A STUDY ON THE POTENTIAL MUTAGENICITY
OF OVERCOOKED HARDBOILED EGGS
USING Allium cepa TEST
__________________
A Research Paper
Presented to the
Science &Mathematics Department
Integrated Developmental School
MSU-Iligan Institute of Technology
__________________
In Partial Fulfillment for the Course
Science Research
__________________
RUSSEL CHRISTINE Bordaje CORCINO
GERMAINE Daniel ARDA
March 2012
APPROVAL SHEET
This research paper entitled “A STUDY ON THE POTENTIAL MUTAGENICITY
OF OVERCOOKED HARDBOILED HEN EGGS USING Allium cepa TEST”
prepared and submitted by RUSSEL CHRISTINE Bordaje CORCINO and
GERMAINE Daniel ARDA
PROF. ODYSSA NATIVIDAD R. M. MOLO
Adviser
__________________
Date
PROF. ALMA GLORIA L. SILVA
Panel Member
_________________
Date
PROF. EVERLITA E. CANALITA
Panel Member
_________________
Date
PROF. MILFE A. ABABA
Technical Consultant
___________________
Date
Accepted and approved in partial fulfillment of the Course in Science
Research.
PROF. VICTORIA A. TARRANZA
Chairperson, Science & Mathematics
________________
Date
PROF. LEILA V. BERNALDEZ
Principal, IDS
_______________
Date
ii
ABSTRACT
Corcino, Russel Christine B. and Arda, Germaine D. (2012). “A Study on the
Potential Mutagenicity of Overcooked Hardboiled Eggs Using Allium cepa test.
Integrated Developmental School, MSU-Iligan Institute of Technology.
Adviser: Prof. Odyssa Natividad R. M. Molo
Technical Consultant: Prof. Milfe A. Ababa
Using microscopic assay of the root tips through the estimation of the mitotic
indices of the root tips of Allium cepa, a study in the potential mutagenicity of hardboiled
chicken eggs cooked in three different time intervals was conducted. Results show a
significant difference in the mitotic indices (MI) of eggs cooked 2 minutes and 5 minutes
beyond standard cooking time, which ranged from 51.3 - 73.9 and 54.6 - 64.03,
respectively, as compared to the eggs cooked in standard time. Chromosomal aberrations
were also detected during the microscopy of root tips exposed to egg cooked 2 minutes
beyond standard.
iii
TABLE OF CONTENTS
Page
TITLE PAGE ………………………………………………………………..i
APPROVAL SHEET ……………………………………….………………ii
TABLE OF CONTENTS ………………………………………………......iii
LIST OF TABLES
LIST OF FIGURES
ACKNOWLEDGMENT
DEDICATION
CHAPTER
I. INTRODUCTION
A. Background of the Study ………………………………………...1
B. Statement of the Problem ………………………………………...2
C. Objectives of the Study ………………………………………......3
D. Hypotheses of the Study ……………………………….………...4
E. Significance of the Study ………………………………………...4
F. Scope and Limitations of the Study ………………………….…..5
G. Definition of Terms ….….…………………….………………...5
II. REVIEW OF RELATED LITERATURE & STUDIES ………………...7
III. METHODOLOGY
A. Research Design …………………………………………………13
B. Materials and Equipment ………………………………………..13
C. Experimental Set-up …………………………………………….14
D. General Procedure ………………………………………………16
E. Procedures in Data Gathering …………………………………...19
F. Statistical Tool for Data Analysis …………………………….....22
IV. RESULTS AND DISCUSSIONS ………………………………………23
V. CONCLUSION AND RECOMMENDATIONS
A. Summary ………………………………………………………...30
B. Conclusion ………………………………………………………30
C. Recommendations ……………………………………………….31
VI. REFERENCES ………………………………………………………….32
VII. APPENDICES
A. Appendix A Preparation of Onion Bulbs ……………………………. 35
B. Appendix B Preparation of Materials .………………………………..36
C. Appendix C Staining Process …….…………………………………..37
D. Appendix D Some Pictures in Microscopy …………………………..38
E. Appendix E ANOVA Table and Post-hoc Test .……………………..39
F. Appendix F Curriculum Vitae ……………………………………….40
iv
LIST OF TABLES
TABLE NO.
1
2
3
4
5
6
7
8
TITLE
List of the Different Treatments
Specimen Set-up
Mitotic Index of the Roots (over 400 cells) in Replicate A
Mitotic Index of the Roots (over 400 cells) in Replicate B
Mitotic Index of the Roots (over 400 cells) in Replicate C
Mitotic Index of the Roots (over 400 cells) in Replicate A
Mitotic Index of the Roots (over 400 cells) in Replicate B
Mitotic Index of the Roots (over 400 cells) in Replicate C
PAGE
14
15
18
18
18
24
24
25
LIST OF FIGURES
FIGURE NO.
1
2
3
4
5
6
TITLE
A Picture of an Overcooked Hardboiled Egg
Allium cepa test Setup
Example of Chromosomal Stickiness
Flowchart of the General Procedure
Mitotic Indices of the Roots Exposed to Treatments
Chromosomal Aberrations Detected
PAGE
7
15
20
22
26
27
(ma’am, wala pa mi acknowledgement ug dedications, ug curriculum vitae. Hehehe..)
v
ACKNOWLEDGMENT
vi
DEDICATION
vii
CHAPTER I
INTRODUCTION
A.
Background of the Study
The egg is a major part of the daily diet of humans. Humans have been
consuming eggs since the dawn of human time. Eggs could be obtained in any part of
the world. Ostrich and chicken are the most common. Eggs are relatively easy to obtain,
excellent protein sources, adaptable to many different types of recipes, from simply
boiled, fried, or stuffed to complicated quiche, custards or meringue, and fit the bill for
meatless fasting days required by some religions. Eggs have been the object of much
socio-religious symbolism and tradition. Over time, some groups have encouraged the
consumption/decoration of eggs in celebration of certain events (Food Timeline, 2000).
It is one of the many sources of vitamins except vitamin C, essential minerals except
calcium, and it also has high biological values of protein (Encyclopedia Americana,
2005). However, in some instances, people cannot manage to cook them properly, which
results to overcooked eggs. Overcooking eggs can alter their composition, which can
lead to the formation of new compounds in the egg, particularly hydrogen sulfide or H2S.
