Comparison of Formalin and Honey with 3-5% Acetic Acid as Histological Fixative

MEMBERS

Roberto Paolo Atienza

Angelo Tadeo Bautista
Amber Justine Chua
Nerissa Jayne Dalay
John Joseph Ericsson Ermitao
Diana Eliza Reyes
Alianna Gaibrielle Tan
Jamille Simon

3 MT-3

2nd Semester

Research I

Submitted to:
Dr. Polly Chua-Chan, MD, MPH, MHA, FPAFP
 ABSTRACT

The purpose of this research is to compare the fixative properties of Honey with 3-5% Acetic acid
and the gold standard, Formalin. Formalin has been a known carcinogenic and is detrimental to the health
on long term use. The first and foremost step in Histotechnique is Fixation. Using 3 controls, with the 1st
using only honey, the 2nd using honey with 3-5% acetic acid, and 3rd being formalin, we, first, compared
the meat used as to gross appearances (color, consistency, size). Following the fixation step would be
dehydration, clearing, impregnation, embedding, trimming, staining, then finally mounting. After
mounting, we will then observe the differently fixed tissues on the microscope to compare the nuclear
and cytoplasmic structures as to their clarity, delineation, and staining. This research, therefore, looks for
a comparable fixative to formalin, one that is safe, accessible, and one that is health friendly for the future
generations to use.
 Comparison of Formalin and Honey with 3-5% Acetic Acid as Histological Fixative

INTRODUCTION

Fixation is the first and most crucial step in tissue processing. The primary aim of this step is to
preserve the morphological and chemical integrity of the cell in the most life-like manner as possible. This
step also aims to harden the tissues for easier cutting and protects the tissue from possible trauma due
to handling. Not only should a fixative be cheap and stable, the following characteristics should also be
taken into account: amount of distortion it can cause to the tissue, inhibition of autolysis, rapidity and
evenness in its penetration of the tissue, tonicity, and its safety property when it comes to handling.
However, as with all other processes there is no single ideal fixative that will adequately fix all types of
structures. Which is why formalin has been the most common fixative used in the histopathology section
since time immemorial due to its ability to satisfactorily fix a variety of structures.

Formalin, otherwise known as formaldehyde, is compatible with many stains and penetrates the
tissues well thus chosen as a standard by many laboratories and hospitals. It is usually prepared as a 10%
concentration of formalin and has a fixation time of 24 hours. Its extreme adaptability and convenience
in handling makes it one of the most ideal fixatives. Despite being the most widely used it also has its
disadvantages, one of which is the main concern of this paper: its carcinogenicity. Chronic exposure to
formalin is believed to cause hematopoietic and possible lymphatic cancers.

Honey, on the other hand, has been proven to adequately fix histopathologic tissues due to its
acidic property as well as its dehydrating ability. When used alone however, it can pose other problems
or concerns such as the longevity of the preserved tissue regarding its storage and its ability to attract
ants. Acetic acid, however, counteracts this problem with its ability to repel ants due to its pungent odor.

STATEMENT OF THE PROBLEM

Formalin is carcinogenic, thereby urging various researchers to explore the possibility of an

alternative that will not have this problem. Studies showed that honey has the capability of preserving
tissues and has no carcinogenic effects. But previous researches faced a major problem, the tissue
preserved by the honey attract ants which can affect the morphology and can generally destroy the
preserved tissue. Acetic acid, on the other hand, has been known as an ant repellant and can also assist
in tissue preservation.

It is therefore hypothesized that a mixture of Honey and Acetic Acid may be used for tissue
preservation augmenting the problem of honey.

RESEARCH QUESTION

Can Honey with 3-5% acetic acid preserve and fix tissue morphology as well as formalin?
 HYPOTHESIS

Null hypothesis

Honey with 3-5% acetic acid is not comparable with formalin

Alternative hypothesis

Honey with 3-5% acetic acid is comparable with formalin

REVIEW OF LITERATURE

Gross Morphology

A key element in assessing tissue pathology is through gross examination. The influence of fixation
on gross morphology is key in measurements such as tumor staging and differentiation. It is also important
in sectioning and embedding as the proper orientation is essential in diagnosing a diseased organ.

Formalin

Known for being the routine fixative of choice for tissues, formalin has a well-studied mechanism
of action consisting of cross-linkages in the protein residues causing the fixation and hardness of the tissue
(Bruce-Gregorios, 2006). In terms of physical appearance, formalin has a relatively neutral effect on the
tissue leading to minimal discoloration and an accurate representation of the colors of the tissue. In
some cases, formalin has been known to form acid-hematin pigments (Pizzolato, 1976) resulting in
changes in color. This may be avoided by the use of neutral buffered formalin (Pizzolato, 1976), which is
known for its pigment preservation qualities. Hardness and consistency is noted to be excellent in
formalin, as it is often used to preserve large organs and tissues for observation. A tendency for formalin-
preserved organs to be brittle has been noted in multiple journals (Kikugawa, 2004). Dilution of formalin
to an appropriate concentration fit for the organ to be preserved may be employed in order to reduce the
lack of bending properties seen in prolonged formalin application. Formalin may also be combined with
other fixatives to counter-act its different disadvantages such as the use of alcohol in order to reduce the
overall time needed for fixation and dehydration (Bruce-Gregorios, 2006).

