgO
compared to those of Zn-doped MgO nanoparticles.61
Monzavi et al (2015) evaluated the antibacterial efficacy of different concentrations
of magnesium oxide nanoparticles (5 mg/L and 10 mg/L), 5.25% sodium
hypochlorite and 2% chlorhexidine against endodontic pathogens such as E. faecalis,
S. aureus and Candida albicans .The results showed no significant differences in the
antimicrobial efficacies of the irrigant solutions used against the tested endodontic
pathogens. However, the inclusion of magnesium oxide nanoparticles in an irrigant
solution produced extended antibacterial activity when compared with sodium
hypochlorite.48
Anicic et al (2016) evaluated nano-texturing of the microrod's surface at calcination
temperatures higher than 700 °C. The prepared particles improved the antibacterial activity
against Escherichia coli (E. coli, ATCC 47076), which was related to the enhanced contact
with the bacteria. The nano-texturing of the MgO microrods was achieved after calcination
at 900 °C. Result showed that these microrods eliminated the bacteria 75% faster than
commercially available MgO nanoparticles.6
REVIEW OF LITERATURE
13
Mirhosseini et al (2016) evaluated the antibacterial action of magnesium oxide
nanoparticles (MgO NP) alone and in combination with nisin (antibacterial peptide)
against Escherichia coli and Staphylococcus aureus. Scanning electron microscopy
was used to characterize the morphological changes of E. coli after antimicrobial
treatment. Results found that MgO NPs along with nisin were able to destroy the cell
wall, resulting in a leakage of intracellular contents and eventually the death of
bacterial cells.45
Iram S et al (2016) compared the antibacterial activity of CaO, MgO and ZnO
particles alone and in combinations with antibiotics ciprofloxacin, erythromycin,
methicilin and vancomycin against E. faecalis and E. faecium. The results showed
that the sizes and concentrations of CaO, MgO and ZnO particles have a significant
role in antibacterial activity. MICs of antibiotics conjugated with nanoparticles
revealed that the ZnO particles effectively enhanced the MICs of antibiotics in low
concentrations in comparison with CaO and MgO nanoparticles.28
Ibrahim et al (2017) evaluated the antibacterial activity of biosynthesized MgO NPs
using A. niger that appeared as a spherical morphology with a particle size ranged
between 40–95 nm. Antibacterial activity performed against S. aureus and P.
aeruginosa confirmed that Zone of Inhibition diameter assay was found to have high
inhibition effects and their effects were more in gram positive bacteria compared
with gram negative bacteria.27
REVIEW OF LITERATURE
14
Sharma et al (2017) synthesized magnesium oxide nanoparticles using aqueous
extract of Swertia chirayaita a phytoassisted method. The stable magnesium oxide
nanoparticles (MgO NPs) formed by this method were spherical particles that were
20 nm in size. MgO NPs tested against Gram-positive - Staphylococcus aureus –
MTCC-9442, Staphylococcus epidermidis – MTCC-2639, Bacillus cereus – MTCC-
9017 and Gram-negative bacteria - Escherichia coli – MTCC-9721, Proteus vulgaris
– MTCC-7299, Klebsiella pneumonia – MTCC-9751 by agar-well diffusion method
were found to be effective against both Gram-negative bacteria and Gram-positive
bacteria.70
ENTEROCOCCUS FAECALIS
Spratt et al (2001) evaluated the effectiveness of NaOCl (2.25%), 0.2%
chlorhexidine gluconate (CHX), 10% povidone iodine, 5 ppm colloidal silver, and
phosphate‑ buffered saline (PBS) solution (as control) against monoculture biofilms
of five root canal isolates including Prevotella intermedia, Peptostreptococcus
micros, Streptococcus intermedius, Fusobacterium nucleatum, and E. faecalis.
Results proved Naocl to be the most effective antimicrobial, followed by the iodine
solution.74
Pruzzo et al (2002) found that E. faecalis is capable of entering and recovering from
the viable but nonculturable (VBNC) state, a survival strategy adopted by bacteria
when exposed to environmental stress. VBNC E. faecalis displayed cell wall
alterations that might provide protection under unfavourable environmental
conditions and maintained adhesive properties to cultured human cells. 57
REVIEW OF LITERATURE
15
Sedgley CM (2006) tested the hypothesis that long term survival of E.faecalis
is dependent on the type of endodontic sealer and the capacity for microbial
gelatinase activity. They also suggested that gelatinase activity plays a role in
long term survival of E.faecalis in obturated root canals.6
Ferrer‑Lugue et al. (2010) evaluated the in vitro capacity of maleic acid (MA) as
well as the combinations of cetrimide (CTR) with MA, citric acid, and EDTA in
eradicating E. Faecalis biofilms. According to their findings, MA eradicated E.
Faecalis biofilms at a concentration of 0.88% after 30 sec and at 0.11% after 2 min
contact time. When combined with 0.2% CTR, it eradicated the biofilms at all three
times of exposure. The combination of 0.2% CTR with either 15% EDTA or 15%
citric acid gave 100% bacterial kill after 1 min of contact with the biofilms.17
Afzal et al (2013) used two strains of E. faecalis ‑ATCC 29212 and clinical isolate
in the study. Naocl, MTAD, CHX were compared for antibacterial action against
both strains. Sodium hypochlorite 5.25% showed the highest antibacterial efficacy
against the E. faecalis biofilm, followed by 2% Chlorhexidine, MTAD and distilled
water.1
Dianat et al (2015) conducted an invitro study to compare the antimicrobial efficacy
of nanoparticle calcium hydroxide against E. faecalis to that of calcium hydroxide by
measuring the minimum inhibitory concentration (MIC) and agar diffusion test
(ADT) in dentin models from different depths. Study concluded that the
antimicrobial activity of Nano Calcium Hydroxide was superior to Calcium
Hydroxide in culture medium. In dentinal tubules the efficacy of NCH was again
better than CH on the 200- and 400-µm samples.12
REVIEW OF LITERATURE
16
Krishnan et al (2015) evaluated antimicrobial efficacy of the silver nanoparticles.
This study shows that silver nanoparticles have potential bactericidal effects against
E. faecalis at a concentration of 5 mg/ml. Silver nanoparticles can be incorporated in
the root canal medicaments, sealers and irrigants, as it possess a good antimicrobial
efficacy against E. Faecalis.39
Fan et al (2016) synthesized the mesoporous calcium-silicate nanoparticles that were
functionalized with chlorhexidine and evaluated their releasing profile, antibacterial
ability, effect on cell proliferation and in vitro mineralization property.
Chlorhexidine was incorporated into mesoporous calcium-silicate nanoparticles by a
mixing-coupling method. The new material could release chlorhexidine as well as
Calcium and Silicate ions in a sustained manner with an alkaline pH value under
different conditions. The antimicrobial ability against planktonic E. faecalis was
dramatically improved after chlorhexidine incorporation. Result concluded that
Mesoporous calcium-silicate nanoparticles with chlorhexidine exhibited release of
ions and excellent antibacterial action and promoted mineralization.14
Dowlatababdi F et al (2017) evaluated the antibacterial activity properties of silver
doped zinc oxide nanoparticles (ZnO: Ag). Silver doped zinc oxide nanoparticles
(ZnO:Ag) were synthesized with wet chemical method in an aqueous solution. Size
of the nanoparticles was obtained as between 12 and 13 nanometers in average. The
results showed that silver doped zinc oxide nanoparticles prevented Escherichia coli,
Staphylococcus aureus, and Pseudomonas aeruginosa, but did not affect
REVIEW OF LITERATURE
17
Enterococcus faecalis. The zone of inhibition diameter increases as the density of the
nanoparticles does.13
Del Carpio-Perochena et al (2017) evaluated the efficacy of chitosan nanoparticles
(CNPs) and ethanolic propolis extract (EPE) incorporated into a calcium hydroxide
paste to kill bacterial biofilms. All evaluated pastes were able to significantly reduce
the E. faecalis colony forming units (CFU) after 7 or 14 days. However, the CFU
reduction was significantly improved when CNPs were incorporated into the
Ca(OH)2 paste.10
MATERIALS AND METHODOLOGY
18
MATERIALS REQUIRED FOR NANOPARTICLE SYNTHESIS
ZINC OXIDE NANORODS (Fig – 1 to 15)
Zinc acetate (Sigma Aldrich)
Sodium hydroxide
Deionized water
Ethanol
Distilled water
ZINC OXDE NANOSPHERE
Zinc chloride
Zinc nitrate (Sigma Aldrich)
Sodium hydroxide
MAGNESIUM OXIDE NANORODS
Magnesium nitrate
Polyethylene glycol (Sigma Aldrich)
Liquid ammonia
Ethanol
Deionized water
MAGNESIUM OXIDE NANOSPHERE
Magnesium nitrate (Sigma Aldrich)
Sodium hydroxide
Methanol
CHARACTERISATION - UV-VISIBLE SPECTROPHOTOMETER (BioTek),
FTIR- FOURIER TRANSFORM INFRARED SPECTROSCOPY (JASCO),
TRANSMISSION ELECTRON MICROSCOPE (TEM) (TECNAI)
MATERIALS AND METHODOLOGY
19
NANOPARTICLE SYNTHESIS
ZINC OXIDE NANOPARTICLES
ZINC OXIDE NANOROD (ZnO-R)
The precursor was prepared by dissolving 5.48 g zinc acetate dehydrate {Zn (CH3
COO)2 ·2H2O} and 10.00 g sodium hydroxide (NaOH) in deionized water to form a
30 ml solution. To the precursor solution, 44mL of ethanol was mixed together in a
beaker under constant stirring. The temperature of the oven was raised to 110º C and
held constantly for 4 to 5 hrs; then it was allowed to cool to room temperature. The
precipitate obtained was washed three times with distilled water and alcohol, then
dried at 60º C.88 (Fig 10a-10f)
ZINC OXIDE NANOSPHERE (ZnO-S)
Zinc Oxide nanosphere was prepared by mixing 50 ml 0.5 M Zinc chloride, and 50 ml
0.5 M Zinc nitrate. To this was added 50 ml 2M sodium hydroxide solution slowly,
drop wise with vigorous stirring which was continued for 45 min. The resulting white
precipitate obtained was filtered and washed thoroughly with deionised water for 3 to
4 times. After washing, the precipitate was allowed to dry at 100°C for 10 hours on
hot air oven. This caused Zinc hydroxide (ZnOH) to decompose into Zinc Oxide
(ZnO). The obtained product was calcined at 400°C for 5 hours. 65(Fig 11a-11f)
MATERIALS AND METHODOLOGY
20
MAGNESIUM OXIDE NANOROD – (MgO-R)
An appropriate amount of magnesium nitrate (Mg (NO3)2.6H2O) was dissolved in 200
ml distilled water in order to form 0.012 M solution. Polyethylene glycol 600 (PEG)
was added separately to the desired amount of 50 ml liquid Ammonia (NH3-H2O) and
mixed well by stirring for 5 min. The prepared solution was added drop wise to the
solution of magnesium nitrate (Mg (NO3)2.6H2O) that was dissolved in 200 ml
distilled water at room temperature under stirring. The mixture was heated to reaction
temperature of 70º C and kept for 10 mins. As the reaction completed, the white
precipitate formed was washed with distilled water and ethanol to remove the ions
possibly remaining in the final products, and finally dried at 60º C overnight. The
product was calcined at 550º C for 2hrs.3 (Fig 12a-12f)
MAGNESIUM OXIDE NANOSPHERE – (MgO-S)
Magnesium oxide nanosphere was synthesized using magnesium nitrate
(MgNO3.6H2O) as a source material with sodium hydroxide. Procedure involves 0.2M
magnesium nitrate (MgNO3.6H2O) dissolved in 100 ml of deionized water. 0.5M 50
ml of sodium hydroxide solution was added drop wise to the prepared magnesium
nitrate (MgNO3.6H2O) solution while stirring it continuously. White precipitate of
magnesium hydroxide appeared in beaker after few minutes. The stirring was
continued for 30 minutes. The precipitate was filtered and washed with methanol three
to four times to remove ionic impurities and dried at room temperature. The dried
white powder samples were dried for two hours at 500º C.87 (Fig 13a-13f)
ZINC OXIDE NANOPARTICLES MAGNESIUM OXIDE
NANOPARTICLES
ZnO nanorods MgO
nanorods MgO
nanosphere
CHARACTERISATION OF NANOPARTICLES
UV- VISIBLE SPECTROPHOTOMETER
FOURIER TRANSFORM
INFRARED SPECTROSCOPY
TRANSMISSION ELECTRON
MICROSCOPE
ZnO nanosphere
SYNTHESIZED AND CHARACTERISED NANOPARTICLES WERE USED FOR MICROBIOLOGICAL ASSAY
SYNTHESIS AND CHARACTERISATION OF NANOPARTICLES
MATERIALS REQUIRED FOR NANOPARTICLE SYNTHESIS
Fig 1-ZINC ACETATE Fig 2- SODIUM HYDROXIDE
Fig 3 ZINC CHLORIDE
Fig 4 MAGNESIUM NITRATE
Fig 5- POLYETHYLENE GLYCOL
Fig 6- LIQUID AMMONIA
Fig 8-ETHANOL Fig 7-METHANOL Fig -9 BEAKERS
ZINC OXIDE NANO- ROD SYNTHESIS
ZINC OXIDE NANO RODS
Fig 10a- Zinc acetate and sodium hydroxide
Fig 10b- Zinc acetate and sodium hydroxide solution prepared
Fig 10c- Zinc acetate + sodium hydroxide
Fig 10d- Ethanol added dropwise
Fig 10e- Precipitate formation Fig 10f –Dried powder ZnO nanorods
ZINC OXIDE NANO- SPHERE SYNTHESIS
ZINC OXIDE NANOSPHERE
Fig 11b-Zinc chloride, zinc nitrate, sodium hydroxide
solution prepared Fig 11a-Zinc nitrate, sodium
hydroxide, zinc chloride
Fig 11c-Zinc chloride + Zinc nitrate
Fig 11d- Sodium hydroxide added dropwise
Fig 11e- Precipitate formation Fig 11f-Dried
powder- ZnO nanosphere
MAGNESIUM OXIDE NANO-ROD SYNTHESIS
MAGNESIUM OXIDE
NANORODS
Fig 12d-Precipitate formation
Fig 12a-Magnesium nitrate, Liquid ammonia. PEG
Fig 12b -Magnesium nitrate, Liquid Ammonia. PEG
solution
Fig 12c-PEG and liquid ammonia mixture added dropwise to magnesium
nitrate
Fig 12e-Precipitate formation
Fig 12f-Dried powder MgO-
nanorods
MAGNESIUM OXIDE NANOSPHERES
Fig 13a-Magnesium nitrate, Sodium Hydroxide
Fig 13b-Magnesium nitrate Sodium Hydroxide solution
Fig 13c- Sodium Hydroxide added drop wise to Magnesium nitrate
Fig 13e- Precipitate formation
Fig 13d- Precipitate formation
MAGNESIUM OXIDE
NANOSPHERE
Fig 13f-Dried powder MgO nanospheres
MATERIALS AND METHODOLOGY
21
CHARACTERISATION OF NANOPARTICLES
The synthesized nanoparticles were characterised by UV spectrophotometer, FTIR
and TEM.
UV-VISIBLE SPECTROPHOTOMETER (Fig 14)
Synthesized nanoparticles were characterized using UV-vis spectrophotometer to
evaluate the characteristic absorbance peak. Absorbance peak for zinc oxide
nanopaticles ranged between 300 to 400nm and for magnesium oxide nanoparticles
between 200 to 300nm. Peak values obtained for Zinc oxide nanorods - 360, zinc
oxide nanosphere – 350, magnesium oxide nanorods - 250, magnesium oxide
nanosphere – 270 (Graph 1- 4).
