PANDIT DEENDAYAL UPADHYAY MEMORIAL HEALTH

SCIENCES AND AYUSH UNIVERSITY OF CHHATTISGARH,

RAIPUR (C.G.)
Maitri College of Dentistry And Research Centre

Anjora, Durg

Department Of Oral Pathology And Microbiology

To,
The Dean,
Maitri College of Dentistry And Research Centre
Anjora, Durg. (Chhattisgarh)
Through: Head of the Department.

Subject: Dissertation Synopsis Submission.

Respected Sir,

I, Dr.Rajeev Kumar, student of M.D.S. 1st year in the Department of Oral Pathology
And Microbiology.Request you to grant me the permission for synopsis of dissertation on
topic "A Comparative study of the efficacy of Cedar wood oil, coconut oil and
dish wash liquid as alternatives to Xylene as Deparaffinizing agents.

Kindly grant me the permission to proceed with the above mentioned subject and
complete my synopsis .

Thanking you in anticipation

Yours Sincerely:
Dr. Rajeev Kumar
Post-Graduate Student
Department Of Oral Pathology and Microbiology

Guided By:
Dr.Sonam Aggarwal
Reader & Guide
 PANDIT DEENDAYAL UPADHYAY MEMORIAL HEALTH
SCIENCES AND AYUSH UNIVERSITY OF CHHATTISGARH,
RAIPUR (C.G.)

1 NAME OF DR.Rajeev Kumar

CANDIDATE

2 PERMANENT Dr. Rajeev kumar, Ujjwal Enclave, Flat no:207 Pugmil Road

ADDRESS Hazaribag. Jharkhand-825301

3 NAMEOF MAITRI COLLEGE OF DENTISTRY AND RESEARCH

INSTITUTION CENTRE, ANJORA, DURG, CHHATTISGARH

4 COURSE OF THE MASTER OF DENTAL SURGERY (M.D.S.), ORAL

STUDY & SUBJECT PATHOLOGY AND MICROBIOLOGY, MAITRI

COLLEGE OF DENTISTRY AND RESEARCH CENTRE,
ANJORA, DURG, CHHATTISGARH.

5 DATE OF 20 February 2022

ADMISSION TO
COURSE
A Comparative study of the efficacy of Cedar wood oil,
6 TITLE OF THE coconut oil and dish wash liquid as alternatives to Xylene as
TOPIC Deparaffinizing agents.

r
 INTRODUCTION

Histopathology is an art of analyzing and interpreting the shapes, sizes and

architectural patterns of cells and tissues within a given specific clinicalbackground

and a science by which the image is placed in the context ofknowledge of

pathobiology, to arrive at an accurate diagnosis.1

The backbone of daily histopathological diagnostic work is the paraffin

section usually stained with Hematoxylin and Eosin. Paraffin sections are still

prepared by methods largely unchanged for over 150 years. The process of

deparaffinization of the slides using Xylene is an important preliminary step before

the staining process, which makes the tissue sections to uptake the Haematoxylin and

Eosin stain properly. This makes Xylene an inevitable compound in histopathology

due to its paraffin solvent action. However it is a toxic compound that is hazardous for

human use and the environment in which it is disposed. Therefore, any substitute that

minimizes the use of Xylene in experiments, reduces tissue staining time and does not

compromise its quality will be efficient for diagnostic reasons and valuable for

maintaining a safe laboratory environment.2

Xylene is an aromatic hydrocarbon widely used in industry and

medical technology as a solvent. It is a colorless, sweet smelling liquid or gas

occurring naturally in petroleum, coal and wood tar, and is so named because it

is found in crude wood spirit (Greekxylon- wood). It has a chemical formula of C6

H4 (CH 3)2 and is referred to as “dimethyl benzene” because it consists of a

six-carbon ring to which two methyl groups are bound. It exists in three

isomeric forms: ortho-, meta- and para- xylene.