Honey

Honey has been historically used on the preservation of organs due to its relative ease of
production. Historic accounts of mummification by immersion in honey has been noted in preservation of
the bodies of Alexander the Great and Agesipolis king of Sparta in Libya (Birx et al, 2005). Certain osmotic
effects due to the high concentration of honey has been known to desiccate small samples of tissues,
though its effects on whole organs has not yet been properly studied (Birx et al, 2005). In terms of physical
appearance, honey is said to possibly impart pigments present in the honey itself affecting the gross
colorization of the organ. Brittleness has also been noted (Patil et al. 2015.) which may affect the
specimens tolerance to the sectioning and embedding process. Noted shrinkage of tissue due to the
osmotic property of honey has affected physical properties such as the size and consistency of the
preserved tissue. Honey also has a relatively low pH which is known to be detrimental to tissue not
physiologically exposed to acidic environments. A noted disadvantage in the use of honey is the formation
of molds, this may be prevented by the addition of thymol crystals (Patil et al. 2015.)

Acetic acid

A noted additive in fixation, acetic acid has been known to enlarge tissues primarily due to its
osmotic property (Bruce-Gregorios, 2006). This may possibly affect gross morphology as tissue swelling
generally leads to a weak consistency due to the expansion of tissue. In processing, this may affect
dehydration and sectioning. Acetic acid does not greatly affect tissue coloration, though it may have
effects on some pigments which are soluble to the acetic medium (Tsukui, 2000). The individual effect of
acetic acid is not well-documented as it is often used in-solution with other fixatives in order to counter-
act their disadvantages such as shrinkage and alkalinity.

Effect of Temperature on Fixation

Variation of temperature could significantly affect the rate of fixation. Increase of temperature
could significantly hasten the fixation process compared to a normal room temperature. On the other
hand, there is no known notable effect in an increase of temperature, assuming that after an increase, it
will stand it at room temperature. The suggested temperature for fixation is at 37C and 45C for an
optimal quality (Geoffrey Rolls, 1997).

pH

One research says that pH doesnt really make any significant changes in light microscope.
However it does affect the slide per se when using Electron microscope; physiological pH must match in
that case. In addition, the suggested pH is about 6.8 to 7.2 (Carson FL., 1997).

Microscopic Effects

Formalin

Formalin is the gold standard in fixation therefore its effect on the microscopic components of th
e tissue is minimal. The problem in formalin is that it requires an optimum time for the fixation or else it
can destroy the tissue structures. When formalin fixation is inadequate, the cytoplasm is coarsely
coagulated as well as the nuclear components, including DNA and strongly basic proteins. This event can
be lessened with the use of acetic acid (Kiernan, 2000). The dilution, pH, temperature, concentration, and
duration are some of the important factors to consider during fixation in formalin, because even a slight
error changes the morphologic characteristics of the tissue under the microscope (Bryant, 2017). A study
that used liver and kidney tissues fixed in formalin showed excellent structural details, even with
preservation of the cytoplasm with its components and cellular outline (Maaini and Bryant, 2006).
Formalins reaction with Hematoxylin and Eosin (H&E) stain shows excellent morphology and the
structures are well demarcated and expressed (Maiini and Bryant, 2008).
Honey

Honey has been compared in various journals to formalin as an alternative fixative. There was a
study comparing formalin (10%), honey (20%), and jaggery (30%) as to its fixative properties as well as its
morphologic and microscopic characteristics. By 6 months, the morphology of tissues were still intact,
however there was deterioration of nuclear and cytoplasmic compartments in all three reagents. Below
is their comparison of the three reagents (pros and cons):

Table 1.1

Their staining characteristics, however, showed that they stain adequately in H&E, Periodic Acid Schiff
(PAS), and Massons Trichrome (MT) although slightly inferior to Formalin fixed tissues. Their conclusion
was that honey and jaggery can be used as an alternate preservative in the long run, although more
studies are still required (Patil, et. al. 2015).

Another study compared honey-fixed tissues and 10% Neutral buffered formalin (NBF), its results
showed similar histomorphology between the two, with slight changes in the tissues stained with honey,
which did not affect its diagnostic capability (Ozkan, et. al. 2012).

Staining capabilities of different percentages of honey in connective tissues were also tested in
another study. The stains used were Van Gieson stain (a stain for collagen and connective tissue for
differentiation), Gordon and Sweets reticulin (stain for reticulin fibers), Millers elastic stain (for elastic
fibers), and Massons Trichrome (a three colored stain for connective tissues). With a formalin control,
1%, 5%, 10%, 20% honey fixed tissues were stained with the specials stains just mentioned. These were
the results, they rated the histomorphology of the tissues in differents stains where 0 = poor and 4 =
excellent.

Table 2.2

Staining method Formalin 1% Honey 5% Honey 10% 20% Honey

control Honey
Van Gieson stain 3 0 4 4 3
Gordon and Sweets 4 0 4 4 3
reticulin
Millers elastic 3 1 4 4 3
Massons Trichrome 4 3 4 4 4

It just shows that honey can be an alternative to formalin as a fixative and does not affects the tissues
staining capabilities (Maiini and Bryant, 2008).
Acetic Acid

As previously mentioned, formalin and honey can cause shrinkage of the tissue upon fixation, we
then thought to use acetic acid to counteract this shrinkage. Acetic acid is commonly used together with
other fixatives and is a component of almost all, if not, all alcoholic fixatives, hence has not much effect
on the staining and histomorphologic properties of the tissues under study. Acetic acid mainly causes
nuclear chromatin coagulation, which facilitates recognition of cell types (Kiernan).