FTIR- FOURIER TRANSFORM INFRARED SPECTROSCOPY
FTIR spectroscopy is frequently used to find the infrared spectrum related to the
vibrations of molecules and is unique for each compound. Characteristic peak
obtained as following, the sharp peak is the characteristic absorption of Zinc to
Oxygen and Magnesium to Oxygen bond stretching vibration and the broad
absorption peak can be attributed to the characteristic absorption of hydroxyl group
(denoting the use of alcohol in synthesis).
TEM- TRANSMISSION ELECTRON MICROSCOPY (Fig- 15)
Synthesized nanoparticles were evaluated using TEM for size and shape, average size
of nanoparticles were 20 nm and morphology (rod and sphere) of the
nanoparticles were analysed. (Fig 16 – fig 19)
NANOPARTICLES SHARP PEAK BROAD RANGE PEAK ZnO –R (Graph 5) 424 cm-1 3369 cm-1
ZnO –S (Graph 6) 411 cm-1 3354 cm-1
MgO-R (Graph 7) 568 cm-1 3430 cm1
MgO-S (Graph 8) 565 cm-1 3328 cm1
TABLE -1
Fig 14.UV-VISIBLE SPECTROPHOTOMETER
Fig 15- TEM- TRANSMISSION ELECTRON MICROSCOPY
MATERIALS AND METHODOLOGY
22
UV-VISIBLE SPECTROPHOTOMETER ANALYSIS
ZINC OXIDE NANORODS (Graph 1)
ZINC OXIDE NANOSPHERE (Graph 2)
300 - 400
360
300 - 400
350
MATERIALS AND METHODOLOGY
23
MAGNESIUM OXIDE NANORODS (Graph 3)
MAGNESIUM OXIDE NANOSPHERES (Graph 4)
200 - 300
250
270
200 - 300
MATERIALS AND METHODOLOGY
24
FTIR- FOURIER TRANSFORM INFRARED SPECTROSCOPY ANALYSIS
ZINC OXIDE NANORODS (Graph 5)
ZINC OXIDE NANOSPHERE (Graph 6)
Characteristic sharp peak at 424 (ZnO-R) and 411 cm-1 (ZnO-S)
Broad range peak at 3369 (ZnO-R) and 3354 cm-1(ZnO-S)
MATERIALS AND METHODOLOGY
25
MAGNESIUM OXIDE NANORODS (Graph 7)
MAGNESIUM OXIDE NANOSPHERES ( Graph 8)
Characteristic sharp peak at 568 (MgO-R) and 565 cm-1 (MgO-S)
Broad range peak at 3430 (MgO-R) and 3328 cm-1(MgO-S)
TRANSMISSION ELECTRON MICROSCOPE ANALYSIS
ZINC OXIDE NANORODS
Fig 16a- SINGLE NANOROD
Fig 16b-NANORODS CLUSTERS
20 nm
ZINC OXIDE NANOSPHERES
Fig 17a- SINGLE NANOSPHERE
Fig 17b- NANOSPHERE CLUSTERS
20 nm
MAGNESIUM OXIDE NANORODS
Fig 18a- NANOROD CLUSTERS
Fig 18b- SINGLE NANOROD
20 nm
MAGNESIUM OXIDE NANOSPHERES
Fig 19a- SINGLE NANOSPHERE
FIG 19b- NANOSPHERE CLUSTERS
20 nm
MATERIALS AND METHODOLOGY
27
METHODOLOGY
MICROBIAL ISOLATES USED IN THE STUDY:
1. STANDARD STRAIN: A freeze dried ampoule of Enterococcus faecalis
ATCC 29212 was procured from HiMedia laboratories Pvt Ltd, India. (Fig -21)
2. CLINICAL ISOLATE: Stock culture of an oral isolate of Enterococcus
faecalis isolated from the root canal of a patient with RCT failure that had been
identified, confirmed and maintained in the Department of Microbiology, Sree Balaji
Dental College & Hospital, Chennai was used for the study.
BACTERIOLOGICAL CULTURE MEDIA USED IN THE STUDY
MacConkey agar:
Ingredients
Peptic Digest of Animal tissue - 20.00 gm
Lactose - 10.00 gm
Sodium taurocholate - 5.00 gm
Neutral Red - 0.04 gm
Agar -20.00gm
Distilled water - 1000 ml
pH - 7.4 ± 0.2
MATERIALS AND METHODOLOGY
26
MICROBIOLOGICAL ANALYSIS MATERIALS REQUIRED (fig 20 – 34)
STRAINS
ATCC 29212 - E.FAECALIS (Hi Media)
CLINICAL ISOLATE- E.FAECALIS
AGAR AND BROTH
MACCONKEY AGAR
MUELLER HINTON AGAR (Hi Media)
MUELLER HINTON BROTH
DIMETHYL SULPHOXIDE
3% SODIUM HYPOCHLORITE – (Septodont)
CORK BORER
STERILE COTTON SWAB
STERILE LOOP
PETRI DISHES
STERILE TEST TUBES
MICRO TITRE PLATES
EPPENDORF TUBES (Eppendorf Research, Germany)
VORTEX MIXER (Remi Motors, Mumbai)
DIGITAL COLONY COUNTER (Deep Vision, India)
INCUBATOR (Technico)
MATERIALS AND METHODOLOGY
28
The dehydrated medium was procured from Hi Media Laboratories Pvt Ltd, India.
The dehydrated medium (5.5 grams) was suspended in 100ml of distilled water.
The medium was dissolved completely by boiling. The medium was sterilized by
autoclaving at 121ºC at 15 lbs pressure for 15 minutes. 20 ml of the medium was
poured into sterile disposable petri plates (Hi Media laboratories Pvt Ltd, India).
Sterility check was performed for each lot by incubating a representative plate at
37ºC. The plates were stored at 4º C until use.(Fig – 36)
Mueller-Hinton agar:
Ingredients
Beef extract - 300.0 gm
Casein Acid hydrosylate - 17.5 gm
Starch - 1.5 gm
Agar - 17.0 gm
Distilled water - 1000 ml
pH - 7.4 ± 0.2
The dehydrated medium was procured from Hi Media Laboratories Pvt Ltd, India.
The dehydrated medium (38 grams) was suspended in 1000 ml of distilled water. The
medium was dissolved completely by boiling. The medium was sterilized by
autoclaving at 121ºC at 15 lbs pressure for 15 minutes. 20 ml of the medium was
poured into sterile disposable petriplates (Hi Media laboratories Pvt Ltd, India).
MATERIALS AND METHODOLOGY
29
Sterility check was performed for each lot by incubating a representative plate at
37ºC. The plates were stored at 4º C until use. (Fig -35)
Mueller-Hinton broth:
Ingredients
Beef extract - 300.0 gm
Casein Acid hydrosylate - 17.5 gm
Starch - 1.5 gm
Distilled water - 1000 ml
pH - 7.4 ± 0.2
The dehydrated medium was procured from HiMedia laboratories Pvt Ltd, India.
The dehydrated medium (2.1 grams) was suspended in 100 ml of distilled water. The
medium was sterilized by autoclaving at 121ºC at 15 lbs pressure for 15 minutes. One
ml of the medium was poured into sterile disposable microfuge tubes (1.5 mL
capacity, Tarsons India) Sterility check was performed for each lot by incubating a
representative tube at 37ºC. The tubes were stored at 4º C until use.
Revival of Enterococcus faecalis:
The freeze-dried culture of Enterococcus faecalis ATCC 29212 was reconstituted with
500 µl of sterile saline. Ten microliters of each of the reconstituted bacterial culture
was pipetted out using sterile micro-pipette (Eppendorf Research, 1- 10 µl variable-
Germany) and was seeded on sterile MacConkey agar plates. The inoculum was
MATERIALS AND METHODOLOGY
30
streaked using sterile Hi-Flexi Loop 4 (HiMedia laboratories Pvt Ltd, India) on the
agar surface for isolation. The stock culture of E. faecalis oral isolate (agar deep) was
revived by dispensing 10µl of sterile MHB and was further sub-cultured on sterile
Mac Conkey agar plates. The plates were incubated at 37˚C for 24 hours. After
incubation, the colony morphology of E. Faecalis was observed. (Fig 37-38)
Inoculum preparation – E. faecalis ATCC 29212 & E. faecalis clinical isolate:
Isolated colonies of E. faecalis ATCC 29212 & E. faecalis clinical (oral isolate) from
MacConkey agar plate cultures were suspended in sterile Muller Hinton Broth (MHB)
in individual test tubes and the cell densities were adjusted to 1. 5 x108 cfu/ml using
0.5 Mcfarland standard (HiMedia laboratories Pvt Ltd, India). (Fig – 39)
Preparation of the test solutions:
Stock solutions of the nanoparticles viz., Zinc Oxide- Rod (ZnO-R), Zinc Oxide-
Sphere (ZnO-S), Magnesium Oxide - Rod (MgO-R) and Magnesium Oxide- Sphere
(MgO-S) were prepared at a concentration of 100mg/mL in 10% Dimethyl sulphoxide
(DMSO). The nanoparticles were suspended uniformly using a vortex mixer. 3%
Sodium Hypochlorite was included as positive control and 10% DMSO as negative
control. (Fig – 40)
Group 1-10% DMSO
Group 2 - ZnO –R
Group 3 – ZnO –S
Group 4 – MgO –R
Group 5 – MgO –S
Group 6 – 3% NaOCl
50, 75,100,125,150 ul
MATERIALS AND METHODOLOGY
31
Screening for antibacterial activity of the test solutions by Agar well diffusion
technique:
Lawn culture of E. faecalis ATCC 29212 & E. faecalis clinical (oral isolate) was made
on separate MHA plates. ETO sterilized cotton swabs (Polymer Medical devices,
Chennai, India) were dipped in the fresh broth cultures of E. faecalis (cell density
adjusted to 1.5 x108 cfu/ml) and excess of broth was drained by pressing against the
inner walls of the test tube and the inoculum was seeded in three different directions
to form a lawn culture. The plates were allowed to dry at room temperature for 10
mins. (Fig – 41)
Wells of 8 mm diameter were punched using sterile cork borer and 25, 50, 100, 150 µl
of the nano-suspension of Zinc Oxide- Rod (ZnO-R), Zinc Oxide- Sphere (ZnO-S),
Magnesium Oxide- Rod (MgO-R) and Magnesium Oxide- Sphere (MgO-S) were
added in the respectively labeled wells and the plates were incubated at 37°C for 24
hours. 10% DMSO and 3% NaOCl was used as the negative and positive control
respectively. After incubation, the diameter of the zone of inhibition around the wells
were measured using Hi Antibiotic zone scale-C (Hi Media laboratories Pvt Ltd,
India) and recorded in milli-meters (mm) (Fig – 43a-43d, 44a-44d, 49a-49b). The
assay was performed in triplicate.18
Determination of Minimum Inhibitory Concentration of the Nano - suspensions
by Broth micro-dilution technique:
Broth dilution is a technique in which incrementally increasing concentrations of
the antimicrobial agent is added to containers holding identical volumes of broth
culture with known density of the test organism. Broth microdilution24 is a
MATERIALS AND METHODOLOGY
32
modification of the broth dilution and is performed on microtitre plates with a
holding capacity of ≤ 300 µl per well. In this procedure double serial dilution of an
antibacterial agent is prepared in the broth medium and the lowest concentration of
the antimicrobial agent under defined in vitro conditions that completely inhibit
visible bacterial growth (turbidity) within a defined period of time is recorded as
the minimal inhibitory concentration (MIC).
Minimum inhibitory concentration of the test solutions was determined by Broth
micro dilution method in sterile disposable 96 well microtitre plates (Zellkulter,
Germany) according to CLSI guidelines, 2017 (Clinical Laboratory Standards
Institute). MIC of the nano-suspensions of Zinc Oxide- Rods (ZnO-R), Zinc Oxide-
Sphere (ZnO-S), Magnesium Oxide-Rod (MgO-R) and Magnesium Oxide- Sphere
(MgO-S) were assessed as follows. Further, stock solution of ZnO-S was added in the
first well of the respectively labeled rows (A1, B1, C1 & D1). Double serial dilutions
of ZnO-S were prepared (say A1 through A11; B1 through B11; C1 through C11
and D1 through D11). Similarly, stock solution of ZnO-R was added in the first well
of the respectively labeled rows (E1, F1, G1 &H1). Double serial dilutions of ZnO-R
were prepared (say E1 through E11; F1 through F11; G1 through G11 and H1
through H11).
The last well of each row served as the culture control (no test solution was added).
10 µl of the respective culture suspension was added to all the wells including the
culture control (E. faecalis ATCC 29212 to rows viz., A, B, E, F & E. faecalis clinical
(oral isolate) to rows C, D, G, H). The plates were incubated for 18 hrs at 37°C. The
MIC was recorded as the lowest concentration of the test solution which inhibits
bacterial growth (no visible turbidity).The same procedure was adopted for
MATERIALS AND METHODOLOGY
33
Magnesium Oxide- Rod (MgO-R) and Magnesium Oxide- Sphere (MgO-S),
Hypochlorite and DMSO. (Fig- 43e, 44e, 49c)
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 Control
B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 Control
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 Control
D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 Control
E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 Control
F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 Control
G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 Control
H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 Control
Schematic representation of Broth Microdilution Assay
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 Control
B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 Control
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 Control
D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 Control
E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 Control
F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 Control
G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 Control
H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 Control
MgO -R
ZnO -R
ZnO -S
ATCC
ORAL ISOLATE
ATCC
ORAL ISOLATE
MgO -S
ATCC
ORAL ISOLATE
ATCC
ORAL ISOLATE
MATERIALS AND METHODOLOGY
34
Determination of Minimum Bactericidal Concentration (MBC):
Minimum bactericidal concentration of the test solutions was determined by spot
inoculation of 5 µl of the culture from each well of the microtitre plate onto a
MHA plate. The plates were incubated for 18 hrs at 37°C. Minimum concentration
of the test solution that completely inhibited (~99%) the growth of the colonies on
the MHA plate was scored as the MBC of the respective test solution. (Fig- 45a,
45b, 46a, 46b, 47, 48a, 48b, 49d)
Time Kill Assay: Time kill assay 98: The rate of killing over a specified period by
exposing a bacterial isolate to a certain concentration of antimicrobial agent in a
broth medium forms the basis of the Time Kill Assay. Killing time (KT) was
determined as the exposure time required to kill a standardized microbial
inoculum. Overnight cultures of E. faecalis ATCC 29212& E. faecalis clinical (oral
isolate) in MacConkey agar plate cultures were suspended in 1mL of the test solutions
in separate Eppendorf tubes and the cell densities were adjusted to 1.5 x108cfu/ml.
The tubes were incubated at 37°C and 10µl of the culture from each tube was
pipetted out at 15 mins intervals – 15 mins, 30 mins, 45 mins, 60 mins, 75 mins
and 90 mins. The inoculum was seeded onto MHA plates by spread plate
technique. After incubation, the number of colony forming units (cfu) was counted
using a digital colony counter. The number of viable bacteria was plotted over time
to calculate the rate of killing. Killing time (KT) values in minutes are reported
according to Gavini et al. (Fig 50-53)
Acquired data were tabulated and Statistical analysis done using S.P.S.S version 16
software. One Way Anova with Tukey’s Post Hoc test were carried out.