Scientists are suspecting that this compound is harmful to the body.
Motivated by these findings, the researchers wish to test if overcooked eggs have
a potential to be mutagenic to the human body using Allium cepa test, a test that uses
onion bulbs as determinants of toxic substances by allowing developing roots to come in
contact with the substance being tested.
2
B.
Statement of the Problem
The potential of overcooked hardboiled eggs as mutagen using the Allium cepa
test. Furthermore, it seeks to answer the following sub-problems:
1.
How long is the normal cooking time of an egg to be considered?
2.
What are the physical characteristics of an overcooked hardboiled egg and
a normal cooked hardboiled egg in terms of:
a) Color of the egg yolk
b) Texture of the egg
3.
What are the mitotic index that can be observed in the Allium cepa of the
following egg samples:
a) Sample A: normally-cooked
b) Sample B: 2 minutes overcooked
c) Sample C: 5 minutes overcooked
4.
Is there a significant difference on the mitotic index among the
overcooked hardboiled egg and normal cooked hardboiled egg?
5.
Is there a significant difference on the observations recorded on the Allium
cepa with the yolk of the overcooked hardboiled eggs at different time
intervals?
6.
Does this result in the mitotic index of Allium cepa test signify that
overcooked hardboiled eggs have the potential to be mutagenic?
C.
Objectives of the Study
The study aims to overcooked hardboiled eggs have the potential to be mutagenic
to the human DNA using the Allium cepa test. Furthermore, it aims to:
3
1.
Determine the normal cooking time of hardboiled eggs.
2.
Determine the physical characteristics of normal hardboiled egg and
overcooked in terms of color and texture of egg.
3.
Determine the mitotic index of normal hardboiled egg and overcooked
hardboiled eggs cooked in different time intervals using Allium cepa test.
4.
Determine if there is a significant difference on the characteristics
observed of normal hardboiled egg and overcooked hardboiled eggs
cooked in different time intervals.
D.
Hypotheses
1.
Overcooked hardboiled eggs have the potential to be mutagenic to the
human body.
2.
There is no difference on the characteristics of overcooked hardboiled
eggs cooked in different time intervals.
3.
There is no significant difference on the characteristics that can be
observed in the roots of the Allium cepa with the egg yolk of overcooked
hardboiled eggs cooked at different time intervals.
E.
Significance of the Study
The results of the study serves as a warning for everyone in the community,
whether child or adult, considering the fact that eggs are a major part of the human diet.
This research is especially for those people who love eating eggs and also those who
4
usually overcook eggs but still prefer to eat them, thinking that it would be a waste. The
result that eggs overcooked 2 minutes and 5 minutes beyond normal cooking time have
the potential to be mutagenic also alerts everyone to be careful not to overcook eggs.
Many people still insist for overcooked eggs to be safe but some say that they are not.
This research can confirm that particular argument. Researchers also can benefit from
this study, especially those who are focused in food nutrition and in dietetics.
F.
Scope and Limitations
The method used in cooking the eggs in this study is limited to boiling. Only the
yolks were used in the study. There were three different time intervals (including the
normally-cooked egg) and only two eggs were cooked in each interval. The Allium cepa
test was used to determine if the eggs are mutagenic for each time interval. The roots tips
in each bulb were examined, and then the microscopic conditions of the cells of the roots
in each time interval were observed, recorded, and compared using statistical tools.
Chemical analysis of the egg was not included in the study. The study is to be conducted
in MSU-IIT IDS Chemistry Laboratory during the school year 2011 – 2012.
G.
Definition of Terms
Allium cepa
Scientific name for onion bulb, which is the test
subject of the study.
Allium cepa test
Efficient test for chemical, genotoxicity, and
mutagenicity screening.
5
Egg yolk
The round yellow portion of the hen egg, which will
be used as the test subject.
Hardboiled cooking
The method used in cooking the eggs.
Mitotic Index
Percentage/part of the total cells counted which are
in mitotic phase; the dependent variable of the
study.
Mutagenicity
The capability of a substance to change genetic
structure in subsequent generations.
Normally Cooked
Food cooked under standard conditions. It is one of
the treatments in the study.
Overcooked
Food cooked beyond the normal conditions. It is
also one of the treatments in the study.
6
CHAPTER II
REVIEW OF RELATED LITERATURE AND STUDIES
Hen eggs are a major article of diet in most developed countries; average
consumption varies between one and seven eggs per week, the highest rates being in
Canada and in the United States (Encyclopedia Americana, 2005). An analysis by the
International Egg Commission’s economist Peter van Horne, presented in the
Commission's International Egg Market Annual Review, broadly reveals that in Europe,
egg consumption falls between 150 and 300 eggs. Uptake is particularly low in Finland
and Ireland but high in Denmark and Hungary. Outside Europe, consumption is
spectacularly high at more than 300 eggs per person in Japan, China and Mexico. While
the uptake/person is low in India, at just 48 eggs, when this figure is applied to the large
human population of 1.2 billion, this country becomes the third largest hen egg producer