Discrepancies on Morphologic and Histologic Structures

There was a study conducted in University of Wales Institute, Cardiff where they used honey as
a histological fixative in comparison to formalin by virtue of cellular outline, nuclear detail and overall
morphology. In this study they used rat kidney and liver as tissue samples. Beforehand they make a
control using the tissue samples aforementioned, that are fixed with 10% buffered neutral formalin
(since 10%BNF is the gold standard for histological fixation). After which they subjected the samples in
the same conditions, same processing schedule and H&E staining schedule are observed. Observing at
room temperature, the liver tissue that are fixed with low concentration honey are well preserved and
well defined in terms of the morphology of their parenchyma and other organelles as that of Formalin.
Whereas when they subjected the rat kidney on a 20% honey solution, the glomeruli and the proximal
convoluted tubule's nuclei are well preserved. However, concerning the cellular outline and cytoplasmic
size, the renal tissue subjected to a high concentration honey shrinked and lost its cellular outlines. (Al-
Maaini and Bryant, 2006)

Histochemical Effects

Background

A major part of histopathology is rooted in the diagnosis of specific diseases. The end-goal of the
process is to specify what is causing the illness. This may be difficult in cases wherein morphological
features may be similar between two separate disease-states. Certain advancements in immunology has
been recently applied to histopathology helping in differentiating similar diseases. This is especially true
for differentiation of malignant and benign states in organs such as the lung. Pathol (2003) noted that the
use of antibodies to discriminate epithelioid mesothelioma and lung adenocarcinoma resulted in better
differentiation. This added specificity may help in giving the proper diagnosis of patients. Antibodies are
known for their specificity and sensitivity for either corresponding antigens. Applying this to the
histopathology laboratory has further boosted the sensitivity and specificity of the tests. Another goal for
histopathology is to achieve a level of objectivity wherein ones diagnosis is as least subjective as it can
be.

Pearse (1975) said that it is impossible to apply a single histochemical technique to a problem
in histopathology without gaining an increase in objectivity. The use of histoimmunochemical processes
has led to an almost purely objective result aided by the action of antibodies. The effects of formalin,
honey and acetic acid to these processes is increasingly important as processes like these are recently
growing more popular.
Formalin

Although being a staple in histologic preservation, formalin like most aldehyde-based fixation
methods has a degrading effect on the immunodetection of antigens. One study showed that formalin
fixation causes denaturation of most molecules (Dapson 2007). The mechanism by which formalin
preserves tissue is by making cross-linked bridges between the molecules. The cross-linkages leads to a
firmer tissue composition in the expense of chemical alterations. This is especially important as most
antigens present in our body can easily be denatured which greatly affects their reaction. The
denaturation of antigens means that antibodies specific for these denatured molecules will have nothing
to bind onto leading to an erroneous result. It can also cause block of antibody access to the epitope
(OLeary et al. 2008). The formalin acts to block antibodies either physically or chemically from the epitope
itself.

Due to the cross-linkages of tissue molecules with formalin, antibodies will have a difficult
binding process with the antigens present on the preserved tissue. Bogen et al. (2008) stated that formalin
binds to the epitopes directly. This prevents any antibodies from reacting with the specific molecules they
were specific for. Under-fixation of <12 hours with formalin inadvertently helps preserve the
histochemical aspects of the tissue (Eltoum et al. 2001a., Eltoum et al. 2001b). In addition to this, a change
in hydrophobicity is often attributed with the dehydrating agents used after formalin fixation (Grizzle et
al. 2008; Spencer and Bancroft 2008). This prevents certain antibodies from binding to their respective
antigen due to the osmotic disruptions introduced by the dehydration process. These mechanisms further
cement the omission of formalin as a fixative for immunostaining.

Honey

Most research on using honey as a substitute for formalin has compared the two in terms of
their microanatomical fixation quality. Little is known on the effects of honey on the antigens present on
the fixed tissue. Amidst this, it is possible to assume the effects caused by the addition of honey from its
chemical characteristics and the knowledge we have on antigen-antibody reactions. Honey is known to
be very acidic due to the high amounts of sugar present (Krushna et. Al, 2006). While some antigens can
withstand the low pH caused by the honey, antibodies which are mostly protein in nature get easily
denatured as a result of extremely low pH levels. This reduces their response and promptly causes them
to be inactivated. This results in a falsely negative result in terms of immunostaining. It is similarly known
that honey contains antibody-enzymes deposited by the bees to protect their food from possible
bacterial infestation (Kwakman & Zaat, 2012).

Antigens in our different tissues are similar in structure to the antigens commonly found in
nature. This has lead theories on why naturally-occurring antibodies may be present in our own blood.
Some of these note that pollen grains and bacteria may be the cause of the bodys antibody production
against the antigens present on them (Harmening, 2012). This leads to the possibility of Bee-defensin-1,
a bee-antibody equivalent, cross-reacting with the antigens present in our tissues leading to a possible
blockage of epitopes by the enzyme. The effect of immunostains will then be negated by the presence of
the defensin-antigen complex. Osmotic pressure is also present in the honey due to the high
concentrations of chemicals leading to a hypertonic solution (Kwakman & Zaat, 2012). This affects the
antibody uptake, as antibodies reactions are hastened by reduced levels of water. The different possible
factors explained are still unclear in terms of their mechanisms. Little is known on the effects of honey in
fixation and lesser still for its effects on the histochemical processes.

Acetic Acid

Commonly used as in conjunction with other fixatives, acetic acid is used in histopathology to
demonstrate the cellular nucleus. Being an acid, it inherently has a low pH which can affect antibody-
binding and antigen presence (Lee et. al,) Protein antigen denaturation is especially possible as it is easily
affected by the pH level of the surrounding medium. The addition of acetic acid disrupts the cytoplasm
leading to the release of cytoplasmic constituents leading to a possibly easier immunostaining reaction.
This can also work against the detection of the antigen-antibody reaction as it disrupts the cell membrane
morphology leading to possible deformation and obstruction of antigens. As acetic acid is only used as an
additive for other fixatives (ie. honey), its effects on histochemistry is very limited primarily due to its low
volume on the composition of the reagent for fixation.

