Running head: RESEARCH PROJECT FINAL PROPOSAL

Research Project Proposal: The effect of pesticides and chemicals such as Acetamiprid,
Azoxystrobin, and Imidacloprid on bacteria found fruit and vegetables such as E.coli.
Hunter Galindo
Silva Topchyan
Biomedical Sciences
Period 8
West Career and Technical Academy

Purpose Statement

Running head: RESEARCH PROJECT FINAL PROPOSAL

The effect of pesticides and chemicals such as Acetamiprid, Azoxystrobin, and Imidacloprid on
bacteria found fruit and vegetables such as E.coli .
This research project is designed to test how pesticides/chemicals Acetamiprid,
Azoxystrobin, and Imidacloprid effect commonly found bacteria-E.Coli. Pesticides/chemicals
are a major use in the commercial farming business, which allows extensive production without
the product spoiling due to insects and other factors. This allows farmers to save money on spoil
crops and decrease amount of food wasted. The use of the pesticides/chemicals has had effect on
the contamination of water sources and the health of people and wildlife. This has launched a
major cultural debate of using organic techniques in growing crops to reduce these hazards. The
goal of this research project is to determine if bacteria is affected by pesticides/chemicals. We
will experiment on E.coli and shigella with the use of the pesticides Acetamiprid, Azoxystrobin,
and Imidacloprid which are commonly used in crops. The main objective of this project is to
determine the effects of pesticides on the effect on the bacterias structure, growth, and behavior.
Findings from this project, may figure out if chemicals/pesticides will have a negative or positive
effect in eliminating potential disease causing bacteria found in major commercial crops.
Should this project find that the effect of pesticides decrease the potential growth and
destroy the structure of the bacterium which would protect consumers from potential biohazards.
Should this project find that the effect of pesticides increase the potential growth and further
allow the bacteria to grow immune to the use of chemical which would negatively affect human
health and increase the chance of widespread diseases by making the bacterium resistant to
chemicals that could have possibly be used to kill them. Other environmental factors that could

Running head: RESEARCH PROJECT FINAL PROPOSAL

be tested that would affect the bacterial growth, structure, and behavior would be type of soil,
temperature conditions where bacteria is stored or food is grown in, sunlight that the bacteria is
exposed in, water that the plant was used for watering, etc.

Methodology

Materials:
E.coli bacteria
Acetamiprid pesticide
Imidacloprid pesticide
Azoxystrobin pesticide
petri dishes
gloves
goggles
spray bottles
surgical/gas mask/fume hood
inoculation loop
bunsen burner
incubator
compound light microscope
Safranin
alcohol
Crystal Violet
slides
Iodine
bibulous paper
distilled water
ruler
notebook
calculator
writing utensil

Procedures :
Part I: Conducting experiment
1. Upon receiving the required materials, begin with recording and observing the basics of
the E.coli using standard classification methods. Before opening the bacteria, cleanse the

Running head: RESEARCH PROJECT FINAL PROPOSAL

workplace with spray disinfectant to rid the area of potential contaminants. Record
drawings and other notes using the compound light microscope within your laboratory
journal. Be sure to be precise and thorough.
2. Incubate the E.coli bacteria for 7 days in a 76 degree fahrenheit (average temperature
environment for annual crops in U.S.) environment in three different petri dishes label
them A,B, C, and D.
*END OF DAY 1*