in the world, behind China and the USA! (The Poultry Site, 2009)
Mr. van Horne stresses that many factors influence egg consumption levels
including culture, tradition, income and religion. On the aspect of income he has shown
that, in many countries (north-west Europe, Canada, New Zealand and Australia) where
the gross national income per person ranges between US$20,000 and $40,000, egg uptake
is between 150 and 300 per person per year (The Poultry Site, 2009).
Hen eggs consist of approximately 66% water and 34% solids, the solids being
composed of 66% organic matter and 34% inorganic matter. The main organic
7
components are proteins (12%), lipids (10.5%), and carbohydrates (1%). (Encyclopedia
Americana, 2005)
An egg has two main parts: the albumen and the yolk. The egg white is known as
the albumen. Four alternating layers of thick and thin albumen contain approximately 40
different proteins, the main components of the egg white in addition to water. The yolk
contains less water and more protein than the white, some fat, and most of the vitamins
and minerals of the egg. These include iron, vitamin A, vitamin D, phosphorous, calcium,
thiamine, and riboflavin. (The Accidental Scientist, 2011)
If a boiled egg is overcooked, a greenish ring sometimes appears around the egg
yolk due to the iron and sulfur compounds in the egg. It can also occur when there is an
abundance of iron in the cooking water. The green ring does not affect the egg’s taste;
overcooking, however, harms the quality of the protein (Egg Safety Center, 2011).
Figure 1. A picture of an overcooked egg*
Good texture and flavor are obtained by taking care not to overcook the eggs,
which coagulates their proteins and generates too much hydrogen sulfide. (McGee, 2004)
Hydrogen sulfide is the chemical compound with the formula 𝐻2 S. It is a
colorless, very poisonous, flammable gas with its foul odor of rotten eggs noticeable at
8
concentrations as low as 0.00047 ppm. It often results from the bacterial breakdown of
organic matter in the absence of oxygen, such as in swamps and sewers; this process is
commonly known as anaerobic digestion. It also occurs in volcanic gases, natural gas and
some well waters. The human body produces small amounts of 𝐻2 S and uses it as a
signaling molecule. (Wikipedia, 2011)
A study shows that hydrogen sulfide 𝐻2 S, a metabolic end product of sulfatereducing bacteria, represents a genotoxic insult to the colonic epithelium, which may also
be linked with chronic disorders such as ulcerative colitis and colorectal cancer. 𝐻2 S was
genotoxic in a concentration range from 250 to 2,000 microM, which is similar to
concentrations found in the large intestine. Overall, the study demonstrates that 𝐻2 S
modulates the expression of genes involved in cell-cycle progression and triggers both
inflammatory and DNA repair responses. (Attene-Ramos, 2010)
However, in a retrospective epidemiological study using the cancer registry from
1981 to 1990, Bates et al. (1998) evaluated the risk of cancer to known target organ
systems of hydrogen sulfide toxicity in residents of Rotorua, a New Zealand city that uses
geothermal energy for industrial and domestic heating purposes. The exposures to
hydrogen sulfide and mercury from geothermal sources could have a health impact. A
significantly increased risk of nasal cancers was found among Rotorua residents
compared with the rest of the population of New Zealand. However, this is a rare cancer,
and this finding was based on only four cases. Because the population of Rotorua has a
higher percentage of Maoris than the rest of New Zealand, these researchers also
examined their data stratified by ethnicity and sex and found a significantly increased risk
of cancers of the trachea, bronchus, and lung among female Maoris in Rotorua compared
9
with female Maoris in the rest of New Zealand. Differences in smoking history between
the two populations were not sufficient to explain the observed differences in risk. The
authors concluded that there are inadequate data on exposure to permit conclusions on
possible causal relationships between hydrogen sulfide and cancer incidence. In total, it is
not possible to evaluate the carcinogenic potential of hydrogen sulfide on the basis of the
human studies. (Chou, 2003)
Germ cell mutagens/genotoxins are substances that cause heritable (passed on to
progeny) changes in the genetic material in germ cells, namely spermatocytes or oocytes.
The term mutagen refers to a substance that induces transmissible changes in DNA
structure involving a single gene or a group of genes. Genotoxins are a broader category
of substances that induce changes to the structure or number of genes via chemical
interaction with DNA and/or non-DNA targets. The term genotoxicity is generally used
unless a specific assay for mutations is being discussed. (The Humane Society of the
United States and Procter & Gamble, 2011)
A mutagen (Latin, literally origin of change) is a physical or chemical agent that
changes the genetic material, usually DNA, of an organism and thus increases the
frequency of mutations above the natural background level. As many mutations
cause cancer, mutagens are typically also carcinogens. Not all mutations are caused by
mutagens: so-called "spontaneous mutations" occur due to errors in DNA replication,
repair and recombination. The first mutagens to be identified were carcinogens,
or cancer-causing substances. Early physicians detected tumors in patients more than
2,000 years before the discovery of chromosomes and DNA. (Wikipedia, 2011)
Carcinogens lead to carcinogenesis or oncogenesis or tumorigenesis, which is literally the