Mechanism of Preservation

The formation of Hydroxymethylfurfural (HMF), a furanic compound; intermediate product of

Mailards reaction, marks the beginning of the preserving action of honey. This reaction occurs naturally
for substances which contain reducing sugars. It is then accelerated with the help of an acidic medium or
heat treatment. Afterwhich, di-Schiff base reaction occurs and cross-linked the proteins in tissues to the
honey which makes it fixed. (Barnali Majumdar 2016)

Formalin

Formalins mechanism of preservation entails the production of methylene hydrate, which

indicates the first step in fixation. Methylene hydrate is simply Formaldehyde gas dissolved in H2O, and it
produces Formalin. The formaldehyde or methylene hydrate binds with the N (Nitrogen)-portion or
other atoms of protein in which they will form a cross-link CH2- called a methylene bridge. The binding
of formaldehyde to protein can be completed in 24 hours, but formation of methylene bridges take a little
longer. The fixative action of formalin can be mainly attributed to its reaction with proteins (Helander,
1994) (Kiernan, 2000).

Honey

Honey is a product of honey bees from the nectar of plants, its excretion and plant-sucking
inasects excreta. Types of Honey varies depends on several factors like: type of flora used, quantity of its
sugars and temperature. However despite of these discrepancies, it has many common components
found in every types of honey with varying composition, these are: The sugars (Glucose, Galactose,
Sucrose, Maltose, Melezitose) Glucoseoxidase, Phenolic, Flavinoids, Terpenes, Hydrogen Peroxide, trace
elements, Ascorbic acid and water. The honey bees then stores honey as their reserve during extreme
climates, weather and food scarcity (Aluko Esther Olusola1* 2013)( Ananthalakshmi Ramamoorthy 2016).
The use of honey as food, preservative and medicine were traced back in the paintings depicted in stone
age. It was found as an ancient treatment for wound due to its bacterial inhibitory effect. It also has an
antioxidant capacity for wound healing as well. (Tahereh Eteraf-Oskouei and Moslem Najafi 2013).

Advantages & Disadvantages

Essentially the Honey, being non-hazardous, makes it a good alternative preservative in terms
of safety compared to Formaldehyde (Group 1 classified carcinogen) (Shankargouda Patil, Roopa S. Rao
205). Honey was known to contain an anti-bacterial property and dehydrating activity due to its ascorbic
acid content, phenol inhibine and hydrogen peroxide content respectively (Dhengar 2016). However in
the presence of acid that accounts for low pH (3 - 4), using honey as a preservative could pose a problem
in preserving cytoplasmic organelles. In the presence of phenol inhibine as its anti-bacterial, honey is more
prone in fungal infection. Honey has an anti-autolysis property noted (Ananthalakshmi Ramamoorthy
2016). Another study was conducted if the honey could contend to formalin as a fixative. The results
shows that the difference in microscopic morphology and staining is insignificant. (B. Sabarinath 2014)

Acetic Acid

Acetic acid (CH3COOH) or in layman term, vinegar has been used for a long time as a preservative
of foods. It has also been implicated in the fixation process in the form of Glacial Acetic Acid. Glacial acetic
acid is concentrated acid without the water component. The concentration normally used varies from 3-
5% (mainly 5%) (Baker, 1958). Acetic acid is considered a non-coagulated fixative. But it causes the
coagulation of nuclear proteins, which stabilizes it and prevents losses of nucleic acids. Aside from being
a fixative, it is also known as a disinfectant, which is great help in the fixation process in preventing
bacterial contamination (Tijare, 2014). Acetic acid does not react much with proteins, it is mainly used in
conjunction with ethanol as a cytological fixative. Its major effect is to precipitate DNA, which aids in the
preservation of nuclei. Acetic acid also causes the swelling of cells, when used alone. Therefore, acetic
acid has been used to counteract the shrinking effects of other fixatives (Huang and Yeung, 2015). Acetic
acid has many benefits to the fixation process and is most used together with other fixatives (Tijare, 2014).

Fixative properties

Honey

The acidity and dehydration properties of honey prove that the latter is an acceptable substitute for
formalin as a histological fixative. In addition, concentration of honey is said to affect the fixation results
due to its acidity. A study conducted in rat liver and kidney tissue that were fixed for 24 hours with 10 to
100% honey diluted in distilled water at 37c and at room temperature. The result showed that the tissues
fixed with 10% and 20% honey demonstrated comaparable results to tissues fixed with formalin (Rahma
Al-Manini & Phil Bryant 2013).

It also contains antimicrobial, antiviral and antimutagenic properties that prevent putrefaction
without harming the medical technologists.

Acetic Acid

Diluted acetic acid inhibits growth of pathogens and prevents the formation of biofilms.
 In addition, acetic acid inhibits Pseudomonas aeruginosa, Acinetobacter baumannii, Staphyloccocus
aureus, Enteroccocus faecalis and other 25 pathogens.

Adverse Morphologic Effects

Formalin

Being regarded as the fixative of choice in most laboratories for general preservation of tissue,
formalin has well-studied and well-known mechanisms of action. It has been reported by Cancer et. al
(2015) that formalin causes the shrinkage of tissue. This is usually a non-issue as it still preserves the
general anatomical structure of the specimen. An instance where it becomes a factor for erroneous results
would be in tumor classification. As tumors are measured by size which is also proportional to the overall
severity of the lesion. Cancer et. al (2015) also called out for the standardization of the tumor-sizing as to
the concentration of formalin used. The shrinkage of tissue may further be studied in order to find an
effective standardized staging criteria which is very important in malignancies as an indicator of prognosis.