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Xylene is used as a solvent in the printing, rubber, paint and leather industries. It is

found in small amounts in airplane fuel, gasoline and cigarette smoke.3

Laboratory-grade xylene iscomposed of m-xylene (40–65%), p-xylene (20%),

o-xylene

(20%), and ethyl benzene (6–20%) and traces of toluene,thiophene, trimethyl

benzene, phenol, hydrogen sulfide, andpyridine. 4

Pathology laboratory being the hub of various activities which involves

translational aspects of histopathology and cytopathology into confirmatory diagnosis

exposes the technicians, research workers and histopathologists to an array of

hazardous chemicals that pose great threat to health. One such commonly encountered

chemical is xylene. It is an aromatic hydrocarbon widely used in tissue processing as

a clearing agent and as a deparaffinizing agent in staining and mounting. Maximum

handling and exposure occurs during deparaffinizing of tissue sections. OSHA

(Occupational Safety and Health Administration) regulation identifies xylene as a

hazardous substance and the quest for nontoxic substitutes thus began.5

The National Institute of Occupational Safety and Health recommended

exposure limits for xylene at 100 ppm as a time‑weighted average for up to a 10 hour

work shift and a 40 hour work week and 200 ppm for 10 min as a short term limit.

After the hazardous effects of xylene became indisputable in the 1970s, many

potential substitutes became available in order to make a xylene‑free environment in

laboratories, such as limonene reagents, aliphatic hydrocarbons, aromatic

hydrocarbons, olive oil, vegetable oils, and mineral oil substitute. However, these

chemicals were used to substitute xylene as a clearing agent during routine

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processing, while the exposure and handling of xylene is maximum during

deparaffinization of the tissue sections.6

Exposure to xylene can affect various organs such as the eyes, nose, throat,

lungs, and nervous system, which can lead to difficulty in breathing, impaired lung

function, and impairment of the central nervous system. Long-term exposure to

xylene can cause anemia, thrombocytopenia, leukopenia, chest pain with

electrocardiogram (ECG) abnormalities, dyspnea, and cyanosis. 7

Despite knowing the demerits of xylene, the preparatory steps in routine

hematoxylin and eosin (H&E) staining procedure mandates the use of xylene. Thus,

xylene being used since ages in laboratories till today reflects our failed attempt in

finding a safer substitute. Green chemistry is a challenging arena which explores safer

and environmentally friendly alternatives to conventionally used toxic chemicals. 5

Substitution is the method of finding a substance that can perform almost

thesame function and which may lessen the hazard. The new substitute should not be

hazardous in nature. Many potential substitutes became available after the hazardous

effects of xylene became indisputable in the 1970s. In general, these substitutes can

be categorized into four classes, but they are marketed under various tradenames.

These areLimonene reagents, Aliphatic hydrocarbon mixtures, Aromatic hydrocarbon

mixtures, Mineral oil mixtures. However Limonene reagents have various

disadvantages also. It is expensive, Claims to be less toxic butthe hazards are not well

documented, Offensive odor, Very oily, Incompatible with some of the mounting

media, Cannot be distilled, Samples take more time to dry thoroughly and it removes

oil from skin.

There are few disadvantages for aliphatichydrocarbon mixtures also. Aliphatic

hydrocarbon mixtures are classified as hazardous waste due to flammability and it is

3|P a g e
also not easily biodegradable and some of them are more expensive than xylene and

less tolerant of contamination than xylene. Aromatic hydrocarbon mixtures are not so

popular because they are just as toxic asxylene.

Mineral oil or paraffin oil mixtures found to be promising in

eliminatingxylene from most of the procedures. Disposal of mineral oiland its

mixtures can be easily accomplished by mixing with theused paraffin and incinerating

the resulting solid.3 However mineral oil mixtures were mainly used as alternatives

for xylene in tissue processing as clearing agents, maximum exposure of xylene

occurs during deparaffinization of sections. Hence a natural alternative for xylene is

required as an effective deparaffinizing agent. 4

So, the present study was conducted to find best biosafe alternative for

Xylene.

In present study we have to use Xylene, Dish wash liquid, Cedar wood oil

and Coconut oil for deparaffinization to find the efficient and best deparaffinizing

agent and the best alternative for xylene.

Falkeholm et al have experimented for the first time on liquid dish

washing soap (DWS) to dewax the tissue sections by eliminating both xylene and

alcohol from H and E staining procedures. The liquid DWS is a mixture of surfactants

composed of sodium laurethsulfate, cocamidopropylbetaine, sodium dodecyl

benzenesulfonate and nonionic surfactants. These anionic surfactants, lowers the

surface tension of water. Detergent dish soap made with ingredients of the highest

quality, high performance and strong power has neutral pH 7 that does not

mistreat hands, leaving with a pleasant aroma.6Another material which will be used

in the present study is Cedar wood oil. Cedarwood oil

is the most well known natural wood oil for clearing tissues. It is obtained

4|P a g e
from juniper and cypress species, both of which are actually cedar trees. For

histological processing a low viscous cedar wood oil variety is needed. The

viscosity of the oil affects the clearing time. Though the oil takes longer time to

process, it does not causesdamage to the tissues.