10
creation of cancer. It is a process by which normal cells are transformed into cancer cells.
It is characterized by a progression of changes on cellular and genetic level that
ultimately reprogram a cell to undergo uncontrolled cell division, thus forming a
malignant mass. (Wikipedia, 2005) Also, mutagens cause changes to the DNA that can
affect the transcription and replication of the DNA, which in severe cases can lead to cell
death. The mutagen produces mutations in the DNA, and deleterious mutation can result
in aberrant, impaired or loss of function for a particular gene, and accumulation of
mutations may lead to cancer. (Wikipedia, 2011)
The chemicals that act on DNA fall into three classes: (1) chemicals that resemble
DNA nucleotides but pair incorrectly when they are incorporated into DNA. (2)
Chemicals that remove the amino group from adenine or cytosine; and (3) chemicals that
add hydrocarbon groups to nucleotide. In the first class, some of the new AIDS
chemotherapeutic drugs are analogs of nitrogenous bases that are inserted into the
viral/infected cell DNA. The second and third classes cause the nitrogenous bases in
nucleotides to pair incorrectly. (Raven, 1996)
On the other hand, among the plant test systems Allium cepaL. is the most
commonly used plant for studies of chromosomal aberrations: its bulbs produce a large
number of roots in a short time interval (the cell cycle is approximately 20 h) and the
chromosomes are of a relatively large size (Evandri et al., 2000). Higher plants including
ferns, club mosses, flowering plants, conifers, and other gymnosperms, are recognized as
excellent genetic models to detect environmental mutagens and are frequently used in
monitoring studies. Among the plant species, Allium cepa or onion has been used to
evaluate DNA damages, such as chromosome aberrations and disturbances in the mitotic
11
cycle. It has been used to this day to assess a great number of chemical agents, which
contributes to its increasing application in environmental monitoring. The Allium cepa
test is characterized as a low-cost test. Since the target is DNA, common to all organisms,
Allium cepa represents consistent results that may serve as a warning to other biological
systems. (Morales, 2009)
Also, the root tip is often the first part of any plant that is likely to come into
contact with chemicals and pollutants found in soil and water supplies. It has been shown
that Allium cepa is particularly sensitive to the harmful effects of environmental
contaminants. Allium test is an important tool for environmental monitoring studies. The
allium test provides a rapid screening procedure for chemicals, pollutants, contaminants,
and so on that may represent environmental hazards. Root growth inhibition and adverse
effects on chromosomes provide an indication of likely toxicity. (Fiskesjo, 2007) A.
cepatest permits the study of different, but complementary aspects of food contamination
and could be helpful to better evaluate cancer hazards related diets (Feretti et al., 2007).
A research was carried out used the Allium cepa test-system to study the
cytotoxicity, genotoxicity and mutagenicity of the Monjolinho River to evaluate the
effects of domestic and industrial effluents in different seasons of the year. In the summer
and intermediate seasons, chromosome aberration, micronuclei, cell death and inhibition
of the mitotic index were observed in water samples collected at different sites (Bianchi,
2010).
In the test, mitotic index is to be observed. Mitotic index reflects cell proliferation
and is regarded as an important parameter when determining the rate of plant root growth
(Liu et al. 1992). The mitotic index and replication index are used as indicators of
12
adequate cell proliferation (Gadano et al. 2002), which can be measured using Allium
cepa. The chromosomal aberration method in Allium roots is validated by the
International Program on Chemical Safety (IPCS), as an efficient test for the analysis and
in situ monitoring of the genotoxicity of environmental substances.
Studies have shown the formation of mutagens in the major cooked protein-rich
foods in the US diet studied in the Ames-Salmonella typhimurium test. Eggs and egg
products produced mutagens only after cooking at high temperatures (the yolk to a
greater extent than the white). Eggs cooked at temperatures above 225°C were said to
have contained mutagens detectable by the Ames/Salmonella assay. (Bjeldanes, 1982)
The researchers will study the mutagenicity of overcooked eggs using the Allium
cepa test. The study is different from other studies in the sense that the test that used is
different from the test used in the existing studies on the mutagenicity of egg. As being
cited in the references, one study used the Ames-Salmonella typhimurium test. However,
Salmonella typhimurium is a prokaryote; therefore it is not a perfect model for humans
(Wikipedia, 2011).
13
CHAPTER III
METHODOLOGY
A.
Research Design
The study used completely randomized design to determine if overcooked
hardboiled eggs have the potential to be mutagenic by comparing the condition of the
roots applied with the yolks of the eggs cooked at four different time intervals. The
research employed the Allium cepa test where onion served as a test subject for
mutagenicity. After 3-4 days of pretreatment, three bulbs were chosen to be subjected to
the treatments through multistage sampling. After exposing some of the roots to the
treatments, microscopy was done. The observations were compared using statistical tools.
B.
Materials and Equipment
Materials