Honey

Little is known on the effects of honey as a preservative and fewer is known on its effects on
gross anatomical structure. It can be inferred however that since honey is a very concentrated mixture
(Kwakman & Zaat, 2012). It causes the general shrinkage of the tissue. This, as does formalin, may cause
shrinkage of the specimen leading to erroneous staging of tumors. It is also known to be of low pH
(Krushna et. Al, 2006) leading to deterioration of acid-labile tissue causing erroneous gross and histological
structure.

Acetic Acid

Acetic acid is widely used in histology to boost the nuclear detail (Marina et. al, 2012). This is
important in investigating tissues wherein the main pathology can be differentiated from another similar
pathology by expression of nuclear differences. Such is the case in precancerous cervical lesions (Marina
et. al, 2012) where it has been used for decades to aid in diagnosis. Acetic acid has been repeated excluded
in cytoplasmic studies such as Golgi (1898) and Lewitsky (1911) for it has a swelling effect on the cell. This
has made it a contraindication for cytoplasmic studies (Bruce-Gregorios, 2006).

SIGNIFICANCE OF THE STUDY

This study will greatly contribute to the laboratory personnel considering that they face a major
problem in tissue fixation and that is the carcinogenic effect the reagents bring. The greater demand for
a nontoxic reagent leads to the creation of many researches that explores other alternatives to replace
formalin in terms of quality and value. Thus, this section will provide a brief explanation on the importance
of the study given to the people involved.

To the students. This study aims to help the students have a better understanding that will guide, identify,
and handle the dangers in the laboratory. It can interest students continue and improve this research that
many researchers were not able to explore.
To the professors. By this study they will be provided knowledge about the properties of honey and acetic
acid, and how it preserves tissue detail.

To the laboratory personnel and pathologists. This proposed study will benefit them the most allowing
them to work in a safer place with no fear of the consequences regarding their health.

GENERAL OBJECTIVE

To determine the comparability of honey and vinegar mixture with formalin in histological fixation

SPECIFIC OBJECTIVES

1. To compare honey and acetic acid mixture with formalin on fixing different:
a. Types of tissue fixed
i. Skeletal muscle of pork
2. To assess the gross appearance of the tissues as to:
a. Color of the tissue
b. Ease with which it is cut
c. Size of the tissue
3. To evaluate the microscopic appearance of the fixed tissues as to:
a. Staining reaction
b. Morphologic feature
c. Inconsistencies in the histologic structures (nuclear and cytoplasmic)
 CONCEPTUAL FRAMEWORK

Autolysis

Chemical contamination of
reagents in production

Fixation of tissue:
Collagen bridging of
Honey with Acetic Acid Gross
Microscopic

Bacterial Contamination:
Exposure to radiation
Acetic acid
bacteria

* Some information is provided by Dr. Sierra Roma S. Hernandez, MD (Anatomic and Clinical
Pathologist)*
 RESEARCH METHODOLOGY

RESEARCH DESIGN

This research will be utilizing analytical methods. Specifically, the experimental method that is
done by comparing the advantages of using the independent variable, which is the Honey with Acetic acid,
instead of formalin as fixative of various tissues, the dependent variable, but in our case, skeletal muscle
is our tissue of choice.

SELECTION OF SUBJECTS

As this research we will be needing tissues in its experiment. Most researches has used pork as its
specimen as it is also known as the translational research model wherein 80-90% of its organ system is
comparable to humans. This greatly imply that if a research performed in pork is working, there is a higher
chance that it will work in humans as well.

OPERATIONAL DEFINITION OF TERMS

Skeletal muscle striated muscle; under voluntary muscle tissue and it is attached to bones by

tendons.

Fixation is the most critical step in the preparation and preservation of histological sections in

order to prevent autolysis or putrefaction.

Autolysis/Putrefaction destruction of cells or tissues

Gross appearance term that refers to the findings that can be seen with the naked eye

Results of fixation using formalin:

A.) Appearance: change from a fresh looking tissue to a dull looking one
B.) Color: change from a fresh color to a dull color of a specimen
C.) Consistency: soft to firm tissue
D.) Size: slight shrinkage from original size
E.) Hardness: does not easily crumble; not friable

Microscopic Appearance structures that can be observed using microscope

Hematoxylin basic dye or mordant that is used to stain acidic structures and seen as purplish

blue

Eosin a counterstain that binds to proteins in the cytoplasm and seen as pink in color
 Acid alcohol simple mixture of an acid and an alcohol that is used to decolorize sample that

contains cells

Staining technique used in microscopy to enhance contrast in the microscopic image

Levels of staining:

1. Primary Stain: uses hematoxylin

2. Decolorizer: uses acid alcohol
3. Secondary Stain: uses Eosin

Results:

1. Nuclei: stains blue/black

2. Cytoplasm: stains pink
3. Muscle fibers: stain deep red
4. Red Blood Cells: stain orange red
5. Fibrin: stain deep pink

Results are obtained from Dr. Gloria Tan, RMT, MD, FPSP, MMHA, MPM

DATA COLLECTION TOOL

The data collected will be obtained by the researchers themselves (1o data). The Method of Data
Collection is through Observation by experimentation. Histotechniques will be the method used for
testing.

DATA PROCESSING AND ANALYSIS

MATERIALS

1. Honey
2. 3-5% Acetic Acid (Datu Puti)
3. Fresh Tissue (Pork)
4. Knife
5. Chopping Board
6. Ruler
7. 10% Formaldehyde
8. Tissue Cassette
9. Paraffin Wax
10. Alcohol Lamp
11. Rotary Microtome
12. Water Bath
 13. Applicator Sticks
14. Glass Slides
15. Xylene
16. Absolute Alcohol
17. 95%, 80%, and 70% alcohol
18. Distilled Water
19. Ammonia Water
20. Hematoxylin and Eosin

PROCEDURE:

Fixation
Honey
Procedure:
1. Observe the quality of the unfixed specimen.
2. Cut the tissue for about 5mm and place it inside the tissue casette together with a paper label.
3. Place the tissue block inside a container with 10-20% honey.
4. Fix the tissue for 24 hours.
5.Observe changes in the fixed specimen.