Coconut oil is another material which we can use for deparaffinization in

the present study.Coconut oil is extracted from the kernel or meat of the

mature coconuts obtained from the coconut palm (Cocosnucifera). Because of its

high saturated fat content, it slowly oxidizes, hence it is resistant to

rancidification. Coconut oil is available all over the tropical world and it is a

commonly used vegetable oil. It is also non-toxic and heat stable.8

Hence this present study will done on natural biosafe materials for

deparaffinization to prove its efficiency in it and to find the best biosafe alternative for

hazardous Xylene in deparaffinization.

5|P a g e
 NEED FOR THE STUDY

Xylene is an aromatic hydrocarbon which is widely used as clearing and

deparaffinizing agent, and it is extremely biohazardous. Exposure to xylene in a

laboratory occurs during tissue processing, deparaffinization of tissue sections, cover

slipping, cleaning tissue processors and recycling. The main effects of inhaling xylene

vapor are depression of the central nervous system and other organs are

also affected. All these effects can cause by depletion of mitochondrial

adenosine triphosphate (ATP) in the affected cells.10

The need of our study is to compare the efficacy of Cedar wood oil,

Coconut oil and Dish wash liquid (DWS) as possible safer replacements for Xylene as

deparaffinizing agents in H and E staining procedures.

6|P a g e
 OBJECTIVES OF THE STUDY

1. To compare the efficacy of Cedar wood oil, Coconut oil and Dish wash liquid

with Xylene as a deparaffinizing agent during tissue processing.

2. To find out the best biosafe alternative to Xylene-Cedar oil, Coconut oil and

Dish wash liquid.

7|P a g e
 Xylene is an aromatic hydrocarbon which is widely
used as a deparaffinizing agent, and it is extremely
biohazardous.
The aim of this study will be to compare the efficacy
of Cedar wood oil, Coconut oil and Dish wash liquid
(DWL) with Xylene as a deparaffinizing agent and to
AIM OF figure out the best biosafe alternative to Xylene. The
EXPEERIMENT: study will consist of 50 samples and results will be
analyzed based on the cellular architecture and total
quality of staining. Xylene and DWS shall show same
result of high-quality staining in case of cellular
architecture. But in case of total quality of staining,
only Xylene will show the best results. Hence, we
may conclude that though Xylene is toxic after
experiment, it could be the best deparaffinizing agent
and more studies will be done in this field to prove
the efficiency of natural agents.

The present study will consist of 50 Formalin fixed

paraffin embedded tissue blocks of diagnosed
random cases, which will be collected from the
departmental archives.
MATERIALS From each paraffin blocks, 4 sections of 3-4 µm will
AND METHODS: be sliced using semiautomatic Microtome
(MICROM model HM340E). Proposed sample size
will be 200, total 200 sections.
Each section of same blocks will be deparaffinized
with Dish wash liquid, Coconut oil, Cedar wood oil
and Xylene respectively at different time in a single
day. Deparaffinizing agents shall change in a week
basis to improve deparaffinization.
 Table 1: - Deparaffinization & H and E staining procedure for
Xylene, Cedar wood oil, DWL & Coconut oil:
Procedure Material used Temperature Time
Deparaffinization Xylene I Xylene II At room temperature At 5 min
room temperature
5 min
Cedar wood oil At room temperature 70°C 4 hrs
Water wash with distilled 1 min
water 5 min

1.7% DWL 90°C 2 min

Water wash with distilled water 2 min
Coconut oil 90°C 2 min
Water wash with distilled water
2 min
Rehydration Descending grades At room temperature At room 10 min
of alcohol Water temperature 10 min
wash
Nuclear staining Harri's Hematoxylin At room temperature At room 8 min
Water wash temperature 2 min
Differentiation 1% acid alcohol At room temperature At room 1 dip
Water wash temperature 10 min
Cytoplasmic staining 1% eosin At room temperature 1 min
Dehydration 100% alcohol At room temperature 5 min
 SUMMARY

This study will aim to introduce best biosafe alternative for xylene as

deparaffinizing agent in histopathology. The methodology will be used to find an

appropriate deparaffinizing agent which can stain effectively and give

satisfactory staining for cellular architecture and good quality staining in routine

histopathology laboratories.