GROUP 1 10% DMSO NEGATIVE CONTROL
GROUP 6 3% NaOCL POSITIVE CONTROL
GROUP 2 ZnO RODS
GROUP 3 ZnO
SPHERE
GROUP 4 MgO RODS
GROUP 5 MgO
SPHERE
ATCC 29212 E. FAECALIS
ORAL ISOLATE E. FAECALIS
MICROBIOLOGICAL ASSAYS
AGAR WELL DIFFUSION ASSAY
MINIMUM INHIBITORY CONCENTRATION
MINIMUM BACTERICIDAL CONCENTRATION
TIME KILL ASSAY
50 ul
75 ul
100 ul
125 ul
150 ul
VOLUME USED
STRAINS EVALUATED
MICROBIOLOGICAL ANALYSIS
MATERIALS REQUIRED FOR MICROBIOLOGICAL ASSAYS
Fig 20 - MACCONKEY AGAR, MUELLER HITON BROTH AND AGAR
Fig 21- E. FAECALIS ATCC 29212 Fig 22-CORK BORER
Fig 23- INCUBATOR Fig 24- DIGITAL COLONY
COUNTER
Fig 29-LAMINAR AIR FLOW CABINET
Fig 25- MICROPIPETTES
Fig 28- STERILE COTTON SWAB Fig 27- STERILE LOOP
Fig 26-VORTEX MIXER
Fig 30- ANTIBIOTIC ZONE SCALE C
Fig 31- DIGITAL ELECTRONIC BALANCE
Fig 32- AGAR PLATES
Fig 33- DIMETHYL SULFOXIDE
Fig 34- 3% SODIUM HYPOCHLORITE
MICROBIOLOGICAL PROCEDURE
Fig 35- MUELLER HINTON AGAR PLATE
Fig 37-ATCC E.FAECALIS REVIVED ON MACCONKEY
AGAR
Fig 36- MACCONKEY AGAR PLATE
Fig 38-ORAL ISOLATEE.FAECALIS
REVIVED ON MACCONKEY AGAR
Fig 39 - BROTH CULTURE OF ATCC E.FAECALIS – A
BROTH CULTURE OF ORAL ISOLATE- C
The same procedure carried out for all the groups
Fig 41 – LAWN CULTURE WITH WELLS PUNCHED AND INOCULATED WITH
TEST AGENTS
Fig 42- BROTH MICRODILUTION FOR THE TEST SOLUTION
Fig 40- DIFFERENT CONCENTRATION OF ZnO AND MgO NANOPARTICLES (RODS
AND SPHERES) PREPARED.
RESULTS
35
Test solution
Vol (µl)
E. faecalis ATCC 29212
E. faecalis Oral isolate
1 2 3 1 2 3
Group -1 DMSO (10%) 150 8 8 8 8 8 8
Group -2
Zinc Oxide- Rod
(ZnO-R)
(100 mg/mL)
50 8 8 8 8 8 8
75 10 10 11 11 11 10
100 12 12 13 13 13 13
125 15 15 15 13 14 13
150 16 16 17 15 15 15
Group -3
Zinc Oxide- Sphere
(ZnO-S)
(100 mg/mL)
50 8 8 8 8 8 8
75 8 8 8 8 8 8
100 10 10 11 8 8 8
125 10 11 11 8 8 8
150 12 12 12 10 10 11
Group 4-
Magnesium Oxide- Rod
(MgO-R)
(100 mg/mL)
50 8 8 8 8 8 8
75 8 8 8 8 8 8
100 10 9 10 8 8 8
125 12 12 11 10 10 10
150 14 14 15 11 12 11
Group -5
Magnesium Oxide- Sphere
(MgO-S)
(100 mg/mL)
50 8 8 8 8 8 8
75 8 8 8 8 8 8
100 10 10 11 11 12 11
125 11 11 11 12 12 12
150 12 13 12 13 13 13
Group 6-
NaOCl (3%)
50 12 12 12 13 13 12 75 14 13 14 14 14 14
100 18 16 18 18 18 17
125 22 21 22 22 21 21
150 24 24 25 24 24 23
TABLE 2 – Zone of inhibition in millimetres tabulated for all Groups
RESULTS
36
GRAPH 9 - HISTOGRAM REPRESENTATION OF THE ZONE OF INHIBITION OF ALL GROUPS AGAINST ATCC STRAIN AT DIFFERENT VOLUMES
GRAPH 9 – showing,
3% NaOCl was effective at 50, 75, 100, 125, 150 μl against ATCC E.FAECALIS
Among the nanoparticles,
At 50 μl , nanoparticles were not effective At 75 μl, ZnO-R was effective At 100μ l, ZnO-R ˃ ZnO-S ˃MgO-S ˃MgO-R At 125μ l ZnO-R ˃ MgO-R ˃ MgO-S ˃ ZnO-S At 150μl ZnO-R ˃ MgO-R ˃ MgO-S = ZnO-S
0
5
10
15
20
25
30
50 μl 75 μl 100 μl 125 μl 150 μl
10%DMSO
ZnO ROD
ZnO SPHERE
MgO ROD
MgO SPHERE
3%NaOCL
Zon
e of
inhi
bitio
n in
mm
Volumes used
RESULTS
37
GRAPH 10:
HISTOGRAM REPRESENTATION OF THE ZONE OF INHIBITION OF ALL GROUPS AGAINST ORAL ISOLATE STRAIN AT DIFFERENT VOLUMES
GRAPH 10 showing:
3% NaOCl was effective at 50, 75, 100, 125, 150 μl against oral isolate E.faecalis.
Among the nanoparticles,
At 50 μl , nanoparticles were not effective At 75 μl, ZnO-R was effective At 100 μl, ZnO-R ˃MgO-S At 125 μl ZnO-R ˃ MgO-S ˃ MgO-R ˃ At 150 μl ZnO-R ˃ MgO-S ˃ MgO-R ˃ ZnO-S
0
5
10
15
20
25
50 μl 75 μl 100 μl 125 μl 150 μl
10%DMSOZnO RODZnO SPHEREMgO RODMgO SPHERE3%NaOCL
RESULTS
38
Test solution
Time (mins)
E. faecalis ATCC 29212 (cfu/mL)
E. faecalis Oral isolate (cfu/mL)
Group- 2
Zinc Oxide- Rod
(ZnO-R)
(100 mg/mL)
15 0 0
30 0 0
45 0 0
60 0 0
75 0 0
90 0 0
Group-3
Zinc Oxide- Sphere
(ZnO-S)
(100 mg/mL)
15 0 0
30 0 0
45 0 0
60 0 0
75 0 0
90 0 0
Group-4
Magnesium Oxide-
Rod (MgO-R)
(100 mg/mL)
15 12,600 0
30 0 0
45 0 0
60 0 0
75 0 0
90 0 0
Group- 4
Magnesium Oxide-
Sphere (MgO-S)
(100 mg/mL)
15 0 0
30 0 0
45 0 0
60 0 0
75 0 0
90 0 0
Group -6
NaOCl (3%)
15 0 0
30 0 0
45 0 0
60 0 0
75 0 0
90 0 0
Culture control > 1,00,000 > 1,00,000
TABLE 3- TIME KILL ASSAY – colony forming units/ml
RESULTS
39
Time kill assay evaluates the time needed for the nanoparticles to kill the
bacteria.
TABLE 3- Time kill assay of all nanoparticle against both strains are tabulated,
All nanoparticles were effective against both strains within 15 mins, except
MgO-R which showed growth at 15 mins against ATCC E.faecalis.
GRAPH 11 HISTOGRAM REPRESENTATION OF TIME KILL CURVE OF ALL THE GROUPS AGAINST ATCC AND ORAL ISOLATE E.FAECALIS
GRAPH –11
Showing ZnO-R, ZnO-S, MgO-S, 3% NaOCl – All were effective within 15 mins (zero cfu/ml) against ATCC strain except MgO-R showed 12600 cfu/ml at 15 mins against ATCC strain, but zero cfu/ml at 30 mins.
ZnO-R, ZnO-S, MgO-R, MgO-S, 3% NaOCl – All were effective within 15 mins against Oral isolate strains. (zero cfu/ml)
ZnO-RZnO-S
MgO-RMgO-S
3% NaOCl
0
5
10
15
20
25
30
ATCCORAL ISOLATE
ZnO-R
ZnO-S
MgO-R
MgO-S
3% NaOCl
STRAINS TIM
E IN
MIN
S T
O K
ILL
BA
CT
ER
IA
RESULTS
40
TABLE – 4 MINIMUM INHIBITORYCONCENTRATION / MINIMUM BACTERICIDAL CONCENTRATION
TABLE – 4
Minimum inhibitory/ Minimum bactericidal concentration of the nanoparticles against both the strains of E.faecalis.
ATCC strain – Among the nanoparticles, Mgo-S (1.17mg/ml) was effective at lesser concentration followed by MgO-R and ZnO-R effective at (18.75 mg/ml), ZnO-S effective at a concentration of (37.5 mg/ml)
MgO-S < MgO-R = ZnO-R < ZnO-S (lesser to higher conc.)
ORAL ISOLATE strain – MgO-R = MgO-S effective at a concentration of 18.5mg/ml, followed by ZnO-R (37.5mg/ml), followed by ZnO-S (75mg/ml).
MgO-R = MgO-S < ZnO-R < ZnO-S
Test solution
MIC/ MBC (mg/mL)
E. faecalis
ATCC 29212
E. faecalis
Oral isolate
Zinc Oxide- Rod
(ZnO-R)
18.75
37.5
Zinc Oxide- Sphere
( ZnO-S)
37.5
75
Magnesium Oxide-
Rod (MgO-R)
18.75
18.75
Magnesium Oxide-
Sphere (MgO-S)
1.17
18.75
3% NaOCl 0.0002
0.0002
RESULTS
41
GRAPH-12
HISTOGRAM REPRESENTATION OF MIC/MBC ALL THE GROUPS FOR BOTH STRAINS
GRAPH -12
Showing MIC/MBC of nanoparticles and 3% NaOCl against both strains of E.faecalis (ATCC and Oral isolate)
3%NaOCl and MgO-S were effective at lower concentration against ATCC strain
3% NaOCl followed by MgO-S and MgO-R were effective at lower concentration against Oral isolate strain.
Considerably, higher concentration of ZnO-S was required to inhibit both the strains.
0
10
20
30
40
50
60
70
80
ATCC E.FAECALISORAL ISOLATE E.FAECALIS
ZnO-R
ZnO-S
MgO-R
MgO-S
3%NaOCL
MIC
/ M
BC
mg/
ml
RESULTS
42
STATISTICAL ANALYSIS
TABLE – 5 ATCC E.FAECALIS - One way ANOVA – Descriptive analysis of all groups (mean, std deviation, std error)
N Mean
Std. Deviation
Std. Error
95% Confidence Interval for Mean
Minimum Maximum
Lower Bound
Upper Bound
50 μl DMSO 10% 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00
ZnO-R 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00
ZnO-S 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00
MgO-R 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00
MgO-S 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00
3% NaOCl 3 12.0000 .00000 .00000 12.0000 12.0000 12.00 12.00
Total 18 8.6667 1.53393 .36155 7.9039 9.4295 8.00 12.00
75μl DMSO 10% 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00
ZnO-R 3 10.3333 .57735 .33333 8.8991 11.7676 10.00 11.00
ZnO-S 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00
MgO-R 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00
MgO-S 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00
3% NaOCl 3 13.6667 .57735 .33333 12.2324 15.1009 13.00 14.00
Total 18 9.3333 2.19625 .51766 8.2412 10.4255 8.00 14.00
100μl DMSO 10% 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00
ZnO-R 3 12.3333 .57735 .33333 10.8991 13.7676 12.00 13.00
ZnO-S 3 10.3333 .57735 .33333 8.8991 11.7676 10.00 11.00
MgO-R 3 9.6667 .57735 .33333 8.2324 11.1009 9.00 10.00
MgO-S 3 10.3333 .57735 .33333 8.8991 11.7676 10.00 11.00
3% NaOCl 3 17.3333 1.15470 .66667 14.4649 20.2018 16.00 18.00
Total 18 11.3333 3.10597 .73208 9.7888 12.8779 8.00 18.00
125μl DMSO 10% 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00
ZnO-R 3 15.0000 .00000 .00000 15.0000 15.0000 15.00 15.00
ZnO-S 3 10.6667 .57735 .33333 9.2324 12.1009 10.00 11.00
MgO-R 3 11.6667 .57735 .33333 10.2324 13.1009 11.00 12.00
MgO-S 3 11.0000 .00000 .00000 11.0000 11.0000 11.00 11.00
3% NaOCl 3 21.6667 .57735 .33333 20.2324 23.1009 21.00 22.00
Total 18 13.0000 4.52444 1.06642 10.7500 15.2500 8.00 22.00
RESULTS
43
Table no 5- Showing the descriptive statistics for all the groups for ATCC E.faecalis.
TABLE-6 ATCC E.FAECALIS - ANOVA Test for Zone of Inhibition of all groups ( Intra-group and Inter-group analysis were statistically significant)
150μl DMSO 10% 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00
ZnO-R 3 16.3333 .57735 .33333 14.8991 17.7676 16.00 17.00
ZnO-S 3 12.0000 .00000 .00000 12.0000 12.0000 12.00 12.00
MgO-R 3 14.3333 .57735 .33333 12.8991 15.7676 14.00 15.00
MgO-S 3 12.3333 .57735 .33333 10.8991 13.7676 12.00 13.00
3% NaOCl 3 24.3333 .57735 .33333 22.8991 25.7676 24.00 25.00
Total 18 14.5556 5.21561 1.22933 11.9619 17.1492 8.00 25.00
Sum of Squares df Mean Square F Sig.
50 μl Between Groups 40.000 5 8.000 . .
Within Groups .000 12 .000
Total 40.000 17
75 μl Between Groups 80.667 5 16.133 145.200 .000
Within Groups 1.333 12 .111
Total 82.000 17
100 μl Between Groups 158.667 5 31.733 71.400 .000
Within Groups 5.333 12 .444
Total 164.000 17
125 μl Between Groups 346.000 5 69.200 415.200 .000
Within Groups 2.000 12 .167
Total 348.000 17
150 μl Between Groups 459.778 5 91.956 413.800 .000
Within Groups 2.667 12 .222
Total 462.444 17
RESULTS
44
Dependent
Variable (I) Groups (J) Groups
Mean Difference (I-
J) Std. Error Sig.