Blade (1 box)

Match (1 box)

Glasses (20 pcs)

Tissue paper

Masking tape

Toothpick (1 box)
Equipments:

Alcohol Lamp

Stove

Beakers

Wash bottle

Graduated cylinder

Cover slips

Microscope

Dropper

Spot plates

Erlenmeyer flask

Stirring rod

Glass slides
14
Chemicals:


C.
Fixative solution (1 part glacial

Acetocarmine (10 mL)
acetic acid, 3 parts methanol)

1M hydrochloric aciD
0.05% colchicine
Experimental Set-up
Table 1. Treatments Done to the Hardboiled Egg
TREATMENT
DESCRIPTION
A
Cooked at normal cooking time (15 minutes)
B
Cooked 2 minutes beyond normal cooking time
C
Cooked 5 minutes beyond normal cooking time
Table 2. List of the Different Sample of Roots
SAMPLE
DESCRIPTION
A
Exposed to Treatment A
B
Exposed to Treatment B
C
Exposed to Treatment C
Positive Control
exposed to 0.05% colchicine
Negative Control
exposed to distilled water
D.
General Procedure
Preparation of Materials
Six eggs were bought from the market, two of which were cooked at normal
cooking time, two cooked 2 minutes beyond normal cooking time, and two cooked 5
minutes beyond normal cooking time. Twenty glasses of the same size were bought,
washed, and dried. These glasses were used for the Allium cepa test. Multistage sampling
was used in choosing the onion bulbs and eggs of approximately the same size that were
purchased from the market.
Preparation of Onion Samples
15
The test subject that was used in the Allium cepa test is the onion. Twenty onions
were purchased in the market. These onions should be fresh, mold-free, and mature onion
bulbs – approximately of the same size. The outer skin of each onion was peeled and the
roots that already grew were removed for new roots to grow.
Pretreatment of Onion Samples
Twenty glasses with distilled water were used to grow twenty onion bulbs. Thus,
the set-up was composed of 20 glasses each with onion bulb on its top. In particular, one
glass with water and onion on its top looked like the figure below:
Onion Bulb
Toothpick
Onion Roots
Distilled Water
Figure 2. Allium cepa Test Setup
Identification of Normal Cooking Time for Hardboiled Egg
Preparation of Egg Samples
The six eggs were divided into three groups, two eggs per group. Each group was
exposed in three different treatments: one group was cooked at normal cooking time, the
other 2 minutes beyond normal cooking time, and the last group 5 minutes beyond
16
normal cooking time, which is considered overcooked. The eggs were cooked after the
pre-treatment of the Allium cepa. Then, the yolks of the eggs were used to make a
solution with water. The ‘yolk solution’ created was used in the Allium cepa test.
Allium cepa Test
After the pre-treatment, multistage sampling was used to select three onion bulbs
which have grown well and are suitable for the test. Through
random
sampling,
five
distinct roots from each onion bulb were chosen to be exposed in the five different
treatments mentioned in the experimental set-up. Three replicate onion bulbs for each
treatment were prepared.
The root cap of the roots was removed, and the root tip (embryonic
region/meristem) was cut since it was the part of the root to be exposed to the treatments
and to be observed under the microscope.
Three spot plates were prepared for staining. Each root tip was first rinsed with
distilled water to remove dirt and impurities. Next, the root tip was dipped in its
corresponding treatment solution in the first “spot” of the plate for 45 minutes. Then, it
was dipped in distilled water found in the second spot. Immediately, it was moved to the
third spot that contained the fixative solution for 30 minutes for the purpose of preserving
or ‘freezing’ the cells, ceasing it to move or divide more. After dipping it in distilled
water, it was moved to the spot with 1 M HCl and left for 10-12 minutes in order to
soften the root tip, making it easier to squash it. Again, the root tip was moved to the
fourth spot which has distilled water.
Staining Process
17
Afterwards, the root tip was placed in a clean glass slide with a few amount of
HCl to prevent it from drying. One to two drops of acetocarmine was added to it for stain.
It was mashed using another glass slide. When it was already squashed, the glass slide
with the specimen was heated for 5-10 minutes by placing it one and a half inches above
a lighted alcohol lamp to allow better absorption of the stain. After heating, the specimen
was left for 5 minutes for more absorption, but was checked so that it must not dry out or
dehydrate. A cover slip was then placed on top of the root cells slowly with a toothpick.
After that, excess stain was removed by placing tissue paper folded fourths on the cover
glass and pressing down slightly with the thumb.
Microscopy
After the staining process was done, the glass slide with the specimen was placed
under the microscope. Mitotic cells and interphase cells in at least 400 cells were
counted, and possible chromosomal aberrations were scanned and recorded.
Disposal of Onion Bulbs
Onion bulbs smell bad when rotten. The onion bulbs were disposed properly in a
compost pit after use.
E.
Procedures in Data Gathering
The microscopic analysis includes mitotic index and chromosomal aberrations in