Honey with 3-5% Acetic Acid

Procedure:
1. Observe the quality of the unfixed specimen.
2. Cut the tissue for about 5mm and place it inside the tissue cassette together with a paper label.
3. Place the tissue block inside a container with an equal amount of 10-20% honey and 3-5% acetic
acid. (1:1 ratio)
4. Fix the tissue for 24 hours.
5. Observe changes in the fixed specimen.

Formalin
Procedure:
1. Observe the quality of the unfixed specimen.
2. Cut the tissue for about 5mm and place it inside the tissue cassette together with a paper label.
3. Place the tissue block inside a container with 10-20% Formalin.
4. Fix the tissue for 24 hours.
5. Observe changes in the fixed specimen.

Decalcification (if skeletal muscle is attached to bones).

Procedure:
1. Use 10% nitric acid by using the N1V1 = N2V2 formula.
2. In a piece of gauze, wrap the tissue.
 3. Tie the thread to the gauze containing the tissue using a wax thread and suspend it in 10%
nitric acid container.
4. Replace the 10% nitric acid for every 3 days.
5. Using the tap water, wash the tissue thoroughly and check for the completeness of
decalcification via physical method.

Checking for the completeness of decalcification by chemical method

Procedure:

1. Place a used amount of 10% nitric acid in a 2.5 mL container.

2. Add strong ammonia water to alkalinize the solution. Use red litmus paper as an indicator.
3. Add 0.25 mL of aqueous solution of ammonium oxalate.
4. Mix thoroughly and let it be for about 15 30 minutes
5. Observe the completeness of decalcification

Dehydration, Clearing and Impregnation

Procedure:

1. Put tissue and label inside a tissue cassette

2. Do dehydration, clearing, and impregnation following the schedule in the clinical laboratory

30 min 80% Alcohol 90 C

15 min 95% Alcohol 90 C

30 min Acetone 90 C

5 min Air Dry Room temp

30 min Chloroform 90 C

45 min Choloropara 90 C

60 min Paraffin 1 and 2 180 C

Embedding
Procedure:

1. Prepare a paper boat mold using a 2/3 index card.

2. Fill up 2/3 of the paper boat with molten paraffin wax.
3. Using forceps, orient the tissue in the paper boat with molten paraffin wax.
4. Place the label at the side of the mold within the paraffin wax.
5. Allow the block to cool and harden.
6. Place it inside the laboratory refrigerator.
Sectioning

Procedure:

1. Trim the tissue block

2. Mount the tissue block on the rotary microtome
3. Do coarse sectioning by setting the thickness of the microtome at 10-15u to expose the
embedding tissue
4. Make a ribbon of thinner tissue sections (4-6u)
5. Using applicator stick, transfer the ribbon to the water bath set at 6 to 10 degrees lower than
the melting point of paraffin wax.
6. Fish out the tissue sections by immersing the glass slide in a near vertical position as close as
possible to the section. When the slide touches the section, it is lifted vertically.
7. Drain the excess water off the glass slide and place it on a slide track.
8. Put the tissue section on a paraffin oven set at a temperature 2-5C higher than the melting
point of paraffin wax to remove the wax and fix the tissue into the glass slide.

Hematoxylin and Eosin staining

Xylene 10 sec

Absolute 10 sec
alcohol

95% Alcohol 10 sec

80% Alcohol 10 sec

70% Alcohol 10 sec

Distilled water 3 dips

Hematoxylin 15 min

Distilled water 3 dips

Acid alcohol 1 quick dip

Distilled water 3 dips

Ammonia Dip until

water the section
turns blue

Distilled water 3 dips

 70% alcohol 10 sec

Eosin 5 min

95% alcohol 10 sec

Absolute 10 sec
alcohol

Xylene 5 min

Clean the slide

then Air Dry

Mounting

1. Place enough amount of mounting medium on the center of tissue section.

2. Place a dry, clean coverslip on the edge of the slide and released gently.
3. Pressed gently on the coverslip allowing the mounting medium to spread quickly.
4. Properly label the slide.
5. Allow the slide to dry up.

Microscopic Examination

*The Pathologists who will observe the results will be blinded as to which fixatives are used*
 STUDY DESIGN

Pork

Skeletal Muscle

Honey Honey with 3-5% Acetic Acid Formalin

Fixed Unfixed Fixed Unfixed Fixed Unfixed

Analysis of Data
Statistical Test

A Chi- Square Test will be used as a statistical method to determine whether there is significant
association between the different variables, under different qualities of fixation.

If the sizes of the specimens are compared before and after fixation. Measures of Central
Tendency can be used to analyze them.

Dummy Table

These tables will be assessed by three or more Anatomic Pathologists. If there are differences
among their perspectives, they will vote among themselves, a majority vote is required.

A Component Bar Diagram will be used to discern the qualitative variable of pork, between
honey, honey with 3-5% Acetic acid and Formalin as to texture and quality of fixation. And the same chart
will be used further to express the quality of fixation according to its gross appearance, consistency, and
ease of cutting.