95% Confidence Interval
Lower
Bound Upper Bound
75 μl DMSO 10% ZnO-R -2.33333* .27217 .000 -3.2475 -1.4192
ZnO-S .00000 .27217 1.000 -.9142 .9142
MgO-R .00000 .27217 1.000 -.9142 .9142
MgO-S .00000 .27217 1.000 -.9142 .9142
3% NaOCl -5.66667* .27217 .000 -6.5808 -4.7525
ZnO-R DMSO 10% 2.33333* .27217 .000 1.4192 3.2475
ZnO-S 2.33333* .27217 .000 1.4192 3.2475
MgO-R 2.33333* .27217 .000 1.4192 3.2475
MgO-S 2.33333* .27217 .000 1.4192 3.2475
3% NaOCl -3.33333* .27217 .000 -4.2475 -2.4192
ZgO-S DMSO 10% .00000 .27217 1.000 -.9142 .9142
ZnO-R -2.33333* .27217 .000 -3.2475 -1.4192
MgO-R .00000 .27217 1.000 -.9142 .9142
MgO-S .00000 .27217 1.000 -.9142 .9142
3% NaOCl -5.66667* .27217 .000 -6.5808 -4.7525
MgO-R DMSO 10% .00000 .27217 1.000 -.9142 .9142
ZnO-R -2.33333* .27217 .000 -3.2475 -1.4192
ZnO-S .00000 .27217 1.000 -.9142 .9142
MgO-S .00000 .27217 1.000 -.9142 .9142
3% NaOCl -5.66667* .27217 .000 -6.5808 -4.7525
MgO-S DMSO 10% .00000 .27217 1.000 -.9142 .9142
ZnO-R -2.33333* .27217 .000 -3.2475 -1.4192
ZnO-S .00000 .27217 1.000 -.9142 .9142
MgO-R .00000 .27217 1.000 -.9142 .9142
3% NaOCl -5.66667* .27217 .000 -6.5808 -4.7525
3% NaOCl DMSO 10% 5.66667* .27217 .000 4.7525 6.5808
ZnO-R 3.33333* .27217 .000 2.4192 4.2475
ZnO-S 5.66667* .27217 .000 4.7525 6.5808
MgO-R 5.66667* .27217 .000 4.7525 6.5808
TABLE- 7 TUKEY POST HOC TEST- Multiple comparison of all the groups
RESULTS
45
MgO-S 5.66667* .27217 .000 4.7525 6.5808
100 μl DMSO
10%
ZnO-R -4.33333* .54433 .000 -6.1617 -2.5050
ZnO-S -2.33333* .54433 .010 -4.1617 -.5050
MgO-R -1.66667 .54433 .082 -3.4950 .1617
MgO-S -2.33333* .54433 .010 -4.1617 -.5050
3% NaOCl -9.33333* .54433 .000 -11.1617 -7.5050
ZnO-R DMSO 10% 4.33333* .54433 .000 2.5050 6.1617
ZnO-S 2.00000* .54433 .029 .1716 3.8284
MgO-R 2.66667* .54433 .004 .8383 4.4950
MgO-S 2.00000* .54433 .029 .1716 3.8284
3% NaOCl -5.00000* .54433 .000 -6.8284 -3.1716
ZnO-S DMSO 10% 2.33333* .54433 .010 .5050 4.1617
ZnO-R -2.00000* .54433 .029 -3.8284 -.1716
MgO-R .66667 .54433 .817 -1.1617 2.4950
MgO-S .00000 .54433 1.000 -1.8284 1.8284
3% NaOCl -7.00000* .54433 .000 -8.8284 -5.1716
MgO-R DMSO 10% 1.66667 .54433 .082 -.1617 3.4950
ZnO-R -2.66667* .54433 .004 -4.4950 -.8383
ZnO-S -.66667 .54433 .817 -2.4950 1.1617
MgO-S -.66667 .54433 .817 -2.4950 1.1617
3% NaOCl -7.66667* .54433 .000 -9.4950 -5.8383
MgO-S DMSO 10% 2.33333* .54433 .010 .5050 4.1617
ZnO-R -2.00000* .54433 .029 -3.8284 -.1716
ZnO-S .00000 .54433 1.000 -1.8284 1.8284
MgO-R .66667 .54433 .817 -1.1617 2.4950
3% NaOCl -7.00000* .54433 .000 -8.8284 -5.1716
3% NaOCl DMSO 10% 9.33333* .54433 .000 7.5050 11.1617
ZnO-R 5.00000* .54433 .000 3.1716 6.8284
ZnO-S 7.00000* .54433 .000 5.1716 8.8284
MgO-R 7.66667* .54433 .000 5.8383 9.4950
MgO-S 7.00000* .54433 .000 5.1716 8.8284
125 μl DMSO
10%
ZnO-R -7.00000* .33333 .000 -8.1196 -5.8804
ZnO-S -2.66667* .33333 .000 -3.7863 -1.5470
MgO-R -3.66667* .33333 .000 -4.7863 -2.5470
MgO-S -3.00000* .33333 .000 -4.1196 -1.8804
RESULTS
46
3% NaOCl -13.66667* .33333 .000 -14.7863 -12.5470
ZnO-R DMSO 10% 7.00000* .33333 .000 5.8804 8.1196
ZnO-S 4.33333* .33333 .000 3.2137 5.4530
MgO-R 3.33333* .33333 .000 2.2137 4.4530
MgO-S 4.00000* .33333 .000 2.8804 5.1196
3% NaOCl -6.66667* .33333 .000 -7.7863 -5.5470
ZnO-S DMSO 10% 2.66667* .33333 .000 1.5470 3.7863
ZnO-R -4.33333* .33333 .000 -5.4530 -3.2137
MgO-R -1.00000 .33333 .091 -2.1196 .1196
MgO-S -.33333 .33333 .909 -1.4530 .7863
3% NaOCl -11.00000* .33333 .000 -12.1196 -9.8804
MgO-R DMSO 10% 3.66667* .33333 .000 2.5470 4.7863
ZnO-R -3.33333* .33333 .000 -4.4530 -2.2137
ZnO-S 1.00000 .33333 .091 -.1196 2.1196
MgO-S .66667 .33333 .395 -.4530 1.7863
3% NaOCl -10.00000* .33333 .000 -11.1196 -8.8804
MgO-S DMSO 10% 3.00000* .33333 .000 1.8804 4.1196
ZnO-R -4.00000* .33333 .000 -5.1196 -2.8804
ZnO-S .33333 .33333 .909 -.7863 1.4530
MgO-R -.66667 .33333 .395 -1.7863 .4530
3% NaOCl -10.66667* .33333 .000 -11.7863 -9.5470
3% NaOCl DMSO 10% 13.66667* .33333 .000 12.5470 14.7863
ZnO-R 6.66667* .33333 .000 5.5470 7.7863
ZnO-S 11.00000* .33333 .000 9.8804 12.1196
MgO-R 10.00000* .33333 .000 8.8804 11.1196
MgO-S 10.66667* .33333 .000 9.5470 11.7863
150 μl DMSO
10%
Zno-R -8.33333* .38490 .000 -9.6262 -7.0405
ZnO-S -4.00000* .38490 .000 -5.2928 -2.7072
MgO-R -6.33333* .38490 .000 -7.6262 -5.0405
MgO-S -4.33333* .38490 .000 -5.6262 -3.0405
3% NaOCl -16.33333* .38490 .000 -17.6262 -15.0405
ZnO-R DMSO 10% 8.33333* .38490 .000 7.0405 9.6262
ZnO-S 4.33333* .38490 .000 3.0405 5.6262
MgO-R 2.00000* .38490 .002 .7072 3.2928
MgO-S 4.00000* .38490 .000 2.7072 5.2928
RESULTS
47
TABLE – 8
3% NaOCl -8.00000* .38490 .000 -9.2928 -6.7072
ZnO-S DMSO 10% 4.00000* .38490 .000 2.7072 5.2928
ZnO-R -4.33333* .38490 .000 -5.6262 -3.0405
MgO-R -2.33333* .38490 .001 -3.6262 -1.0405
MgO-S -.33333 .38490 .948 -1.6262 .9595
3% NaOCl -12.33333* .38490 .000 -13.6262 -11.0405
MgO-R DMSO 10% 6.33333* .38490 .000 5.0405 7.6262
ZnO-R -2.00000* .38490 .002 -3.2928 -.7072
ZnO-S 2.33333* .38490 .001 1.0405 3.6262
MgO-S 2.00000* .38490 .002 .7072 3.2928
3% NaOCl -10.00000* .38490 .000 -11.2928 -8.7072
MgO-S DMSO 10% 4.33333* .38490 .000 3.0405 5.6262
ZnO-R -4.00000* .38490 .000 -5.2928 -2.7072
ZnO-S .33333 .38490 .948 -.9595 1.6262
MgO-R -2.00000* .38490 .002 -3.2928 -.7072
3% NaOCl -12.00000* .38490 .000 -13.2928 -10.7072
3% NaOCl DMSO 10% 16.33333* .38490 .000 15.0405 17.6262
ZnO-R 8.00000* .38490 .000 6.7072 9.2928
ZnO-S 12.33333* .38490 .000 11.0405 13.6262
MgO-R 10.00000* .38490 .000 8.7072 11.2928
MgO-S 12.00000* .38490 .000 10.7072 13.2928
The mean difference is significant at the 0.05 level.
TABLE 7- Results of Tukey Post hoc for multiple comparison shows (For ATCC E.faecalis)
At 75 μl the mean differences between ZnO-R and 3% NaOCl was statistically significant with all other groups.
At 100 μl ul the mean differences between ZnO-R and 3% NaOCl was statistically significant with all other groups.
At 125 μl the mean differences between ZnO-R and 3% NaOCl was statistically significant with all other groups.
At 150 μl the mean differences between ZnO-R, MgO- R and 3% NaOCl was statistically significant with all other groups.
RESULTS
48
ORAL ISOLATE E.FAECALIS - One way ANOVA – Descriptive analysis of all
groups (mean, std deviation, std error)
N Mean Std.
Deviation Std.
Error
95% Confidence Interval for Mean
Minimum Maximum
Lower Bound
Upper Bound
50 μl DMSO 10% 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00
ZnO-R 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00
ZnO-S 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00
MgO-R 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00
MgO-S 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00
6.3% NaOCl 3 12.6667 .57735 .33333 11.2324 14.1009 12.00 13.00
Total 18 8.7778 1.80051 .42438 7.8824 9.6731 8.00 13.00
75 ul DMSO 10% 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00
ZnO-R 3 10.6667 .57735 .33333 9.2324 12.1009 10.00 11.00
ZnO-S 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00
MgO-R 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00
MgO-S 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00
6.3% NaOCl 3 14.0000 .00000 .00000 14.0000 14.0000 14.00 14.00
Total 18 9.4444 2.33193 .54964 8.2848 10.6041 8.00 14.00
100 μl DMSO 10% 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00
ZnO-R 3 13.0000 .00000 .00000 13.0000 13.0000 13.00 13.00
ZnO-S 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00
MgO-R 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00
MgO-S 3 11.3333 .57735 .33333 9.8991 12.7676 11.00 12.00
6.3% NaOCl 3 17.6667 .57735 .33333 16.2324 19.1009 17.00 18.00
Total 18 11.0000 3.66221 .86319 9.1788 12.8212 8.00 18.00
125 μl DMSO 10% 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00
ZnO-R 3 13.3333 .57735 .33333 11.8991 14.7676 13.00 14.00
ZnO-S 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00
MgO-R 3 10.0000 .00000 .00000 10.0000 10.0000 10.00 10.00
MgO-S 3 12.0000 .00000 .00000 12.0000 12.0000 12.00 12.00
6.3% NaOCl 3 21.3333 .57735 .33333 19.8991 22.7676 21.00 22.00
Total 18 12.1111 4.70155 1.10817 9.7731 14.4491 8.00 22.00
RESULTS
49
Table 8- Showing the descriptive statistics for all the groups for Oral isolate
E.faecalis.
150μl 150 ul
DMSO 10% 3 8.0000 .00000 .00000 8.0000 8.0000 8.00 8.00
ZnO-R 3 15.0000 .00000 .00000 15.0000 15.0000 15.00 15.00
ZnO-S 3 10.3333 .57735 .33333 8.8991 11.7676 10.00 11.00
MgO-R 3 11.3333 .57735 .33333 9.8991 12.7676 11.00 12.00
MgO-S 3 13.0000 .00000 .00000 13.0000 13.0000 13.00 13.00
6.3% NaOCl 3 23.6667 .57735 .33333 22.2324 25.1009 23.00 24.00
Total 18 13.5556 5.17030 1.21865 10.9844 16.1267 8.00 24.00
TABLE -9 ANOVA Test for Zone Of Inhibition of all the Groups
(Intra-group and Inter-group analysis were statistically significant)
Sum of Squares df Mean Square F Sig.
50 μL Between Groups 54.444 5 10.889 196.000 .000
Within Groups .667 12 .056
Total 55.111 17
75μl Between Groups 91.778 5 18.356 330.400 .000
Within Groups .667 12 .056
Total 92.444 17
100μl Between Groups 226.667 5 45.333 408.000 .000
Within Groups 1.333 12 .111
Total 228.000 17
125μl Between Groups 374.444 5 74.889 674.000 .000
Within Groups 1.333 12 .111
Total 375.778 17
150μl Between Groups 452.444 5 90.489 542.933 .000
Within Groups 2.000 12 .167
Total 454.444 17
RESULTS
50
Dependent Variable (I) Groups (J) Groups
Mean Difference (I-J)
Std. Error Sig.