1. Begin observations with describing the colony and content of the petri dish. Begin Gram
staining to observe structure of E.coli .
Get sample for the Gram stain.
Transfer a drop of the sample to be examined on a slide with an inoculation loop. Add a
drop distilled water on the slide and aseptically transfer a small amount of a colony from
the petri dish. You only need a very small amount of sample. Add 1-2 drops of the
sample onto a glass slide. Spread it evenly on the slide to form a thin smear with an
inoculation loop, which can be done by sliding the edge of another glass slide across the
glass slide. Allow it to air dry.
Heat fix the smear, by quickly passing it two to three times through a bunsen burner.
Allow it to air dry.
Flood the smear with crystal violet, wait thirty seconds.
Gently rinse off the crystal violet with distilled water. Do not rinse too much, it will
remove the stain from the bacteria.
Flood the smear with iodine, Leave three seconds.
Gently rinse iodine with distilled water.
Decolorize by adding alcohol to the smear while holding the slide at an angle to drain the
coloring. Stop when runoff becomes clear, within seconds.
Gently rinse off excess color with distilled water.
Flood the smear with safranin counterstain.
Gently rinse off excess safranin with distilled water. Gently blot slide with bibulous paper
to remove distilled water and leave it to air dry. The Gram stain is done.
1. Once initial observations have been taken, separate the E.coli into four separate petri
dishes. Be sure to divide the bacteria into equal amounts.
2. Use petri dish A. Within the first dish, take the spray bottle containing acetamiprid
pesticide, and spray the bacteria three times to ensure that every inch of the bacteria is
fully washed by the acetamiprid pesticide. Immediately close the lid afterwards to
prevent contamination.
3. Use petri dish B. Within the second petri dish, take the spray bottle containing
imidacloprid pesticide, and spray the bacteria three times to ensure that every inch of the
bacteria is fully washed by the imidacloprid pesticide. Immediately close the lid
afterwards to prevent contamination.
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Running head: RESEARCH PROJECT FINAL PROPOSAL

4. Use petri dish C.Within the third petri dish, take the spray bottle containing azoxystrobin
pesticide, and spray the bacteria three times to ensure that every inch of the bacteria is
fully washed by the imidacloprid pesticide. Immediately close the lid afterwards to
prevent contamination.
5. Leave petri dish D unaltered by any pesticides, as this dish will be used as the control
throughout the course of this experiment. Like the other three, petri dish D will be
growing E.coli as well, just in an unadjusted way. It will be used as a reference point
when taking down observations of the other three dishes.
6. After step 5 has been properly completed, place all four sealed petri dishes in the
incubator.
The temperature of the incubator will be set to 76 degrees fahrenheit.
The bacteria will incubate for 7 days.
1. Clean up the workspace by properly putting instruments away and disposing of
components correctly, re-sterilize the area by using the spray disinfectant once again,
wash hands thoroughly before leaving the laboratory.
2. repeat these steps two more times to have an overall of three trials
*END OF DAY 7*

1. After seven days take out the petri dish and begin collecting results.
2. Collect sample from each of the tree petri dishes and begin gram staining (refer to # 2).
*END OF DAY 14*

Party II: How to Collect and Analyze Data

1. When examining the petri dish, draw the results in your lab note book. Record the
number and type of colony; also, describe any abnormalities of the bacteria such as
locations of where the bacteria grew on the dish or if the bacteria were more spread out or
clustered in the dish.
2. After gram staining, draw the image of the microscope in your lab book and create a
scale bar.This will give one of the variables of what is being tested-size.
Steps for Scale Factor:
1. Once you have drawn an accurate representation of the specimen, at the bottom of your
drawing, draw a reference bar scale of at least 2 cm, under the bar scale write that 2 cm =
20 mm
2. Pick out the largest component of the specimen and measure it out in centimeters, then
draw a bar scale right under it along with its length. Convert its length in centimeters to
millimeters. For example, if the length was 0.7 cm, one would write under the bar scale
0.7 cm = 7 mm.
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Running head: RESEARCH PROJECT FINAL PROPOSAL

3. Now the scale bar variables need to be found: to find the field approximation, you take
the component that has the bar scale under it and physically count how many times it
could fit within that field of view - while taking its size into account. You already have
the length of the specimen. Lastly the approximate field diameter is always 0.13 mm.
4. Lastly, you need to calculate the variables to get the scale factor for that specific
specimen. To do this, the reference bar scale (20mm) is multiplied by the approx. field
diameter (0.13mm) which is divided by the field approx. (17) which is then again divided
by the components length (7mm). Rounding to the hundreds, this new value (in this case
0.021) is multiplied by 1000, thus you have your scale factor - 21UM
1. Also describe and list the type of structure the bacteria has. If recording the data after the
pesticides have been sprayed then the description of any abnormalities must be recorded.