This table will be used to distinguish the quality of fixation by tallying the percentage of the fixed
tissue in Honey, Honey with 3-5% Acetic acid and Formalin as to the following variables:

TABLE 1.3 Quality of Fixation

Honey Honey with Formalin Total

acetic acid
Cut section Number % Number % Number % Number %
Crushed
Bad fixation
Good fixation
Excellent
TOTAL

TABLE 1.4 Consistency

Honey Honey with Formalin Total

acetic acid
Cut section Number % Number % Number % Number %

Rigid
Soft
Firm
TOTAL
 TABLE 1.5 Ease of Cutting

Honey Honey with Formalin Total

acetic acid
Cut section Number % Number % Number % Number %

Brittle
Friable
Firm
TOTAL

TABLE 1.6 Gross Appearance

Honey Honey with acetic acid Formalin

Number % Number % Number %

Color

a) Brown

b) Black

c) Flesh

Thickness

a) Insubstantial

b) Dense

c) Perfect

Texture

a) Rough

b) Rubbery

c) Smooth
 ASSESSMENT BY PATHOLOGIST

Grade 1 Grade 2 Grade 3 Grade 4

Gross and Soft. The tissue is Soft. Once Very hard. The Hard but not rigid.
Consistency tender to touch. pressed, it will not Microtome The microtome
Once pressed, it go back to its blades arent able blades must be
will be back to its original shape. to cut the tissue able to cut the
original shape. as neat as tissue as neat as
possible. possible.
Color Brown Beige (Light Grey Pale Grey
Brown)
Size and Shape Prominent tissue Tissue shrinkage is Tissue shrinkage Tissue shrinkage is
shrinkage is observed but not is minimally minimally
observed. Shape is prominent. Shape observed. Shape observed. Shape is
distorted. is slightly is slightly not distorted.
distorted. distorted.
Microscopic Stains that doesnt Stains that doesnt Stains that absorb Stains that absorb
Stain absorb light absorb light light but doesnt light and renders
rendering the cells rendering the cells render the image the cells visible in
transparent where transparent where visible because of the microscope.
differentiation only minimal undesirable
isnt able. differentiation is contrast.
allowed.
Microscopic Light Blue Blue Purple Purplish-blue
Nuclear
Structure
Microscopic Pale Pink Pale Red Magenta Vibrant Pink
Cytoplasmic
Structure

*Grade 1- LOWEST and Grade 4- HIGHEST*

LIMITATION OF THE STUDY

The limitations of the study are the unexpected contamination, autolysis, chemical contamination
problem with the reagents, and radiation that may affect the procedure. Other than that, there are no
other limitations to our study.

ETHICAL CONSIDERATIONS

There are no ethical violations. The proper handling of the procedure is the problem which can
affect the researcher and also its proper disposal.
 GANTT CHART

DAY SCHEDULE OF ACTIVITIES

The tissue is cut and its appearance is recorded before any fixation is
D-1
done.

The tissue will be fixed in a 10% buffered neutral formalin for 8 hours.

The fixated tissue is placed in 10% nitric acid that is prepared

D-2
beforehand.

D-3 The 10% nitric acid is continuously changed for 3 days.

D-4 The 10% nitric acid is continuously changed for 3 days.

D-5 The 10% nitric acid is continuously changed for 3 days.

The last changed solution is kept for checking of complete decal while
the tissue is being washed using tap water.

Observation is recorded
The tissue is placed back in the cassette where dehydration, clearing
D-6
and impregnation is done.

A paper boat using index card is prepared that will be filled with
D-7
melted paraffin wax with the tissue.
The block is cooled and hardened and is placed inside the laboratory
refrigerator.

D-8 The hardened block is placed in the microtome to produce ribbons.

These ribbons are placed in the water bath.

The ribbons are fished out using a slide and heated to melt the paraffin
wax.
Staining is made by following the H & E procedure and will be
D-9
mounted.

The mounted slide is pressed to prevent bubble formation.

It is labeled and dried afterwards.

D - 10 The slides are checked under the microscope.
All observations seen are recorded.
The recorded observations are compared when using formalin as
fixative.
 REFERENCES

Fixation

Baker, J. (1958). Principle of Biological Microtechnique. London, Methuen, United Kingdom. Retrieved
2017

Clark, R. (1983). Formaldehyde in pathology departments. Laboratory for Aerobiology, 839-846.

Drury RAB, W. E. (1980). Carleton's Histological Technique (5th ed.). New York: Carleton. Retrieved 2017

Fischer, M. (1905). The Toxic Effects of Formalydehyde and Formalin. The Journal of Experimental
Medicine, 487.

Guelrud, M., & I, H. (1998). Acetic acid improves indentification of remnant islands of Barrett's
epithelium after endoscopy. Gastrointestinal Endoscopy, 512.

Harrison, P. (1984). An ehtanol-acetic acid-formalin saline fixative for routine use with special
application to the fixation of non-perfused rat lung.

Huang, B., & Yeung, E. (n.d.). Chemical and Physical Fixation of Cells and Tissues: An Overview. Retrieved
September 20, 2015

Jayalakshmi, K., Ravikumar, H., Naidu, J., & Raghavendra, R. (2011). A silent killer in the laboratory
Formaldehyde: Review of effects and management. International Journal of Oral & Maxillofacial
Pathology., 13-19. Retrieved 2017

Kiernan, J. (2000). Formaldehyde, formalin, paraformaldehyde and glutaraldehyde: What they are and
what they do. Microscopy Today 00-1, 8-12.

Rodger, G., R, H., Cryne, C., & Bailey, J. (1984, May). Diffusion Properties of Salt and Acetic Acid into
Herring and Their Subsequent Effect on the Muscle Tissue. Journal of Food Science, 49(3), 714-
720.

Thavarajah, R., Mudimbaimannar, V., Elizabeth, J., Rao, U., & Ranganathan, K. (2012). Chemical and
physical basic of routine formalydehyde fixation. 400-405. Retrieved 2017

Tijare, M., Smitha, D., Kasetty, S., Kallianpur, S., Gupta, S., & Amith, H. (2014, April 18). Vinegar as a
disinfectant of extracted human teeth for dental educational use. Journal of Oral and
Maxillofacial Pathology, 14-18. doi:10.4103/0973-029X. 131883

Carson FL. Histotechnology. 2nd ed. Chicago: ASCP Press, 1997.