95% Confidence Interval
Lower Bound Upper Bound
50 μl DMSO 10% ZnO-R .00000 .19245 1.000 -.6464 .6464
ZnO-S .00000 .19245 1.000 -.6464 .6464
MgO-R .00000 .19245 1.000 -.6464 .6464
MgO-S .00000 .19245 1.000 -.6464 .6464
3% NaOCl -4.66667* .19245 .000 -5.3131 -4.0202
ZnO-R DMSO 10% .00000 .19245 1.000 -.6464 .6464
ZnO-S .00000 .19245 1.000 -.6464 .6464
MgO-R .00000 .19245 1.000 -.6464 .6464
MgO-S .00000 .19245 1.000 -.6464 .6464
3% NaOCl -4.66667* .19245 .000 -5.3131 -4.0202
ZnO-S DMSO 10% .00000 .19245 1.000 -.6464 .6464
ZnO-R .00000 .19245 1.000 -.6464 .6464
MgO-R .00000 .19245 1.000 -.6464 .6464
MgO-S .00000 .19245 1.000 -.6464 .6464
3% NaOCl -4.66667* .19245 .000 -5.3131 -4.0202
MgO-R DMSO 10% .00000 .19245 1.000 -.6464 .6464
ZnO-R .00000 .19245 1.000 -.6464 .6464
ZnO-S .00000 .19245 1.000 -.6464 .6464
MgO-S .00000 .19245 1.000 -.6464 .6464
3% NaOCl -4.66667* .19245 .000 -5.3131 -4.0202
MgO-S DMSO 10% .00000 .19245 1.000 -.6464 .6464
ZnO-R .00000 .19245 1.000 -.6464 .6464
ZnO-S .00000 .19245 1.000 -.6464 .6464
MgO-R .00000 .19245 1.000 -.6464 .6464
3% NaOCl -4.66667* .19245 .000 -5.3131 -4.0202
6.3% NaOCl DMSO 10% 4.66667* .19245 .000 4.0202 5.3131
ZnO-R 4.66667* .19245 .000 4.0202 5.3131
ZnO-S 4.66667* .19245 .000 4.0202 5.3131
MgO-R 4.66667* .19245 .000 4.0202 5.3131
TABLE 10 - Tukey Post Hoc Test –Multiple Comparison of all the Groups
RESULTS
51
MgO-S 4.66667* .19245 .000 4.0202 5.3131
75 μl DMSO 10% ZnO-R -2.66667* .19245 .000 -3.3131 -2.0202
ZnO-S .00000 .19245 1.000 -.6464 .6464
MgO-R .00000 .19245 1.000 -.6464 .6464
MgO-S .00000 .19245 1.000 -.6464 .6464
3% NaOCl -6.00000* .19245 .000 -6.6464 -5.3536
ZnO-R DMSO 10% 2.66667* .19245 .000 2.0202 3.3131
ZnO-S 2.66667* .19245 .000 2.0202 3.3131
MgO-R 2.66667* .19245 .000 2.0202 3.3131
MgO-S 2.66667* .19245 .000 2.0202 3.3131
3% NaOCl -3.33333* .19245 .000 -3.9798 -2.6869
ZnO-S DMSO 10% .00000 .19245 1.000 -.6464 .6464
ZNo-R -2.66667* .19245 .000 -3.3131 -2.0202
MGO-R .00000 .19245 1.000 -.6464 .6464
MGO-S .00000 .19245 1.000 -.6464 .6464
3% NaOCl -6.00000* .19245 .000 -6.6464 -5.3536
MgO-R DMSO 10% .00000 .19245 1.000 -.6464 .6464
ZnO-R -2.66667* .19245 .000 -3.3131 -2.0202
ZnO-S .00000 .19245 1.000 -.6464 .6464
MgO-S .00000 .19245 1.000 -.6464 .6464
3% NaOCl -6.00000* .19245 .000 -6.6464 -5.3536
MgO-S DMSO 10% .00000 .19245 1.000 -.6464 .6464
ZnO-R -2.66667* .19245 .000 -3.3131 -2.0202
ZnO-S .00000 .19245 1.000 -.6464 .6464
MgO-R .00000 .19245 1.000 -.6464 .6464
3% NaOCl -6.00000* .19245 .000 -6.6464 -5.3536
3% NaOCl DMSO 10% 6.00000* .19245 .000 5.3536 6.6464
ZnO-R 3.33333* .19245 .000 2.6869 3.9798
ZnO-S 6.00000* .19245 .000 5.3536 6.6464
MgO-R 6.00000* .19245 .000 5.3536 6.6464
MgO-S 6.00000* .19245 .000 5.3536 6.6464
100 μl DMSO 10% ZnO-R -5.00000* .27217 .000 -5.9142 -4.0858
ZnO-S .00000 .27217 1.000 -.9142 .9142
MgO-R .00000 .27217 1.000 -.9142 .9142
MgO-S -3.33333* .27217 .000 -4.2475 -2.4192
RESULTS
52
3% NaOCl -9.66667* .27217 .000 -10.5808 -8.7525
ZnO-R DMSO 10% 5.00000* .27217 .000 4.0858 5.9142
ZnO-S 5.00000* .27217 .000 4.0858 5.9142
MgO-R 5.00000* .27217 .000 4.0858 5.9142
MgO-S 1.66667* .27217 .001 .7525 2.5808
3% NaOCl -4.66667* .27217 .000 -5.5808 -3.7525
ZnO-S DMSO 10% .00000 .27217 1.000 -.9142 .9142
ZnO-R -5.00000* .27217 .000 -5.9142 -4.0858
MgO-R .00000 .27217 1.000 -.9142 .9142
MgO-S -3.33333* .27217 .000 -4.2475 -2.4192
3% NaOCl -9.66667* .27217 .000 -10.5808 -8.7525
MgO-R DMSO 10% .00000 .27217 1.000 -.9142 .9142
ZnO-R -5.00000* .27217 .000 -5.9142 -4.0858
ZnO-S .00000 .27217 1.000 -.9142 .9142
MgO-S -3.33333* .27217 .000 -4.2475 -2.4192
3% NaOCl -9.66667* .27217 .000 -10.5808 -8.7525
MgO-S DMSO 10% 3.33333* .27217 .000 2.4192 4.2475
ZnO-R -1.66667* .27217 .001 -2.5808 -.7525
ZnO-S 3.33333* .27217 .000 2.4192 4.2475
MgO-R 3.33333* .27217 .000 2.4192 4.2475
3% NaOCl -6.33333* .27217 .000 -7.2475 -5.4192
6.3% NaOCl DMSO 10% 9.66667* .27217 .000 8.7525 10.5808
ZNo-R 4.66667* .27217 .000 3.7525 5.5808
ZNO-S 9.66667* .27217 .000 8.7525 10.5808
MGO-R 9.66667* .27217 .000 8.7525 10.5808
MGO-S 6.33333* .27217 .000 5.4192 7.2475
125 μl DMSO 10% ZnO-R -5.33333* .27217 .000 -6.2475 -4.4192
ZnO-S .00000 .27217 1.000 -.9142 .9142
MgO-R -2.00000* .27217 .000 -2.9142 -1.0858
MgO-S -4.00000* .27217 .000 -4.9142 -3.0858
3% NaOCl -13.33333* .27217 .000 -14.2475 -12.4192
ZnO-R DMSO 10% 5.33333* .27217 .000 4.4192 6.2475
ZnO-S 5.33333* .27217 .000 4.4192 6.2475
MgO-R 3.33333* .27217 .000 2.4192 4.2475
MgO-S 1.33333* .27217 .004 .4192 2.2475
RESULTS
53
6.3% NaOCl -8.00000* .27217 .000 -8.9142 -7.0858
ZnO-S DMSO 10% .00000 .27217 1.000 -.9142 .9142
ZnO-R -5.33333* .27217 .000 -6.2475 -4.4192
MgO-R -2.00000* .27217 .000 -2.9142 -1.0858
MgO-S -4.00000* .27217 .000 -4.9142 -3.0858
3% NaOCl -13.33333* .27217 .000 -14.2475 -12.4192
MgO-R DMSO 10% 2.00000* .27217 .000 1.0858 2.9142
ZnO-R -3.33333* .27217 .000 -4.2475 -2.4192
ZnO-S 2.00000* .27217 .000 1.0858 2.9142
MgO-S -2.00000* .27217 .000 -2.9142 -1.0858
3% NaOCl -11.33333* .27217 .000 -12.2475 -10.4192
MgO-S DMSO 10% 4.00000* .27217 .000 3.0858 4.9142
ZnO-R -1.33333* .27217 .004 -2.2475 -.4192
ZnO-S 4.00000* .27217 .000 3.0858 4.9142
MgO-R 2.00000* .27217 .000 1.0858 2.9142
3% NaOCl -9.33333* .27217 .000 -10.2475 -8.4192
3% NaOCl DMSO 10% 13.33333* .27217 .000 12.4192 14.2475
ZnO-R 8.00000* .27217 .000 7.0858 8.9142
ZnO-S 13.33333* .27217 .000 12.4192 14.2475
MgO-R 11.33333* .27217 .000 10.4192 12.2475
MgO-S 9.33333* .27217 .000 8.4192 10.2475
150μl DMSO 10% ZnO-R -7.00000* .33333 .000 -8.1196 -5.8804
ZnO-S -2.33333* .33333 .000 -3.4530 -1.2137
MgO-R -3.33333* .33333 .000 -4.4530 -2.2137
MgO-S -5.00000* .33333 .000 -6.1196 -3.8804
3% NaOCl -15.66667* .33333 .000 -16.7863 -14.5470
ZnO-R DMSO 10% 7.00000* .33333 .000 5.8804 8.1196
ZnO-S 4.66667* .33333 .000 3.5470 5.7863
MgO-R 3.66667* .33333 .000 2.5470 4.7863
MgO-S 2.00000* .33333 .001 .8804 3.1196
3% NaOCl -8.66667* .33333 .000 -9.7863 -7.5470
ZnO-S DMSO 10% 2.33333* .33333 .000 1.2137 3.4530
ZnO-R -4.66667* .33333 .000 -5.7863 -3.5470
MgO-R -1.00000 .33333 .091 -2.1196 .1196
MgO-S -2.66667* .33333 .000 -3.7863 -1.5470
RESULTS
54
TABLE- 10 Results of Tukey Post hoc for multiple comparison shows (For Oral isolate E.faecalis)
At 75 μl the mean differences between ZnO-R and 3% NaOCl was statistically significant with all other groups.
At 100 μl the mean differences between ZnO-R, MgO-S and 3% NaOCl was statistically significant with all other groups.
At 125 μl the mean differences between ZnO-R, MgO-R, MgO-S and 3% NaOCl was statistically significant with all other groups.
At 150 μl the mean differences between ZnO-R, MgO-S and 3% NaOCl was statistically significant with all other groups
3% NaOCl -13.33333* .33333 .000 -14.4530 -12.2137
MgO-R DMSO 10% 3.33333* .33333 .000 2.2137 4.4530
ZnO-R -3.66667* .33333 .000 -4.7863 -2.5470
ZnO-S 1.00000 .33333 .091 -.1196 2.1196
MgO-S -1.66667* .33333 .003 -2.7863 -.5470
3% NaOCl -12.33333* .33333 .000 -13.4530 -11.2137
MgO-S DMSO 10% 5.00000* .33333 .000 3.8804 6.1196
ZnO-R -2.00000* .33333 .001 -3.1196 -.8804
ZnO-S 2.66667* .33333 .000 1.5470 3.7863
MgO-R 1.66667* .33333 .003 .5470 2.7863
3% NaOCl -10.66667* .33333 .000 -11.7863 -9.5470
3% NaOCl DMSO 10% 15.66667* .33333 .000 14.5470 16.7863
ZnO-R 8.66667* .33333 .000 7.5470 9.7863
ZnO-S 13.33333* .33333 .000 12.2137 14.4530
MgO-R 12.33333* .33333 .000 11.2137 13.4530
MgO-S 10.66667* .33333 .000 9.5470 11.7863
The mean difference is significant at the 0.05 level.
RESULTS
AGAR WELL DIFFUSION ASSAY-
ZINC OXIDE NANORODS - ZOI
ZINC OXIDE NANOSPHERE-ZOI
Fig 43e- MIC determination of ZnO nanorods and nano sphere
Fig 43a- E. faecalis ATCC 29212
Fig 43b- E. faecalis clinical (oral) isolate
Fig 43c- E. faecalis ATCC 29212
Fig 43d- E. faecalis clinical (oral) isolate
75μl 50μl
100μl
50μl 75μl
100μl
100μl
100μl 50μl
50μl 75μl 75μl
125μl
MAGNESIUM OXIDE NANOROD-ZOI
MAGNESIUM OXIDE NANOSPHERE-ZOI
Fig 44e- MIC determination of MgO SPHERE AND RODS
Fig 44a- E. faecalis ATCC 29212
Fig 44b- E. faecalis clinical (oral) isolate
Fig 44c- E. faecalis ATCC 29212
Fig 44d- E. faecalis clinical (oral) isolate
MBC OF ZINC OXIDE NANORODS
MBC OF ZINC OXIDE NANOSPHERE
Fig 46a- E. faecalis ATCC 29212- NO GROWTH UNTIL
2nd WELL
Fig 45b- E. faecalis clinical (oral) isolate- NO GROWTH
UNTIL 2nd WELL
Fig 45a- E. faecalis ATCC 29212- NO GROWTH UNTIL
3rd WELL
Fig 46b- E. faecalis clinical (oral) isolate – NO GROWTH
UNTIL 1st WELL
MBC OF MAGNESIUM OXIDE NANORODS
Against E. faecalis ATCC 29212 & E. faecalis clinical (oral) isolate
MBC OF MAGNESIUM OXIDE SPHERE
ORAL ISOLATE
ATCC 29212
Fig 48a- E. faecalis ATCC 29212- NO GROWTH
UNTIL 7 WELL
Fig 48b- E faecalis clinical (oral) isolate NO GROWTH
UNTIL 3RD WELL
Fig 47- NO GROWTH UNTIL 3RD WELL
3% SODIUM HYPOCHLORITE (POSITIVE CONTROL)
10 % DMSO (NEGATIVE CONTROL- ZOI
Fig 49c- MIC determination of 3%NaOCl
MBC OF 3% NaOCl
Fig 49d- NO GROWTH
UNTIL 7 WELLS
Fig 49a- E. faecalis ATCC 29212
Fig 49b- E. faecalis clinical (oral) isolate
ORAL ISOLATE
ATCC 29212
100μl NaOCl
150 μl DMSO
150 μl NaOCl 125μl NaOCl
100μl NaOCl 150 μl NaOCl
125μl NaOCl 150 μl DMSO
TIME KILL CURVE – ZINC OXIDE NANORODS
Fig 50a- E. faecalis (ATCC 29212 ) - NO GROWTH
Fig 50b- E.faecalis (Oral isolate) – NO GROWTH
15 MINS
30 MINS 45 MINS
60 MINS
90 MINS
15 MINS
30 MINS 45 MINS
60 MINS
90 MINS
ZINC OXIDE NANOSPHERE
Fig 51a- E. faecalis (ATCC 29212) – NO GROWTH
Fig 51b- E.faecalis (Oral isolate)-NO GROWTH
15 MINS
30 MINS 45 MINS
60 MINS
90 MINS
15 MINS
30 MINS 45 MINS
60 MINS
90 MINS
MAGNESIUM OXIDE NANORODS
Fig 52a- E. faecalis (ATCC 29212)- GROWTH AT 15 MINS
Fig 52b- E.faecalis (Oral isolate) - NO GROWTH
GROWTH AT 15 MINS
15 MINS
30 MINS 45 MINS
60 MINS
90 MINS
15 MINS
30 MINS 45 MINS
60 MINS
90 MINS
MAGNESIUM OXIDE NANOSPHERE
Fig 53a- E. faecalis (ATCC 29212) - NO GROWTH
Fig 53b- E.faecalis (Oral isolate) -NO GROWTH
15 MINS
30 MINS 45 MINS
60 MINS
90 MINS
15 MINS
30 MINS 45 MINS
60 MINS
90 MINS
DISCUSSION
55
Microbial elements are the most common cause of pulpal and periapical
pathosis. All endodontic infections are poly-microbial in nature with differences
between the types of micro-organisms isolated from primary and secondary root
canal infections. Endodontic infection is eventually established as a biofilm mediated
pathosis.19 Hence a new stratergy of disinfection is needed to address the
Endodontic pathosis. This could be accomplished by the domain of Nanotechnology.
Nanotechnology is defined as a science that deals with the development of new
materials with new properties and functions through controlling and restructuring of
the materials on a nanometer scale of “less than 100 nm” and hence the name
nanomaterials. Nanomaterials exist in different forms and shapes.64
They are categorized according to their dimensions: Categories are based on the
number of dimensions, which are not confined to the nanoscale range
Zero-dimensional (0-D) - Materials where in all the dimensions are measured within
the nanoscale (no dimensions, or 0-D). Ex nanospheres.
One-dimensional (1-D) - One-dimensional nanomaterials have one dimension that is
outside the nanoscale. This leads to needle like-shaped nano materials. Ex
nanorods, nanotubes, nanowires, nanoneedles
Two-dimensional (2-D) - In two-dimensional (2-D), Two of the dimensions are not
confined to the nanoscale. 2-D nanomaterials exhibit plate-like shapes, nanofilms,
nanolayers, and nanocoatings
Three-dimensional (3-D) - Three-dimensional (3-D) are Bulk nanomaterials.82
DISCUSSION
56
The use of nanoparticles as antimicrobial agents has recently attracted considerable
attention in the field of medicine as a result of their superior antibacterial properties
compared with those of other antimicrobial agents together with a low potential of
developing microbial resistance.69 The efficacy of the nanoparticles to eliminate
bacterial cells is attributed to the concurrent effect of two different mechanisms. One
involves the binding of nanoparticles to the bacterial cell membrane through
electrostatic forces, causing an alteration in the membrane potential,
depolarization and eventually loss of membrane integrity.54 The second
mechanism includes the production of oxygen free radicals such as reactive-oxygen
species (ROS) that can influence survival of the bacterial cell by blocking the
protein function, metabolism and destroying DNA.84 Different types of
nanoparticles silver, chitosan, BAG, zinc oxide have been investigated recently in
invitro studies to evaluate their efficacy against endodontic pathogens.37
In this invitro study Zinc oxide and Magnesium oxide nanoparticles with two
distinct morphologies nanorod and nanosphere were chosen to explore their
antimicrobial efficacy against the endodontic pathogen (ATCC 29212 and Oral
isolate E.faecalis).
E. faecalis was chosen for this study because it is potentially important
microorganism to colonize in endodontic infections and being the dominant microbe
in post-treatment apical diseases.77 In mixed infections, E. faecalis is typically the
dominant isolate. This organism is extremely adaptable to the environment and it can
withstand harsh conditions in the root filled tooth. It is commonly resistant to various
antimicrobial agents and is considered as the offender in post-treatment diseases.62
DISCUSSION
57
It is therefore important to develop novel disinfection approaches against E.faecalis.
ATCC 29212 is a standard strain for research use and in order to mimic clinical
scenario, an oral isolate E.Faecalis from a root canal failure case was isolated
and used in this study. Further effectiveness of nanoparticles was also compared
between the strains.