Safety:
Be sure to wear surgical gloves at all times when handling either E.coli bacteria or
pesticides as either one can cause harmful effects if brought into contact with the skin. If
so, wash your hands immediately and intensely, afterwards, get your instructors attention
for further instructions.
Wear goggles at all times as well when experimenting with one or both substances to
avoid getting either into your eyes. Contacts will need to be removed as potential mixing
with pesticide chemicals can cause detrimental effects. If pesticides make contact with
your eyes, flush them thoroughly with distilled water and seek medical attention
immediately
When working with the two types of pesticides, wear a mask that will protect you from
the strong fumes created from the application process to the bacteria - these pesticides are
hazardous and may cause severe harm if inhaled for an extended period of time.
Tie back long hair and avoid loose fitting clothing/jewelry when working with both the
bacteria and pesticides along with running the bunsen burner. Use all fire safety cautions
when using the bunsen burner, i.e never reach across an open flame, never leave a burner
unattended, make sure to turn the gas off when finished with all fire related proceedings,
etc.
Make sure to properly wash your hands upon entering and leaving the laboratory.
Washing your hands before getting started will protect samples from being exposed to
contamination, and washing your hands after all experimental proceedings will protect
you and other students who come into the vicinity from being exposed to potential
harmful agents within the pesticides and bacteria.

Running head: RESEARCH PROJECT FINAL PROPOSAL

Literature Review
Pesticides have been an issue in the consumer market that use it for mass production of crops.
They have included issues such as contaminating water projects and causing health issues in
people and animals that consume it. Bacteria in food has also been an issue causing sickness
such as the E.coli bacteria that plagued fruits and vegetables through time.
Wilson and Otsuki (2004), concerned with how governments regulate food safety and
environmental protection, along with pesticide residue levels, their paper explores the food safety
standards and whether food pesticide levels have an affect on international trade flows. In order
to begin their research, they examined regulatory trade data from 11 Organization for Economic
Cooperation and Development importing countries, along with trade data from 21 exporting
countries, such as Latin America, Asia, and Africa. Their finally findings suggested that an
increase by 1% in regulatory stringency, meaning tighter restrictions on pesticide chlorpyrifo ultimately lead to the decrease in banana imports by 1.63%. Their findings demonstrate a
significant impact on trade to countries that rely on agricultural commodity exports such as
bananas. The teams findings deduced that the lack of international consensus standards and
national regulations gone divergent on pesticides is quite costly.
It has been prominent that the use of chemical inputs, much like common pesticides has
significantly increased agricultural production and productivity; however, negative effects from
this use has increased as well. These effects included damage to the agricultural land, fisheries,
flora, and fauna. Another downfall to the use of such pesticides is the unintentional destruction
of the beneficial predators to pests, which then in turn increases the livelihood of many species
of agricultural pests. In addition, such externalities threaten and increase the mortality and
morbidity of humans due to the exposure to pesticides is being recorded, especially in