Rolls GO, Farmer NJ, Hall JB. Artefacts in histological and cytological preparations. In Woods A and Ellis R
eds. Laboratory histopathology. New York: Churchill Livingstone, 1994;5.3-1 - 5.3-29.
Mechanism

Dhengar, Y., Palve, D., Thakur, M., Bhagwatkar, T., Bhondey, A., & Chaturvedi, S. (2016, January-March).
Natural Substitutes for Formalin - Chemical verus Natural: Comparative Study. Annals of Dental
Speciality, 4(1). Retrieved 2017

Eteraf-Oskouei, T., & Najafi, M. (2013, June 16). Traditional and Modern Uses of Natural Honey in
Human Diseases: A Review. Iranian Journal of Basic Medical Sciences, 6, 731-742.

Majumdar, B., Rao, R., & Patil, S. (2016, April-June). Tissue Preservation with Natural Fixatives: An
Immunohistochemical Evaluation. World Journal of Dentistry, 87-91. doi:10.5005/jp-journals-
10015-1371

Mandal, M., & Mandal, S. (2011, April 1). Honey: its medicinal property and antibacterial activity. Asian
Pacific Journal of Tropical Biomedicine, 154-160. doi:10.1016/S2221-1691(11)60016-6

Olusola, A., Helen, O., Enobong, I., & Ezekiel, A. (2013). Comparative Study of Effect of Honey on Blood
Pressure and Heart Rate in Healthy Male and Female Subjects. British Journal of Medicine &
Medical Research, 3(4), 2214-2221.

Patil, S., Rao, R., & Majumdar, B. e. (2015, January-June). Natural sweetners as fixatives of
histopathology: A longituidinal study. Journal of Natural Science, Biology, and Medicine, 1, 67-
70.

Ramamooorthy, A., Ravi, S., Jeddy, N., Thangavelu, R., & Janardhanan, S. (2016, November). Natural
Alternatives for Chemicals Used in HIstopathology Lab - A Literature Review. Journal of Clinical
and Diagnostic Research, 10, 1-4. doi:10.7860/JCDR/2016/23420.8860

Sabarinath, B., & Sathyakumar, M. (2014, March). Fixative properties of honey in comparison with
formalin. Journal of Histotechnology, 37, 21-25. doi:10.1179/2046023613Y.0000000037

The Composition and Biological Activity of Honey: A Focus on Manuka Honey. (n.d.).

Morphological effects

Baker, J. (1957). The Effect of Acetic Acid on Cytoplasmic Inclusions. Quarterly Journal of Microscopical
Science, 98(4), 425-429.

Bruce-Gregorios, J. (2012). Histopathologic Techniques. Manila.

Krushna, e. a. (n.d.). Correcting the Shrinkage Effects of Formalin Fixation and Tissue Processing for Renal
Tumors: toward Standardization of Pathological Reporting of Tumor Size. Retrieved from
NISCAIR Online Periodicals Repository:
http://nopr.niscair.res.in/bitstream/123456789/5238/1/IJEB%2045

Marina, O., Sanders, C., & Mourant, J. (2012, August 8). Effects of acetic acid on light scattering from
cells. Journal of Biomedical Optics. doi:10.1117/1.JBO.17.8.085002
Tran, T., Sundaram, C., & Cheng, L. (2015). Correcting the Shrinkage Effects of Formalin Fixation and
Tissue Processing for Renal Tumors: toward Standardization of Pathological Reporting of Tumor
Size. Journal of Cancer, 759-766.

Histochemical effects

Bee Defensin-1. (2012, 04 07). Retrieved from Santangelo, ECF:

http://flipper.diff.org/apprulesaccount/items/info/4619

Krushna, e. a. (n.d.). Correcting the Shrinkage Effects of Formalin Fixation and Tissue Processing for Renal
Tumors: toward Standardization of Pathological Reporting of Tumor Size. Retrieved from
NISCAIR Online Periodicals Repository:
http://nopr.niscair.res.in/bitstream/123456789/5238/1/IJEB%2045

Kwakman, P., & Zaat, S. (2012). Antibacterial Component of Honey. IUBMB Life, 48-55.

Lee, C., & Shau-Mei, O. (n.d.). Investigation of the Physiochemical Properties of Concentrated Fruit
Vinegar.

Ordonez, N. (2003). The immunohistochemical diagnosis of mesothelioma: a comparative study of

epithelioid mesothelioma and lung adenocarcinoma. Americal Journal of Surgical Pathology,
1031-1051.

Pearse, A. (1975). The role of histochemistry in increasing objectivity in histopathology. Postgraduate

Medical Journal, 708-710.

The Combined Effects of Formalin Fixation and Individual Steps in Tissue Processing on Imunno-
recognition. (2009). Biotechnical HIstochemistry, 223-247.
 APPENDIX

BUDGET

Honey...340.10

Acetic Acid (Datu Puti) .13

Pathologist fee2,100

Stains (H&E) .790.47

Pork (Muscle) ..........220/kilo

TOTAL: 3,463.57

BSC (BIOSAFETY CONCERNS)

Proper handling and disposal is very important. Disposal is by incineration.

 PEER EVALUATION

Leader: Diana Eliza Reyes

Roberto Paolo Atienza

Angelo Tadeo Bautista

Amber Justine Chua

Nerissa Jayne Dalay

John Joseph Ermitao

Alianna Gaibrielle Tan

Jamille Simon

0 - no contribution --------> 10 - most contribution