50 random paraffin blocks will be collected from the Department of

Oral pathology and Microbiology, A J Institute of Dental sciences. Each block will be

cut into 4 sections and respective slides were made. Each section of same

blocks were deparaffinized with Dish wash liquid, Coconut oil, Cedar wood

oil and Xylene respectively at different time in a single day. Other than Xylene,

remaining agents can deparaffinized separately using incubator and can be

stained by using the same procedure. Xylene deparaffinization can be done using

same protocol given in previous studies. This will study will be done in daily basis.

Deparaffinizing agents will be changed in a weekly basis to accelerate

deparaffinization.

The deparaffinized sections will be seen under microscope. The scores

will be obtained, tabulated and statistically analysed. Cellular architecture will be

efficiently stained in both Dish wash liquid and Xylene deparaffinized sections and

statistically very high significant difference will be obtained between other two

agents. Quality of staining will be good for xylene followed by Cedar wood and

Dish wash liquid will show satisfactory result and Coconut oil will show

comparatively poor result.

58 | P a g e
Considering above data, here we can suggest keeping the toxic effects of Xylene

aside, it would be the best deparaffinizing agent to assess both cellular architecture

and staining quality.

Further studies are required to rule out the efficiency of natural deparaffinizing

agents, especially DWS, since it gave excellent result for cellular architecture, so that

Xylene can be avoided completely during routine histopathological deparaffinization.

59 | P a g e
 Maxillary canine paramaeters

MMDWC VALUE
MICAW VALUE
MCI VALUE

MMDWC: mean mesiodistal width of canine, MICAW:

maxillary intercanine arch width, MCI: maxillary canine
index

7.

INFORMED CONSENT

I, ______________________________ the
undersigned hereby give my consent for the oral –
examination and the procedures to be performed on me
. I have been informed about the procedures very
clearly.

Date:

Signature of the Patient

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 determination in north Indians using mandibular canine
index. JIAFM. 2004;26:45–49.
5. Boaz K, Gupta C. Dimorphism in human maxillary

and mandibular canine in establishment of gender. J Forensic

Dent Sci. 2009;1:42–4.

6. Khangura RK, Sircar K, Singh S, Rastogi V.Sex

determination using mesiodistal dimension of permanent

maxillary incisors and canines. J Forensic Dent Sci

2011;3:81-5.

7. Kiran CS, Pachigolla R, Swathi E, Smitha B,

Shankaran S. Discriminant canine index - a novel approach

in sex determination. Ann Stomatol 2015;6(2):43-46.

8. Shetty SJ, Ratnaparkhi I, Pereira T, Acharya S,

Gotmare S, Kamath P. Odontometric Analysis of Canines to

Establish Sexual Dimorphism in an Urban Population .

Indian J Dent Res. 2019 Nov-Dec;30(6):855-859.

8.1 NAME OF DR. Rajeev Kumar

CANDIDATE

8.2 SIGNATURE OF
CANDIDATE

9. SIGNATURE AND
REMARKS OF THE
GUIDE
 DR.SONAM AGRAWAL
AGARWAL,READER, DEPARTMENT
9.1 NAME AND
OF ORAL PATHOLOGY AND MICROBIOLOGY,
DESIGNATION OF MAITRI COLLEGE OF DENTISTRY AND
THE GUIDE RESEARCH CENTRE, ANJORA, DURG(C.G.)

10. HEAD OF THE Dr. ANSHUTA SAHU, PROFESSOR AND HEAD,

DEPARTMENT DEPARTMENT OF ORAL PATHOLGY AND

MICROBIOLOGY

MAITRI COLLEGE OF DENTISTRY AND

RESEARCH CENTRE,ANJORA, DURG,(C.G.)

10.1 SIGNATURE OF
HOD

11. REMARKS OF THE

DEAN

11.1 NAME OF THE DR. ANIL GOVINDRAO GHOM, M.D.S.

DEAN

11.2 SIGNATURE OF
THE DEAN