anaphase and early telophase cell score. Mitotic index in at least 400 cells in each root tip
was recorded in Table 3, 4, and 5 for replicates A, B & C, respectively.
The mitotic index was computed by dividing the # of mitotic cells to the total # of
cells counted (interphase cells + mitotic cells), then multiplied by 100%.
M. Index = (number of mitotic cells / total number of cells counted) x 100%
18
Fig. 3: Example of Chromosomal Stickiness (Barberio)
Table 3. Mitotic Index of the Roots (over 400 cells) in Replicate A/B/C
# of Cells in Division (over 400 cells)
Treatment
Mitotic
Interphase
Total
Mitotic index
cells
cells
cells
(%)
Positive Control
Negative Control
Normally Cooked
Overcooked
(+2 minutes)
Overcooked (+5
minutes)
Table 4. Mitotic Index of the Roots (over 400 cells) in Replicate B
Treatment
# of Cells in Division (over 400 cells)
Mitotic
Interphase
Total
Mitotic index
cells
cells
Cells
(%)
Positive Control
Negative Control
Normally Cooked
Overcooked
(+2 minutes)
Overcooked (+5
minutes)
Table 5. Mitotic Index of the Roots (over 400 cells) in Replicate C
Treatment
# of Cells in Division (over 400 cells)
Mitotic
Interphase
Total
Mitotic index
cells
cells
cells
(%)
19
Positive Control
Negative Control
Normally Cooked
Overcooked
(+2 minutes)
Overcooked (+5
minutes)
F.
Statistical Tool for Data Analysis
For data analysis, one-way Analysis of Variance (ANOVA) was used to identify
if one of the mitotic indices differ significantly or not. Post-hoc test was then used in
determining which among the mitotic indices of the treatments differ from the rest, and if
the difference is significant.
Figure 4. Flowchart of the General Procedure
CHAPTER IV
RESULTS AND DISCUSSIONS
In this chapter, results and discussions of the data gathered are presented. Allium
cepa test was employed to find out if there overcooked hardboiled eggs have the potential
to be mutagenic. Using the mitotic indices obtained from the experiment (microscopy),
the researchers were able to compare the potential mutagenicity of the eggs cooked in
different time intervals.
A. Data Gathered
The following tables contain the recorded mitotic cells, interphase cells, total cells
and mitotic indices of the roots from the three different replicates exposed to the five
treatments.
21
Table 6. Mitotic Index of the Roots (over 400 cells) in Replicate A
Treatment
# of Cells in Division (over 400 cells)
Mitotic Interphase
Total
M. Index
Cells
Cells
Cells
(%)
Positive
Control
Negative
Control
Normally
Cooked
Overcooked
(+2 minutes)
Overcooked
(+5 minutes)
210
343
553
37.97468354
146
298
444
32.88288288
167
274
441
37.86848073
220
209
429
51.28205128
223
185
408
54.65686275
Table 7. Mitotic Index of the Roots (over 400 cells) in Replicate B
Treatment
# of Cells in Division (over 400 cells)
Mitotic Interphase
Total
M. Index
Cells
Cells
Cells
(%)
212
229
441
48.07256236
Positive
Control
153
320
473
32.34672304
Negative
Control
158
279
437
36.15560641
Normally
Cooked
408
144
552
73.91304348
Overcooked
(+2 minutes)
390
219
609
64.03940887
Overcooked
(+5 minutes)
22
Table 8. Mitotic Index of the Roots (over 400 cells) in Replicate C
Treatment
# of Cells in Division (over 400 cells)
Mitotic Interphase
Total
M. Index
Cells
Cells
Cells
(%)
234
253
487
48.04928131
Positive
Control
137
313
450
30.44444444
Negative
Control
132
280
412
32.03883495
Normally
Cooked
275
152
427
64.40281030
Overcooked
(+2 minutes)
330
214
544
60.66176471
Overcooked
(+5 minutes)
Table 4, 5, and 6 show the # of mitotic cells, interphase cells, total cells, and the
mitotic index (in percent) of each treatment in Replicate A, B, and C, respectively. The
mitotic index was computed by dividing the # of mitotic cells to the total # of cells
counted (interphase cells + mitotic cells), then multiplied by 100%.
M. Index = (number of mitotic cells / total number of cells counted) x 100%
The following is the bar graph of the mitotic indices of the roots:
23
80.00000
75.00000
70.00000
Mitotic ndex (%)
65.00000
60.00000
Replicate 1
55.00000
Replicate 2
50.00000
Replicate 3
45.00000
40.00000
35.00000
30.00000
Positive
Control
Negative
Control
Normally
Cooked
Overcooked
(+ 2 mins)
Overcooked
(+ 5 mins)
Treatments
Figure 5. Mitotic Indices of the Onion Roots Exposed to the Different Treatments
The horizontal axis corresponds to the treatments where the meristems were
exposed while the vertical axis corresponds to the mitotic index in percentage. The bars
correspond to the replicates where the meristems of roots were obtained: blue for
replicate 1, red for replicate 2, and green for replicate 3. Notice the difference between
the mitotic indices of the roots. The mitotic indices of the roots of the replicates exposed
to the yolk solution of eggs overcooked two minutes beyond normal cooking time were
higher compared to that of the positive control, negative control, and of the yolk solution
with normally-cooked egg. The same observations were noticeable of that of the mitotic
indices of the roots of the replicates exposed to the yolk solution of eggs overcooked five
minutes beyond normal cooking time. Also, several chromosomal aberrations were
detected during the microscopy.
24
Figure 6. Chromosomal aberrations detected in Replicate 2 dipped in yolk solution
