MASTER OF DENTAL SURGERYrepository-tnmgrmu.ac.in/9948/1/240402018srilekha.pdf · I hereby declare...

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


ZINC OXIDE NANOSPHERE

22

3


MAGNESIUM OXIDE NANORODS

23

4


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


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


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


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


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


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+


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


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


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


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


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


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 nitrate

Polyethylene glycol (Sigma Aldrich)

Liquid ammonia

Ethanol

Deionized water


Magnesium nitrate (Sigma Aldrich)

Sodium hydroxide

Methanol

CHARACTERISATION - UV-VISIBLE SPECTROPHOTOMETER (BioTek),

FTIR- FOURIER TRANSFORM INFRARED SPECTROSCOPY (JASCO),

TRANSMISSION ELECTRON MICROSCOPE (TEM) (TECNAI)


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)


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


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


21


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


22

UV-VISIBLE SPECTROPHOTOMETER ANALYSIS

ZINC OXIDE NANORODS (Graph 1)

ZINC OXIDE NANOSPHERE (Graph 2)

300 - 400

360

300 - 400

350


23

MAGNESIUM OXIDE NANORODS (Graph 3)

MAGNESIUM OXIDE NANOSPHERES (Graph 4)

200 - 300

250

270

200 - 300


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)


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


Fig 18a- NANOROD CLUSTERS

Fig 18b- SINGLE NANOROD

20 nm

MAGNESIUM OXIDE NANOSPHERES

Fig 19a- SINGLE NANOSPHERE

FIG 19b- NANOSPHERE CLUSTERS

20 nm


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


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)


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


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).


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


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


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


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


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


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


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



Total 164.000 17



Total 348.000 17



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 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


Total 55.111 17

75μl Between Groups 91.778 5 18.356 330.400 .000


Total 92.444 17



Total 228.000 17



Total 375.778 17



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 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 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



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


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


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


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 (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


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 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.

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