ZINC OXIDE NANOPARTICLE
Zinc oxide nanoparticle (ZnO) is described as a functional, strategic and versatile
inorganic material with a broad range of applications. ZnO nanoparticle holds unique
optical, chemical sensing, semiconducting, electric, and piezoelectric properties. It is
characterized by a wide band gap (3.3 eV) in the near-UV spectrum, a high
excitation binding energy (60 meV) at room temperature, and a natural n-type
electrical conductivity. These properties make ZnO nanoparticle a unique material
and enable it to have astounding applications in diverse disciplines.15
Basic ZnO nanoparticle Properties
1. Molecular Weight 81.38 g/mol
2. Crystal Structure Wurtzite
3 Lattice Constant a = 3.25 Å, c = 5.2 Å
4 Band Gap 3.3 eV
5 Refractive Index (μD) 2.0041
6 Density 5.606 g/cm3
7 Melting Point 1975°C
DISCUSSION
58
Due to the splendid properties such as high thermal conductivity, high refractive
index, binding energy, UV protection and antibacterial capabilities of Zinc Oxide
NPs, it is extensively applied in various fields including medicine, cosmetics, solar
cells, rubber, concrete and foods technologies. Due to the antimicrobial and
antitumor activities, Zinc Oxide (ZnO) among nano-sized metal oxides has been
extensively applied in medicine. 51
An assortment of ZnO nanostructures with different growth morphologies such as
nanorods, nanoflowers, nanocombs, nanoplates, nanosphere, nanotubes,
nanowires, nanoneedles and nanorings have been successfully synthesized. These
different morphologies displayed striking antibacterial effect toward the targeted
microbe.91, 46
ANTIBACTERIAL MECHANISM OF ZINC OXIDE NANOPARTICLES
Various mechanisms have been put forward by researchers such as: direct contact of
ZnO-NPs with cell walls, resulting in destructing bacterial cell wall integrity,
liberation of antimicrobial ions mainly Zinc ions and Reactive oxygen species
(ROS). The antimicrobial effect is varied depending on size, shape and doping
characteristics.97
The shape (morphology) dependent activity was explained in terms of the percent
of active facets in the NPs. Synthesis and growth techniques markedly influences the
active facets present in NPs. Nano-rod-structures of ZnO have 111 facets, whereas
spherical nanostructures have 100 facets. The presence of high-atomic-density
facets on the surface of NPs exhibit higher antimicrobial action. The facet-
dependent ZnO NPs antibacterial activity has been evaluated by few studies, and
DISCUSSION
59
nanostructured ZnO with different morphologies have different active facets leading
to enhanced antibacterial activity.60,59,67 In this regard, the shape of ZnO
nanostructures can influence their mechanism of interaction, such as rods (more
facet) and wires penetrating into cell walls of bacteria more easily than spherical
ZnO-NPs.41
Size dependent activity ZnO-NPs of smaller sizes, less than 50 nm can easily
penetrate into bacterial cell wall due to their large interfacial area, thus enhancing
their antibacterial efficiency. Considering the impact of particle size on the
antibacterial activity, researchers found that controlling ZnO-NPs size was a critical
key to achieve optimum bactericidal response, and ZnO-NPs with smaller size
(higher surface areas) showed highest antibacterial activity. The dissolution of ZnO-
NPs into Zinc ions was reported as size dependent, and few experiments suggested
that this dissolution of Zn2+ is responsible for toxicity of ZnO-NPs. Smaller size and
higher concentration promoted better activity.98 Owing to above advantages ZnO
nanoparticles synthesized and evaluated in this study was average size of 20 nm.
SCHEMATIC REPRESENTATION OF ZnO NPs ANTIMICROBIAL MECHANISM
Bacterial cell O2-
OH-
Nucleus DNA
NANO RODS
NANOSPHERE
SPHERES CELL WALL DAMAGE
DISCUSSION
60
ZnO antibacterial activity was due to the increased yield of reactive oxygen species
(ROS) such as superoxide anion (O2), hydrogen peroxide (H2O2), and hydroxyl
ion (OH-). The toxicity of these species involves the destruction of cellular
constituents such as lipids, DNA and proteins, as a result of their introjection into
the bacterial cell membrane. The super-oxides and hydroxyl radicals cannot penetrate
into the membrane due to their negative charges. Thus, these species are found on the
outer surface of the bacteria; by disparity, Hydrogen peroxide (H2O2) molecules are
able to pass through the bacterial cell wall and get internalized subsequently leading
to destruction of organelles and finally triggering cell death. 58
In this study zinc oxide nanosphere was synthesized by Sol-Gel method because
it is a simple and versatile technique to control the size and morphology, while
zinc oxide nanorod was synthesized by Hydrothermal method to eventually get
desired nanorod with better crystalline, thermal and chemical stability.
MAGNESIUM OXIDE NANOPARTICLES
Magnesium oxide is odorless and non-toxic. MgO NPs possess high hardness, purity
and a high melting point. MgO NP is an important inorganic material with a wide
band-gap. Magnesium oxide nanoparticles appear in a white powder form. Among
the inorganic metal oxide nanoparticles, MgO NPs is particularly interesting due
to its strong antibacterial activity, high thermal stability and low cost.4 In
medicine, MgO is used for the relief of heartburn, sore stomach, and for bone
regeneration Recently, MgO nanoparticles have shown a promising application in
tumor treatment and also is being explored for their antimicrobial action.11
DISCUSSION
61
Chemical symbol MgO
Group Magnesium2 Oxygen 16
Band gap 7.2 eV
Density 3.58g/cm3
Molar mass 40.3 g/mol
Melting point 2852°C
Boiling point 3600°C
ANTIBACTERIAL MECHANISM OF MgO NANOPARTICLES
Size dependent action: Many reports have shown that the antibacterial activity of
MgO nanoparticles is size-dependent. The antibacterial activity was increased
with the decrease of the particle size of MgO NPs. For particles within the size
range of 45-70 nm, the bactericidal efficacy of nano-MgO increased slowly with
decreasing particle size. Below ~ 45 nm however, the bactericidal efficacy was much
stronger and good. Generally, the surface area of MgO nanoparticles increases as the
size of the nanoparticles decreases. The increase in surface area determines the
potential number of reactive groups on the particle surface, which is expected to
show high antibacterial activity.32 Hence in this study 20 nm sized MgO NPs was
synthesized and evaluated for antibacterial efficacy.
Concentration dependent: In addition to size-dependent antibacterial effect of the
nanoparticles, MgO nanoparticles show concentration dependent activity also. The
antibacterial effect was dose dependent. An et al. (2011) and Zhang et al. (2011) also
MgO NANOPARTICLES - BASIC PROPERTIES
DISCUSSION
62
found that high MgO nanoparticle concentrations resulted in greater bacterial
inactivation. 96 MgO nanoparticles have better activity towards gram-positive
bacteria than towards gram negative bacteria. The reason is probably due to the
difference in cell wall structure. The cell wall of gram-positive bacteria is made of
peptidoglycan, while for gram negative organism cell wall is thick
Lipopolysaccharahide.5
Morphology dependent activity: Various morphologies like, plates, rods, disk,
needles, tubes, spherical shaped MgO nanoparticles can be prepared by controlling
reaction precursors and parameters. The antimicrobial effectiveness also varied
according to the morphology of the MgO NPs; nanorods exhibiting increased surface
area, which aid in better interaction with bacteria.80 Number of mechanisms
proposed, such as the formation of reactive oxygen species (ROS), the interaction of
nanoparticles with bacteria, subsequently damaging the bacterial cell wall, and an
alkaline effect have been proposed to explain the antibacterial mechanism of MgO
nanoparticles. The antibacterial mechanism of MgO nanoparticles is due to the
formation of ROS such as superoxide anion (O2-). 40 It has been reported that the
increase of the surface area of MgO particles leads to an increase of the O2-
concentration in solution, thus resulting in a more effective destruction of the cell
wall of the bacteria.
The mechanism of the antibacterial activity of MgO nanoparticles is due to lipid per-
oxidation, ROS production and the presence of defects on surface of
nanoparticles. Nano-MgO particles could take up halogen gases due to the
defective nature of their surface and its positive charge, which resulted in a
DISCUSSION
63
strong interaction with bacteria, which are negatively charged. The superoxide
anion will attack the carbonyl carbon atom in the peptide linkages of proteins in
microbes, which eventually lead to destruction of the bacteria. 76
The alkaline effect has been considered as another prominent factor in the
antibacterial action of MgO nanoparticles. It was proposed that the possible
antibacterial mechanism was the adsorption of water moisture on the MgO
nanoparticle surfaces, which could form a thin water layer around the particles. The
local pH of this thin water layer formed around the nanoparticles might be much
higher than its equilibrium value in solution. When the nanoparticles are in contact
with the bacteria, the high pH in this thin surface water layer could damage the
membrane, resulting in cell death. 66
SCHEMATIC REPRESENTATION OF MgO NPs ANTIMICROBIAL MECHANISM
In this study Magnesium oxide nanosphere was synthesized by mild and
efficient Sol-Gel method and MgO nanorods by Co-precipitation method proven
to be efficient procedure to produce highly reactive nanoparticles.
H202 O2- O2- O2-
BACTERIAL CELL
DNA
NANO RODS
NANOSPHERE SPHERES CELL WALL
DISCUSSION
64
CHARACTERISATION OF NANOPARTICLES
The synthesized zinc oxide and magnesium oxide nanoparticles with rod and sphere
morphology were characterised by UV spectrophotometer, FTIR and TEM.
UV- VISIBLE SPECTROPHOTOMETER: UV-vis spectrophotometer is a very
useful and reliable technique for the primary characterization of synthesized
nanoparticles. In addition, UV-vis spectrophotometer is fast, easy, simple, sensitive,
selective for different types of NPs, and needs only a short period time for
measurement.21 Absorbance peak for zinc oxide nanopaticles ranged between 300 to
400nm and for magnesium oxide nanoparticles 200 to 300nm. Synthesized
nanoparticles were evaluated and peak values obtained for Zinc oxide
nanorods- 360nm, Zinc oxide nanosphere – 350nm, Magnesium oxide nanorods-
250nm, Magnesium oxide nanosphere –270nm.
FTIR- FOURIER TRANSFORM INFRARED SPECTROSCOPY: Fourier
transform infrared (FTIR) spectroscopy is commonly employed to use the expression
of characteristic spectral bands to reveal nano-material. The infrared spectrum is
related to the vibrations of molecules and is unique for each compound. The
absorption of Infrared radiation transfers energy to the molecules, inducing
corresponding covalent bond stretching, bending or twisting. Generally in a
molecule, the vibrations involve various coupled pairs of atoms or covalent bonds,
each of which must be considered as a combination of the normal modes; therefore,
the IR spectrum, illustrating absorption or transmission versus incident IR frequency,
can offer a fingerprint of the structure of the molecule of interest. 8
DISCUSSION
65
TEM-TRANSMISSION ELECTRON MICROSCOPY: TEM is a valuable,
frequently used, and important technique for the characterization of nanomaterials,
used to obtain quantitative measures of particle and/or grain size, size distribution,
and morphology. The magnification of TEM is mainly determined by the ratio of the
distance between the objective lens and the specimen and the distance between
objective lens and its image plane. TEM has two advantages over SEM: it can
provide better spatial resolution and the capability for additional analytical
measurements.2 Synthesized nanoparticles of average size of 20 nm and
morphology - sphere and rod obtained for both ZnO and MgO NPs was
evaluated using TEM.
EVALUATION OF ANTIMICROBIAL EFFICACY OF NANOPARTICLES
Following synthesis and characterisation, the antimicrobial efficacy of nanoparticles
was determined by Agar well diffusion assay for determining Zone of Inhibition
and Broth microdilution method was chosen for determining MIC/ MBC. Time
kill assay was done to evaluate the time needed for the nanoparticles to destroy the
bacteria.
Agar well diffusion method is widely used to evaluate the antimicrobial activity of
anti-microbial agents. The agar plate surface is inoculated by spreading a volume of
the microbial inoculum over the entire agar surface. Then, a hole with a diameter of 8
mm is punched aseptically with a cork borer and a volume (20–100 ml) of the
antimicrobial agent at desired concentration is introduced into the well. Then, agar
plates are incubated under suitable conditions depending upon the test
DISCUSSION
66
microorganism. The antimicrobial agent diffuses in the agar medium and inhibits the
growth of the microbial strain tested, later zone of inhibition can be analysed. This
procedure is easy and simple to evaluate.83
Broth micro-dilution is one of the most basic antimicrobial susceptibility testing
methods. The procedure involves preparing two-fold dilutions of the antimicrobial
agent in a liquid growth medium dispensed with smaller volumes using 96-well
microtitration plate (microdilution). The MIC Minimum inhibitory concentration
is the lowest concentration of antimicrobial agent that completely inhibits growth of
the organism in microdilution wells (containing antimicrobial agent and bacterial
inoculums) as detected by the unaided eye. The reproducibility and space that occurs
due to the miniaturization of the test are the major advantages of the microdilution
method.9
The determination of Minimum bactericidal concentration (MBC) also known as
the minimum lethal concentration (MLC),is the most common estimation of
bactericidal activity. The MBC is defined as the lowest concentration of
antimicrobial agent needed to kill 99.9% of the final inoculum after incubation for
24 hr under a standardized set of conditions, in which the MBC can be determined
after broth microdilution by sub-culturing a sample from wells, yielding a negative
microbial growth after incubation on the surface of non-selective agar plates to
determine the number of surviving cells (CFU/ml) after 24 h of incubation. The
bactericidal endpoint (MBC) has been subjectively defined as the lowest
concentration, at which 99.9% of the final inoculum is killed. 20
DISCUSSION
67
Time-kill assay/test is the most pertinent method for determining the bactericidal or
fungicidal effect. It is a strong tool for obtaining information about the dynamic
interaction between the antimicrobial agent and the microbial strain. The time-kill
test reveals a time-dependent or a concentration-dependent antimicrobial effect. 56
In this experiment, the Zone of inhibition on plates inoculated with ATCC
29212 and ORAL ISOLATE E. faecalis was investigated to determine the extent
of antibacterial activity of the Zinc oxide (ZnO) and Magnesium oxide (MgO)
nanoparticles with two varied morphologies nanorods and nanosphere.
The results of the agar well diffusion test investigated zone of inhibition of
nanoparticles (ZnO nanorod, ZnO nanosphere, MgO nanorod, MgO nanosphere)
with positive (3% NaOCl) and negative control (10% DMSO). Antibacterial activity
of nanoparticles of average size of 20 nm was evaluated at five different volumes 50,
75, 100, 125, 150 microliters.
ZINC OXIDE NANOPARTICLE
Zinc oxide nanorod exhibited good activity at 75 μl against both strains of
E.faecalis. Antibacterial effectiveness of Zinc oxide Nanorod is because of
increased surface area, presence of more number of facets, which enhances the
antibacterial efficacy by promoting penetration of rods inside the cell and
production of ROS, eventually leading to cell death.
DISCUSSION
68
In addition to the enhancement of internalization of nanoparticles, it has been found
that the contribution of the polar facets of ZnO nanoparticles to the antibacterial
action, that the higher number of polar surfaces (the facets) possess higher
amount of oxygen vacancies. Oxygen vacancies are known to increase the generation
of ROS and subsequently affecting the photocatalytic activity of ZnO. 41
Zinc oxide nanosphere was effective against ATCC E.faecalis at 100 μl but was
effective against oral isolate only at 150 μl. The antibacterial effectiveness of
nanosphere is due to its interaction with bacterial cell wall, production of ROS-
reactive oxygen species which is lethal to cell because it disturbs the cellular
metabolism. This considerable variation in requirement of increased volume of NPs
for antimicrobial action is probably due to variation in synthesis parameters.