with yolk from egg overcooked 2 mins. beyond normal cooking time: (A)
chromosomal bridging during early anaphase and (B) chromosomal stickiness.
This can be explained by carcinogenicity, a type of mutagenicity where the
substance has the qualities to cause cancer to the cells and therefore causes uncontrolled
cell division. There is a possibility that the chemicals/compounds formed in overcooking
the eggs have caused the dramatic rise of the mitotic index.
25
B. One-Way ANOVA
To verify this, one-way Analysis of Variance (ANOVA) was applied to test the
null hypothesis that there is no significant difference between the mitotic indices of the
roots exposed to positive control (0.05% colchicine), negative control (distilled water),
normally-cooked egg, overcooked egg (2 minutes beyond normal cooking time), and
overcooked egg (5 minutes beyond normal cooking time) against the alternative
hypothesis that there is one of the mitotic indices that differ from the others. The test is
done using the mitotic indices obtained from the experiment with 10 degrees of freedom
and 0.05 level of significance. The results of the one-way ANOVA is found in Appendix
E.
Group 1 refers to the mitotic indices of the roots exposed to positive control.
Group 2 refers to the mitotic indices of the roots exposed to negative control. Group 3
pertains to the mitotic indices of the roots exposed to the yolk solution with normallycooked egg. Group 4 speaks of the mitotic indices of the roots exposed to the yolk
solution with overcooked egg (2 minutes beyond normal cooking time). Group 5 relates
to the mitotic indices of the roots exposed to the yolk solution with overcooked egg (5
minutes beyond normal cooking time).
According to the results of the one-way ANOVA, it was shown in the ANOVA
table that the p-value is equal to 0.0003, which is less than the level of significance α=
0.05. Thus, the null hypothesis is rejected. Therefore, there is sufficient evidence to
support the claim that at least one of the mitotic indices differ significantly from the
others. The post-hoc analysis was used to determine which of the groups differ.
C. Post-hoc Analysis
26
Referring to the results of the post-hoc analysis (p-values for pairwise t-tests) found in
Appendix E, group 1 (positive control) has a significant difference with group 2 (negative
control) since their p-value= 0.0312 which is less than α= 0.05. Group 5 (exposed to egg
cooked 5 minutes beyond normal cooking time) has a significant difference with group 2
since their p-value= 0.0003 is less than α= 0.05. Group 5 also has a significant difference
with group 3 since their p-value= 0.0007 is less than α= 0.05. It also has a significant
difference with group 1 since p-value= 0.0145 is less than α= 0.05. Group 4 (exposed to
egg cooked 2 minutes beyond normal cooking time) has a significant difference with
group 1 since their p-value= 0.0047 is less than α= 0.05. Group 4 also has a significant
difference with group 3 since their p-value= 0.0003 is less than α= 0.05. Similarly, it has
a significant difference with group 2 because their p-value= 0.0001 is less than α= 0.05.
Therefore, the researchers can conclude that group 1, 4, and 5 differ from the rest of the
groups. In other words, there is enough evidence to say that the mitotic indices of the
roots exposed to yolk solution containing the yolk of the eggs cooked 2 minutes and 5
minutes beyond normal cooking time differ among the others.
CHAPTER V
CONCLUSION AND RECOMMENDATIONS
A. Summary
Each of the mitotic indices of the roots of the replicates exposed to every
treatment namely positive control (0.05% colchicine), negative control (distilled
water), and the yolk solutions with yolk from (a) normally-cooked egg, (b) egg
overcooked 2 minutes beyond standard cooking time, and (c) egg overcooked 5
minutes beyond standard cooking time were used in the test. One-way ANOVA
was the statistical tool used in the test. The test on the analysis of variance
(ANOVA) at 0.05 level of significance (α) resulted to the acceptance of the
alternative hypothesis that there is at least one of the mitotic indices that differ
significantly from the others.
B. Conclusion
1. The Allium cepa test shows that:
a. There is a significant difference on the characteristics that can be observed in
the roots of the Allium cepa with the egg yolk of overcooked hardboiled eggs
cooked at different time intervals.
2. There is a significant difference on the characteristics of overcooked hardboiled
eggs cooked in different time intervals.
28
3. Overcooked hardboiled eggs have the potential to be mutagenic to the human
body.
C. Recommendations
The researchers recommend the following:
a. For those people who still consume overcooked eggs, it is better not to
do so to prevent possible disorders to develop. When cooking eggs,
make sure to cook it properly to prevent overcooking and wasting of
money, time, and food.
b. For future researchers, it is recommended to do more study about this
research using other tests like Drosophila, etc.
c. For future researchers, it is also recommended to test the possible
effects of raw eggs.
29
REFERENCES:
A. BOOKS