Running head: RESEARCH PROJECT FINAL PROPOSAL

developing countries. The cost in damage from these pesticides are large and affect the farmers
returns. However, despite the large costs that the effects bring, farmers continue to spray
pesticides on crops in increasing quantities. This paper examines the harmful effects of
pesticides, along with the statistical data of farmer use of common pesticides.
The Bacteria E.Coli is known to provoke food poisoning in people. These symptoms
include hemorrhagic colitis which is diarrhea with very little stool and large amounts of blood,
this occurs up to three days after eating contaminated food. The ways of being infected by the
E.Coli causing food poisoning include undercooked meat such as hamburgers, unpasteurized
apple juice and cider, raw milk, contaminated water or ice, vegetables fertilized by cow manure,
or spread from person to person. Other ways of catching the bacteria is from raw vegetables or
cool, moist foods (such as potato and egg salads) that are handled after cooking. People who are
infants and the elderly are more prone to getting food poisoning, having sickle cell anemia,
taking antibiotics and other medicines, having pre-existing medical condition, and people who
have weakened immune system such as pregnant women also increase the risk of getting food
poisoning.(UMMC, 2013)
Some organochlorine pesticides prevent the growth of nitrogen-fixing bacteria from
restoring natural nitrogen fertilizer in soil, resulting in lower crop harvest and growth. This has
resulted in the need of for more chemical additives to boost production. This effect is caused
from pesticides interfering with flavonoid directing from the family of leguminous plants like
alfalfa, peas, and soybeans to soil bacteria that fix nitrogen. People assume that endocrine
disruption by pesticides occurs only in humans and animals with estrogen receptors, but we find
there are nontraditional targets affected by pesticides, says Jennifer Fox, a researcher at the
University of Oregon for the Center for Ecology and Evolutionary Biology ( Potera, C. 2007).

Running head: RESEARCH PROJECT FINAL PROPOSAL

Studies from researchers Savino MJ1, Snchez LA, Saguir FM, de Nadra MC. found that
lactic acid bacteria (LAB) secluded from two apples, use arginine at different initial pH values.
The apples exterior contained an average level of bacteria ranging from log 2.49 0.53 to log
3.73 0.48 cfu/ml . Thirty-one strains were able to develop in presence of arginine at low pH.
They were phenotypically and genotypically classified as Lactobacillus, Pediococcus and
Leuconostoc division. Overall, they did not provoke the presence of ammonia from arginine
when cultured in pH level 4.5 or 5.2 in basal medium with arginine.(Savino MJ1, Snchez LA,
Saguir FM, de Nadra MC. ,2012).
In Germany in May, June, and July, 2011 a study was conducted to describe an outbreak
of gastroenteritis and the hemolyticuremic syndrome that is= caused by Shiga-toxinproducing
Escherichia coli Sprouts were identified as the leading cause of the infection, since it carried the
bacteria. The data came from reports in Germany of Shiga-toxinproducing E. coli
gastroenteritis and hemolyticuremic syndrome. Clinical information on the people affected by
the syndrome was given to Hamburg University Medical Center (HUMC) to study the cause of
it. An outbreak case occurred of the hemolyticuremic syndrome or of gastroenteritis in patients
infected by Shiga-toxinproducing E. coli. This outbreak was caused by an abnormal E. coli
strain. The cases of the hemolyticuremic syndrome occurred frequently in adult females. The
hemolyticuremic syndrome occurred in more than 20% of the cases .( Frank C., 2011).

Reference:

Running head: RESEARCH PROJECT FINAL PROPOSAL

Frank, C. (2011, November 16). Epidemic Profile of Shiga-ToxinProducing Escherichia
coli O104:H4 Outbreak in Germany NEJM. Retrieved October 6, 2014, from
http://www.nejm.org/doi/full/10.1056/NEJMoa1106483
Food poisoning. (2013, January 1). Retrieved October 6, 2014, from
http://umm.edu/health/medical/altmed/condition/food-poisoning
Savino MJ1, Snchez LA, Saguir FM, de Nadra MC. (2012, March 23). Lactic acid
bacteria isolated from apples are able to catabolise arginine. Retrieved October 6, 2014, from
http://www.ncbi.nlm.nih.gov/pubmed/22805821
Wilson, J., & Otsuki, T. (n.d.).(2004) To Spray Or Not To Spray: Pesticides, Banana
Exports, And Food Safety. Food Policy, 131-146.
Wilson, C., & Tisdell, C. (n.d.). Why farmers continue to use pesticides despite
environmental, health and sustainability costs. Ecological Economics, 449-462.
Potera, C. (2007, May 28). Agriculture: Pesticides Disrupt Nitrogen Fixation. Retrieved
October 6, 2014, from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2137115/

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