Padmavathy et al suggested that the antibacterial action of ZnO-NPs is due to the
cell membrane damage caused by defects such as edges and corners, which
results from the abrasive nature of the ZnO surface. Enhancement in antibacterial
action of ZnO-NPs can be established by controlling the defects, impurities, and the
associated charge carriers.52
Additionally smaller size of nanoparticles, i.e 20nm was used in this study may
have contributed to enhanced antibacterial action. Decreased size explained the
increased production of ROS (OH-, H2O2, and O2-) on ZnO surface and proposed a
correlation between photon reactions and the antibacterial activity as follows. The
electron and hole (on surface of NPs) interacts with water (H2O) to produce OH
(hydroxyl ions) and H+ (hydrogen ion). In addition, O2 molecules (suspended within
DISCUSSION
69
the mixture of bacteria and ZnO) yield superoxide anion (O2-), which reacts with H+
to produce HO2. Further HO2 interferes with electrons generating hydrogen peroxide
(•HO2); which combines with H+ giving hydrogen peroxide molecules. Hydrogen
peroxide is capable of entering the cell membrane to ultimately kill the bacteria.29,95
Additionally Zhang et al explained the morphology- dependent release of Zinc
ions, in which spherical nanoparticles release more of Zn2+ ions than rod
structures. Zinc ions interact with protein metabolism thus disrupting the
enzyme system.98
MAGNESIUM OXIDE NANOPARTICLE
Magnesium oxide nanorod was effective against ATCC at 100 μl but against oral
isolate only at 125 μl. The antibacterial property of MgO nanoparticles is mainly
because of production of superoxide anion which interacts with carbonyl carbon
atoms in the peptide linkages of proteins in microbes, which eventually leads to cell
death.
Krishnamoorthy et al explained that sequential oxidation– reduction reactions may
occur at MgO NPs surface to produce reactive oxygen species such as superoxide
radical (O2-), hydrogen peroxide (H2O2) and hydroxyl radical (OH•). The
mechanism of the antibacterial activity of the MgO NPs also depends on the
presence of defects or oxygen vacancy at the surface of the nanoparticle.
Oxygen vacancies result in more ROS production and lipid peroxidation.
Smaller the size of nanoparticles greater the surface area, which is required for
DISCUSSION
70
interaction with the bacteria. As the surface area increases, reactive group or species
formation is more which contributes to antibacterial action.38
Another proposed mechanism of MgO NPs is ALKALINE EFFECT. This is
because of adsorption of water on to the surface of MgO NPs forming a thin water
film on to the surface resulting in rise of local pH on surface of NPs. When these
NPs (with water film) interact with microorganism, the local alkaline pH results in
cell wall disruption ultimately resulting in bacterial collapse.66
Magnesium nanosphere showed activity against ATCC at 125 μl but against oral
isolate showed activity at 100 μl. The antibacterial property of MgO nanosphere is
because of the above mechanism as exihibited by MgO-nanorods. Probable
mechanism is production of reactive oxygen species and alkaline effect that
interferes with various cellular functions, ultimately resulting in bacterial death.
3% NaOCl was effective against both strains at 50 μl. Zinc oxide nanorods activity
was comparable with 3% NaOCl. Considerable antibacterial activity was present in
all nanoparticles. Difference in their antibacterial action may be attributed due to
variation in synthesis parameters (pH, calcination temperature, chemical used).
MIC/MBC-Broth microdilution was performed to determine the MIC/MBC of
nanoparticles against both strains
ATCC strain – Among the nanoparticles, MgO-S (1.17mg/ml) was effective at
lesser concentration followed by MgO-R and ZnO-R effective at (18.75 mg/ml),
ZnO-S effective at a concentration of (37.5 mg/ml)
DISCUSSION
71
ORAL ISOLATE strain – MgO-R = MgO-S effective at a concentration of
18.5mg/ml, followed by ZnO-R (37.5mg/ml), followed by ZnO-S (75mg/ml).
This effective action of MgO at lower concentration is because of presence of more
oxygen vacancies in the surface of the NPs as described earlier.
Time kill assay was performed to evaluate the time needed for the nanoparticles to
kill the organisms. All the nanoparticles were effective within 15 mins comparable
with 3% NaOCl against both strains except Magnesium oxide nanorod which showed
bacterial growth at 15 mins against ATCC strain but killed the bacteria within 30
mins. This rapid action of these nanoparticles within 15 mins may be because of
production of ROS and increased number of facets (Zno nanorods and sphere);
superoxide anion and alkaline effect (MgO-nano rods and sphere).
Synthesized nanoparticles were in an average size of 20 nm which could have
greatly aided and enhanced the antibacterial property. Smaller size promotes
better penetration of nanoparticles into the bacterial cell and greater interaction
with organelles, ultimately leading to cell death.
This preliminary study evaluated the effectiveness of two nanoparticles with two
different morphologies to attribute the difference in antibacterial action that is
considerably influenced by their shape and size.
Further, next leap in the study would be analysing cytocompatibility assay of Zinc
oxide and Magnesium oxide nanoparticles, Enhancement of surface
characteristics of nanoparticles by doping with suitable agents and can be
further explored as a carrier for bioactive agents (delivery systems).
DISCUSSION
72
Understanding the domain of Nanoscience may pave a novel way in the field
of Endodontics, providing a new era in the stratergy of disinfection of the root
canal system, achieving complete sterility of root canal providing success of
endodontic procedure.
These nanoparticles gained noteworthy attention because of their
unique properties which can be manipulated and incorporated into
endodontic materials like sealers; can be coated on gutta percha and post
systems which can eventually avoid the post treatment diseases.
SUMMARY
73
The purpose of this invitro study is to evaluate the antibacterial efficacy of Zinc
oxide and Magnesium oxide nanoparticles with two different morphologies -
nanorods and nanosphere against E. faecalis (ATCC 29212 AND ORAL ISOLATE).
The synthesized nanoparticles were characterized using UV-visible
spectrophotometer, FTIR (analysing absorbance peaks for Zno and MgO NPs). TEM
was used to evaluate the size and shape of synthesized nanopaticles. The ZnO and
MgO nanoparticles reaped were of average size 20 nm and nanorod and nanosphere
morphology was perceived for both. Antimicrobial activity was assessed by Agar
well diffusion assay (ZOI), Broth microdilution (MIC and MBC) and Time Kill
assay.
The nanoparticles with positive and negative control were grouped as follows,
GROUP 1- 10% DMSO (negative control)
GROUP 2- ZnO-R
GROUP 3- ZnO-S
GROUP 4- MgO-R
GROUP 5- MgO-S
GROUP 6- 3% NaOCl (positive control)
The agar well diffusion test investigated zone of inhibition of nanoparticles.
MIC and MBC were evaluated by Broth microdilution method. Antibacterial
activity at lower concentration was achieved with MgO-S and ZnO-R.
Antibacterial activity of nanoparticles was evaluated at five different volumes 50, 75,
100, 125, 150 microliters.
SUMMARY
74
Zinc oxide nanorod exhibited good activity at 75 μl against both strains of
E.faecalis. Result of ZnO-R was statistically significant with all other groups.
Zinc oxide nanosphere was effective against ATCC E.faecalis at 100 μl but was
effective against oral isolate only at 150 μl.
Magnesium oxide nano-rods was effective against ATCC at 100 μl but against oral
isolate only at 125 μl. Magnesium nanosphere showed activity against ATCC at
125 ul but against oral isolate showed activity at 100 ul. 3% NaOCl was effective
against both strains at 50 μl.
Zinc oxide nanorods activity was in par with 3% NaOCl. Considerable antibacterial
activity was present in all nanoparticles. Difference in their antibacterial activity
may be because of difference in synthesis methods and parameters. Statistically
significant results were obtained between groups and within groups.
Time kill assay was performed to evaluate the time needed for the nanoparticles to
kill the organisms. All the nanoparticles were effective within 15 mins against both
strains except Magnesium oxide nanorod showed bacterial growth at 15 mins against
ATCC but killed the bacteria within 30 mins.
NANOPARTICLES AGAINST E.FAECALIS ATCC STRAIN
At 75 ul, ZnO-R was effective At 100 ul, ZnO-R ˃ ZnO-S ˃MgO-S ˃MgO-R At 125 ul ZnO-R ˃ MgO-R ˃ MgO-S ˃ ZnO-S At 150 ul ZnO-R ˃ MgO-R ˃ MgO-S and ZnO-S
NANOPARTICLES AGAINST E.FAECALIS ORAL ISOLATE
At 75 ul, ZnO-R was effective At 100 ul, ZnO-R ˃MgO-S At 125 ul ZnO-R ˃ MgO-S ˃ MgO-R ˃ At 150 ul ZnO-R ˃ MgO-S ˃ MgO-R ˃ ZnO-S
CONCLUSION
75
Within the limitations of this study the following conclusions were drawn,
Zinc oxide and magnesium nanoparticles with two distinct morphologies
showed considerable activity against both ATCC AND ORAL ISOLATE
E.faecalis.
Zinc oxide nanorods exhibited good activity against both strains of
E.faecalis.
All the nanoparticles were considerably effective at different volumes
and concentration against E.faecalis.
So thorough understanding and incorporation of few key parameters
in synthesis will give birth to more potential nanoparticles that would
illuminate the discipline of Endodontics to achieve complete root canal
sterility.
BIBLIOGRAPHY
i
1. Afzal A, Gopal VR, Pillai R, Jacob AS, U-Nu S, Shan S. Antimicrobial
activity of various irrigants against E. faecalis biofilm: An in vitro study. J
Interdiscip Dentistry 2013;3:103-8.
2. Akbari B, Tavandashti M P, Zandrahimi M. Particle Size Characterisation
of Nanoparticles - A Practicle Approach. Iran J Mater Sci Eng. 2011; 8: 48-
56.
3. Alaei M, Jalali M, Rashidi A. Simple and Economical Method for the
Preparation of MgO Nanostructures with Suitable Surface Area. Iran. J.
Chem. Chem. Eng. 2014;33(1):21-28
4. Al-Gaashani, R, Radiman, S, Al-Douri Y, Tabet N. and Daud AR.
Investigation of the optical properties of Mg(OH)2 and MgO nanostructures
obtained by microwave-assisted methods. Journal of Alloys and Compounds.
2012; 52:71-76.
5. An Y, Zhang K, Wang F, Lin L, Guo H., Removal of organic matters and
bacteria by nano-MgO/GAC system. Desalination. 2011; 281(1): 30- 34.
6. Aničić N, Vukomanović M, Suvorov D . The nano-texturing of MgO
microrods for antibacterial applications. RSC Adv. 2016; 6(104): 102657- 64
7. Avanzato CT, Follieri JM and Banerjee IA. Biomimetic synthesis and
antibacterial characteristics of magnesium oxide-germanium dioxide
nanocomposite powders. J Composite Mater. 2009; 43: 897-910.
8. Baer D R. Surface characterization of nanoparticles: critical needs and
significant challenges. J Surf Anal. 2011; 17 (3), 163-169.
BIBLIOGRAPHY
ii
9. Balouiri M, Sadiki, M, Ibnsouda, SK. Methods for in vitro evaluating
antimicrobial activity: A review. J Pharm Anal. 2016; 6(2): 71–79.
10. Del Carpio-Perochena A, Kishen A, Felitti R, Bhagirath AY, Medapati
MR, Lai C, Cunha RS. Antibacterial Properties of Chitosan Nanoparticles
and Propolis Associated with Calcium Hydroxide against Single- and
Multispecies Biofilms: An In Vitro and In Situ Study. J Endod. 2017;
43(8):1332 – 1336.
11. Di DR, He ZZ, Sun ZQ, Liu J. A new nano-cryosurgical modality for tumor
treatment using biodegradable MgO nanoparticles. Nanomedicine. 2012;8
(8):1233-41.
12. Dianat O, Saedi S, Kazem M, Alam M. Antimicrobial Activity of
Nanoparticle Calcium Hydroxide against Enterococcus Faecalis: An In Vitro
Study. Iran Endod J. 2015;10 (1):39-43.
13. Dowlatababdi F, Amiri G, Mohammadi-Sichani M. Investigation of the
Antimicrobial Effect of Silver Doped Zinc Oxide Nanoparticles. Nanomed J.
2017; 4(1): 50-54.
14. Fan W, Li Y, Sun Q, Fan B. Calcium-silicate mesoporous nanoparticles
loaded with chlorhexidine for both anti- Enterococcus faecalis
and mineralization properties. J Nanobiotechnol. 2016; 14:72
15. Fan Z, Lu JG. Zinc oxide nanostructures: synthesis and properties. J.
Nanosci. Nanotechnol. 2005; 5(10):1561–73.
16. Ferraz CC, Gomes BP, Zaia AA, Teixeira FB, Souza Filho FJ. In vitro
assessment of the antimicrobial action and the mechanical ability of
chlorhexidine gel as an endodontic irrigant. J Endod 2001; 27(7):452-5.
BIBLIOGRAPHY
iii
17. Ferrer‑Luque CM, Arias‑Moliz MT, Gonzalez‑Rodriguez MP, Baca P.
Antimicrobial activity of maleic acid and combinations of cetrimide with
chelating agents against Enterococcus faecalis biofilm. J Endod. 2010;
36(10):1673‑ 5.
18. Geethapriya N, Subbiya A, Padmavathy K, Mahalakshmi K,
Vivekanandan P, Sukumaran VG. Effect of chitosan ethylenediamine tetra-
acetic acid on Enterococcus faecalis dentinal biofilm and smear layer
removal. J Conserv Dent. 2016; 19:472-7.
19. Gomes BP, Pinheiro ET, Gadê-Neto CR, Sousa EL, Ferraz CC, Zaia AA
et al. Microbiological examination of infected dental root canals. Oral
Microbiol Immunol. 2004;19(2): 71-6.
20. Gupta P, Khare V, Kumar D, Ahmad A, Banerjee G, Singh M.
Comparative evaluation of disc diffusion and E-test with broth micro-dilution
in susceptibility testing of amphotericin B, voriconazole and caspofungin
against clinical Aspergillus isolates. J Clin Diagn. Res. 2015; 9(1): DC04–
DC07
21. Gupta V, Gupta AR, Kant V. Synthesis, characterization and biomedical
Application of Nanoparticles. Science International. 2013; 1(5); 167-174.
22. Gutierrez FM, Olive PL, Banuelos A, Orrantia E, Nino N, Sanchez EM
et al. Synthesis, characterization, and evaluation of antimicrobial and
cytotoxic effect of silver and titanium nanoparticles. Nanomedicine.
2010;6(5):681-8.
23. Haapasalo M, Shen Y, Qian W, Gao Y. Irrigation in endodontics. Dent Clin
North Am. 2010;54:291-312.
BIBLIOGRAPHY
iv
24. He W, Kim HK, Wamer WG, Melka D, Callahan JH, Yin JJ.
Photogenerated charge carriers and reactive oxygen species in ZnO/Au
hybrid nanostructures with enhanced photocatalytic and antibacterial activity.
J Am Chem Soc. 2014;136(2):750–7.
25. Huang L, Li DQ, Lin Y J, Evans DG, Duan X. Influence of nano-MgO
particle size on bactericidal action against Bacillus subtilis var. niger. Chin.
Sci. Bull. 2005; 50: 514-519.
26. Hubble TS, Hatton JF, Nallapareddy SR, Murray BE, Gillespie MJ.
Influence of Enteroccocus faecalis proteases and the collagen-binding
protein, Ace, on adhesion to dentin. Oral Microbiol Immunol. 2003;
18(2):121– 6.