Raven, P. & Johnson, G. (1996). Biology (4thed.). Molecular Genetics (p. 404).
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Dr
C.-H. Selene J. Chou. (2003). Concise International Chemical
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(2005) Encyclopedia Americana Vol 9.USA : Scholastic Library Publishing
Incorporated
(2002) Grolier Encyclopedia of Knowledge. USA: Grolier Incorporated
C. PERIODICALS
L.F. Bjeldanes, M.M. Morris, J.S. Felton, S. Healy, D. Stuermer, P. Berry,
H.
Timourian& F.T. Hatch (1982).Mutagens from the cooking
of food. II.
Survey by Ames/Salmonella test of mutagen formation
in the major protein- rich foods of the American diet.
Retrieved July
20, 2011 from Food and
Chemical Toxicology Vol.20 Issue 4
Attene-Ramos
MS, Nava
GM, Muellner
MG, Wagner
ED, Plewa, MJ, Gaskins HR.(2010).
DNA damage and
toxicogenomic analyses of hydrogen sulfide in
human intestinal epithelial FHs 74 Int cells. Retrieved September 8, 2011
from Environmental and Molecular Mutagenesis Volume 51 Issue 4 page
304-314)
Bianchi J, Espindola EL, Marin-Morales MA.(2010). Genotoxicity and mutagenicity of
water samples from the Monjolinho River (Brazil) after receiving
untreated effluents.Retrieved September 22, 2011 from Ecotoxicology and
Environmental Safety 2011 May;74(4):826-33
30
Feretti D., Zerbini I., Zani C., Ceretti E., Moretti M., Monarca S. (2007).
Allium cepachromosome aberration and micronucleus tests applied to
study genotoxicity of extracts from pesticide-treated vegetables and
grapes.Retrieved from Food Additives and Contaminants. 24(6): 561572.
Evandri M.G., Tucci P. and Bolle P. (2000).Toxicological evaluation of
commercial mineral water bottled in polyethylene terephthalate: a
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Addit.Contam.17: 1037-1045.
D. INTERNET ACCESS
Leme, D. & Marin M. (July 2, 2009) Allium cepa test in environmental
monitoring: a review on its application. Retrieved July 20, 2011 from
http://www.ncbi.nlm.nih.gov/pubmed/19577002
Dr. Fiskesjo, G. (2006). Allium test. Retrieved July 20, 2011 from
http://masters.donntu.edu.ua/2006/feht/fomina/library/article5.htm
The Accidental Scientist: Science of Cooking. Anatomy of an Egg. Retrieved
July 20,
2011
from
http://www.exploratorium.edu/cooking/eggs/eggcomposition.html
Egg Safety Center.Frequently Asked Questions About Egg Food Safety: What is
an adequate Temperature to cook an egg? Retrieved July 20, 2011 from
http://web.archive.org/web/20080822035116/http://www.eggsafety.org/f_
a
_q.htm#8
Wikipedia: the Free Encyclopedia (2011). Ames test.Retrieved October 17,
2011 from http://en.wikipedia.org/wiki/Ames_test#Limitations
The Poultry Site.(2009) Good news on Global Egg Consumption. Retrieved
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17,
2011
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Olver, Lynne. (2000). Food Timeline: FAQ's: Eggs. Retrieved October 17,
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The Humane Society of the United States and Procter & Gamble
(2011).Genotoxicity.
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September
21,
2011
fromhttp://www.alttox.org/ttrc/toxicity-tests/genotoxicity/
31
Wikipedia: The Free Encyclopedia (2004). Genotoxicity.Retrieved September
21, 2011 fromhttp://en.wikipedia.org/wiki/Genotoxicity
Economics Newspaper (2011).Demand for Eggs and Poultry – organic Food
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Wikipedia: The Free Encyclopedia (2011). Mutagen. Retrieved September 8,
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9, 2012 from http://en.wikipedia.org/wiki/Carcinogenesis
32
APPENDIX A
Pretreatment of Onion Bulbs
Attaching toothpicks in the onion bulb
The Twenty Onion Bulbs in pretreatment
APPENDIX B
33
Preparation of Materials
Some Materials for Microscopy and
Some Chemicals and Materials for
Staining
Staining and Microscopy
The yolk solutions
34
APPENDIX C
Staining Process
Placing Cover Slip on the Specimen
Staining Process
Mashing the Root tip
APPENDIX D
Some Pictures in Microscopy
Chromosomal Bridging Detected in Root tip of R2 dipped in solution with yolk from
egg cooked 2 minutes beyond standard cooking time
Normal Series of Interphase Cells inside Root tip of R2 with negative control
36
APPENDIX E
ANOVA TABLE AND POST-HOC TEST
One factor ANOVA
ANOVA table
Source
Treatment
Error
Total
Mean
n
Std. Dev
44.69884
3
5.823304
Group 1
31.89135
3
1.281413
Group 2
35.35431
3
2.996290
Group 3
63.19930
3
11.363396
Group 4
59.78601
46.98596
3
15
4.752184
14.085817
Group 5
Total
SS
2,385.261840
392.481336
2,777.743176
df
4
10
14
MS
596.3154600
39.2481336
F
15.19
p-value
.0003
Post hoc analysis
p-values for pairwise t-tests
Group 2
Group 3
Group 1
Group 5
Group 4
31.89135
35.35431
44.69884
59.78601
63.19930
Group 2
31.89135
Group 3
35.35431
Group 1
44.69884
Group 5
59.78601
Group 4
63.19930
.5138
.0312
.0003
.0001
.0977
.0007
.0003
.0145
.0047
.5197
Group 2
Group 3
Group 1
Group 5
Group 4
31.89135
35.35431
44.69884
59.78601
63.19930
Tukey simultaneous comparison t-values (d.f. = 10)
Group 2
31.89135
Group 3
35.35431
0.68
Group 1
44.69884
2.50
1.83
Group 5
59.78601
5.45
4.78
2.95
Group 4
63.19930
6.12
5.44
3.62
critical values for experimentwise error rate:
0.05
0.01
3.29
4.34
0.67