27. Ibrahem EJ, Thalij K, Badawy AS. Antibacterial Potential of Magnesium
Oxide Nanoparticles Synthesized by Aspergillus niger. Biotechnology
Journal International. 2017; 18(1): 1-7.
28. Iram S, Akbar Khan J, Aman N, Nadhman A, Zulfiqar Z, Arfat Yameen
M. Enhancing the Anti-Enterococci Activity of Different Antibiotics by
Combining With Metal Oxide Nanoparticles. Jundishapur J Microbiol. 2016;
9(3): e31302
29. Jalal R, Goharshadi E K, Abareshi M, Moosavi M, Yousefi A,
Nancarrow P. ZnO nanofluids: green synthesis, characterization, and
antibacterial activity. Mater.Chem. Phys. 2010; 121(1), 198–201.
30. Jett BD, Huycke MM, Gilmore MS. Virulence of Enterococci. Clin
Microbiol Rev 1994; 7(4):462–78.
BIBLIOGRAPHY
v
31. Jiang LM, Hoogenkamp MA, Van der Sluis LW, Wesselink PR,
Crielaard W, Deng DM. Resazurin metabolism assay for root canal
disinfectant evaluation on dual-species biofilms. J Endod. 2011; 37: 31-5.
32. Jin J, Zhang , Ma H, Lu X, Chen J, Zhang Q, Zhang, H. and Ni Y.
Surface modification of spherical magnesium oxide with ethylene glycol.
Materials Letters. 2009; 63:1514-1516.
33. Jin T, He YP. Antibacterial activities of magnesium oxide (MgO)
nanoparticles against foodborne pathogens. J. Nanopart. Rsc. 2011; 13:6877-
6885.
34. Jones N, Ray B, Ranjit KT, Manna AC. Antibacterial activity of ZnO
nanoparticles suspensions on a broad spectrum of microorganisms. FEMS
Microbiol. Lett. 2008; 279 (1): 71–76
35. Kasemets K, Ivask A, Dubourguier HC, Kahru A. Toxicity of
nanoparticles of ZnO, CuO and TiO2 to yeast Saccharomyces cerevisiae.
Toxicol. In Vitro. 2009; 23(6): 1116–22.
36. Kishen A, Shi Z, Shrestha A, Neoh KG. An investigation on the
antibacterial and antibiofilm efficacy of cationic nanoparticulates for root
canal disinfection. J Endod. 2008; 34:1515–20.
37. Kishen A. Advanced therapeutic options for endodontic biofilms. Endod
Topics 2010; 22:99-123.
38. Krishnamoorthy K, Manivannan G, Kim SJ, Jeyasubramanian K,
Premanathan M. Antibacterial activity of MgO nanoparticles based on lipid
peroxidation by oxygen vacancy. J. Nanopart Rsc. 2012; 14: 1063-6.
BIBLIOGRAPHY
vi
39. Krishnan R, Arumugam V, Vasaviah SK. The MIC and MBC of Silver
Nanoparticles against Enterococcus faecalis - A Facultative Anaerobe. J
Nanomed Nanotechnol. 2015; 6(3): 285-289.
40. Leung YH, Ng AM, Xu X, Shen Z, Gethings LA, Wong MT et al.
Mechanisms of antibacterial activity of MgO: non-ROS mediated toxicity of
MgO nanoparticles towards Escherichia coli. Small. 2014; 10(6):1171–83.
41. Li G, Hu T, Pan G, Yan T, Gao X, Zhu H. Morphology - function
relationship of ZnO: polar planes, oxygen vacancies, and activity. J. Phys.
Chem C. 2008;112 (31):11859–11864.
42. Love RM. Enterococcus faecalis: a mechanism for its role in endodontic
failure. Int Endod J. 2001;34(5):399–405.
43. Makhluf S, Dror R, Nitzan Y, Abramovich Y, Jelinek R. and Gedanken
A. Microwave-assisted synthesis of nanocrystalline MgO and its use as a
bacteriocide. Adv Funct Mater. 2005; 15: 1708-1715.
44. McHugh CP, Zhang P, Michalek S, Eleazer PD. pH required to kill
Enterococcus faecalis in vitro. J Endod. 2004; 30(4):218 –9.
45. Mirhosseini M, Afzali M. Investigation into the antibacterial behavior of
suspensions of magnesium oxide nanoparticles in combination with nisin and
heat against Escherichia coli and Staphylococcus aureus in milk. Food
Control. 2016; 68: 208 – 215.
46. Moezzi A, McDonagh AM, Cortie MB. Zinc oxide particles: synthesis,
properties and applications. Chem. Eng. J. 2012; 185:1–22.
47. Mohammadi Z, Abbott PV. The properties and applications of
chlorhexidine in endodontic: review. Int Endod J. 2009; 42:288-302.
BIBLIOGRAPHY
vii
48. Monzavi A, Eshraghi S, Hashemian R, Momen-Heravi F. Invitro and ex
vivo antimicrobial efficacy of nano-MgO in the elimination of endodontic
pathogens. Clin Oral Investig. 2015; 19:349-56.
49. Nair PN, Henry S, Cano V, Vera J. Microbial status of apical root canal
system of human mandibular first molars with primary apical periodontitis
after “onevisit” endodontic treatment. Oral Surg Oral Med Oral Pathol Oral
Radiol Endod. 2005: 99(2): 231–252.
50. Narayanan P, Wilson W S, Abraham A T, Sevanan M. Synthesis,
characterization, and antimicrobial activity of zinc oxide nanoparticles
against human pathogens. Bio NanoScience. 2012; 2(4), 329–335.
51. Ozgur U, Hofstetter D, Morkoc H. ZnO Devices and Applications: A
Review of Current Status and Future Prospects. Proceeds of the IEEE. 2010;
98(7):1255-68.
52. Padmavathy N, Vijayaraghavan R. Enhanced bioactivity of ZnO
nanoparticles an antimicrobial study. Sci Technol Adv Mater. 2008; 9(3):
035004.
53. Parak W J, Gerion D, Pellegrino T, Zanchet D, Micheel C, Williams CS
et al. Biological applications of colloidal nanocrystals. Nanotechnology.
2003; 14(7):15-27.
54. Pelgrift RY, Friedman AJ. Nanotechnology as a therapeutic tool to combat
microbial resistance. Adv Drug Deliv Rev. 2013; 65:1803-15.
55. Peters OA, Laib A, Rüegsegger P, Barbakow F. Three dimensional
analysis of root canal geometry by highresolution computed tomography. J
Dent Res. 2000: 79(6): 1405–9.
BIBLIOGRAPHY
viii
56. Pfaller MA, Sheehan DJ, Rex JH. Determination of fungicidal activities
against yeasts and molds: lessons learned from bactericidal testing and the
need for standardization. Clin Microbiol Rev. 2004; 17:268–280.
57. Pruzzo C, Tarsi R, Lleo MM, Signoretto C, Zampini M, Colwell R R et
al.. In vitro adhesion to human cells by viable but nonculturable
Enterococcus faecalis. Curr Microbiol. 2002; 45(2): 105–10.
58. Raghupathi KR, Koodali RT, Manna AC. Size-dependent bacterial
growth inhibition and mechanism of antibacterial activity of zinc oxide
nanoparticles. Langmuir. 2011; 27(7), 4020–8.
59. Rago I, Chandraiahgari CR, Bracciale MP, De Bellis G, Zanni E, Guidi
MC et al. Zinc oxide microrods and nanorods: diferent antibacterial activity
and their mode of action against Gram-positive bacteria. Rsc Adv. 2014;
4:56031–40.
60. Ramani M, Ponnusamy S, Muthamizhchelvan C, Marsili E. Amino acid-
mediated synthesis of zinc oxide nanostructures and evaluation of their facet-
dependent antimicrobial activity. Colloids Surf. B Biointerfaces. 2014; 117:
233–9.
61. Rao Y, Wang W, Tan F, Cai Y, Lu J, Qiao X. Influence of different ions
doping on the antibacterial properties of MgO nanopowders. App Surf Sci.
2013; 284:726–31.
62. Rôças IN, Siqueira JF, Santos KR. Association of Enterococcus faecalis
with different forms of periradicular diseases. J Endod. 2004; 30(5):315–20.
BIBLIOGRAPHY
ix
63. Rucucci D, Siqueira Jr JF. Biofilms and apical periodontitis: study of
prevalence and association with clinical and histopathologic findings. J
Endod. 2010; 36:1277-88.
64. Sanchez F, Sobolev K. Nanotechnology in concrete–a review. Constr Build
Mater. 2010; 24: 2060-71.
65. Sarkar S, Sarkar R. Sol-gel Synthesis and Meticulous Characterization of
Zinc Oxide Nanoparticles. J Nanosci Curr Res. 2017; 2(2): 109-112
66. Sawai J, Shuga S, Kojima H. Kinetic analysis of death of bacteria in CaO
powder slurry. International Biodeterioration & Biodegradation. 2001; 47:23-
26.
67. Sawai J. Quantitative evaluation of antibacterial activities of metallic oxide
powders (ZnO, MgO and CaO) by conductimetric assay. J Microbiol
Methods. 2003:54(2): 177–182.
68. Sedgley CM. The influence of root canal sealer on extended intracanal
survival of E feacalis in root canals. J Endod.2006; 32(3):173-177.
69. Seil JT, Webster TJ. Antimicrobial applications of nanotechnology:
methods and literature. Int J Nanomedicine. 2012;7:2767-81.
70. Sharma G, Soni R, Jasuja ND. Phytoassisted synthesis of magnesium oxide
nanoparticles with Swertia chirayaita. Journal of Taibah University for
Science. 2017; 11(3): 471–477.
71. Shrestha A, Kishen A. Antibacterial Nanoparticles in Endodontics:
A Review. J Endod. 2016; 42(10):1417-26.
BIBLIOGRAPHY
x
72. Shrestha A, Shi Z, Neoh KG, Kishen A. Nanoparticulates for Antibiofilm
Treatment and Effect of Aging on Its Antibacterial Activity. J Endod. 2010;
36(6):1030-5.
73. Siqueira JF Jr, Rôças IN, Santos SR, Lima KC, Magalhães FA, de Uzeda
M. Efficacy of instrumentation techniques and irrigation regimens in
reducing the bacterial population within root canals. J
Endod. 2002;28(3):181–4
74. Spratt DA, Pratten J, Wilson M, Gulabivala K. An in vitro evaluation of
the antimicrobial efficacy of irrigants on biofilms of root canal isolates. Int
Endod J. 2001; 34(4):300‑ 7.
75. Stanković A, Dimitrijević S, Uskoković D. Influence of size scale and
morphology on antibacterial properties of ZnO powders hydrothermally
synthesized using different surface stabilizing agents. Colloids Surf B. 2013;
102: 21–28.
76. Stoimenov PK, Klinger RL, Marchin GL, Klabunde, KJ. Metal oxide
nanoparticles as bactericidal agents. Langmuir. 2002; 18(17): 6679-6686.
77. Sundqvist G, Figdor D, Persson S, Sjögren U. Microbiologic analysis of
teeth with failed endodontic treatment and the outcome of conservative
retreatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1998;
85(1):86-93.
78. Sundqvist G, Figdor D. Life as an endodontic pathogen. Ecological
differences between the untreated and root-filled root canals. Endod Topics.
2003; 6:3-28.
BIBLIOGRAPHY
xi
79. Sundrarajan M, Suresh J. and Gandhi RR. A comparative study on
antibacterial properties of MgO nanoparticles prepared under different
calcinations temperature. Digest Journal of Nanomaterials and Biostructures.
2012; 7: 983-989.
80. Sutradhar N, Sinhamahapatra A, Pahari SK, Pal P, Bajaj HC,
Mukhopadhyay I et al. Controlled Synthesis of Different Morphologies of
MgO and Their Use as Solid Base Catalysts. J Phys. Chem. C. 2011; 115(25):
12308-12316
81. Talebian N, Amininezhad SM, Doudi M. Controllable synthesis of ZnO
nanoparticles and their morphology-dependent antibacterial and optical
properties. J Photoch Photobio B. 2013; 120: 66–73.
82. Tiwari JN, Tiwari RN, Kim KS. Zero-dimensional, one-dimensional, two-
dimensional and three-dimensional nanostructured materials for advanced
electrochemical energy devices. Prog Mater Sci. 2012; 57: 724-803.
83. Valgas C, De Souza SM, Smânia EFA, Smânia A. Screening methods to
determine antibacterial activity of natural products. Braz. J Microbiol. 2007;
38(2):369–380.
84. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free
radicals and antioxidants in normal physiological functions and human
disease. Int J Biochem Cell Biol. 2007; 39(1):44-84.
85. Vidic J, Stankic S, Haque F, Ciric D, Le Goffic R, Vidy A et al. Selective
antibacterial effects of mixed ZnMgO nanoparticles. J Nanopart Res.
2013;15(5):1595-4
BIBLIOGRAPHY
xii
86. Wahab R, Absari S G, Dar M A, Kim Y S, Shin H S. Synthesis of
magnesium oxide nanoparticles by sol-gel process. Mater sci. Forum Vols.
2007; 558: 983-986.
87. Wahab R, Mishra A, Yun SI, Kim YS, Shin HS. Antibacterial activity of
ZnO nanoparticles prepared via non-hydrolytic solution route. Appl.
Microbiol. Biotechnol. 2010; 87(5): 1917–25.
88. Wei H, Wu Y, Lun N, Hu C. Hydrothermal synthesis and characterization
of ZnO nanorods. Materials Science and Engineering A. 2005; 398:80–82
89. Whitesides GM. The 'right' size in Nano-biotechnology. Nat Biotechnol.
2003; 21(10):1161-5.
90. Wu J.M, Kao WT. Heterojunction nanowires of AgxZn1-xO–ZnO
photocatalytic and antibacterial activities under visible-light and dark
conditions. J. Phys. Chem. C. 2015; 119(3):1433–1441.
91. Yahya N, Daud H, Tajuddin NA, Daud HM, Shafie A, Puspitasari P.
Application of ZnO nanoparticles EM wave detector prepared by sol–gel and
self-combustion techniques. J Nano Res. 2010; 11:25–34.
92. Yamamoto O, Ohira T, Alvarez K. and Fukuda M. Antibacterial
characteristics of CaCO3-MgO composites. Mater Sci Eng B. 2010; 173,
208-212.
93. Yang H, Liu C, Yang D, Zhang H, Xi Z. Comparative study of cytotoxicity,
oxidative stress and genotoxicity induced by four typical nanomaterials: the
role of particle size, shape and composition. J Appl Toxicol 2009;29(1):
69–78.
BIBLIOGRAPHY
xiii
94. Zanni E, Chandraiahgari CR, De Bellis G, Montereali MR, Armiento G,
Ballirano P et al. Zinc Oxide Nanorods-Decorated Graphene Nanoplatelets:
A Promising Antimicrobial Agent against the Cariogenic
Bacterium Streptococcus mutans. Nanomaterials. 2016; 6(10):179.
95. Zhang H, Chen B, Jiang H, Wang C, Wang H, Wang X. A strategy for
ZnO nanorod mediated multi-mode cancer treatment. Biomaterials.
2011;32(7): 1906-14
96. Zhang K, An Y, Wang F, Lin L, Guo H. Experimental investigation on
water treatment by the combined nano MgO-nanofiltration technique. Water
Sci Technol. 2011; 63(11): 2542-2546.
97. Zhang L, Ding Y, Povey M, York D. ZnO nanofluids-a potential
antibacterial agent. Prog. Nat. Sci. 2008; 18(8):939–944.
98. Zhang L, Jiang Y, Ding Y, Povey M, York D. Investigation into the
antibacterial behaviour of suspensions of ZnO nanoparticles (ZnO
nanofluids). J. Nanopart. Res. 2007; 9(3): 479–489.