Download Pesticide Effects on Yeast Survivorship

Pesticide Effects on Yeast
Survivorship
JACK LEECH
PITTSBURGH CENTRAL CATHOLIC
HIGH SCHOOL
GRADE 11
2010
Pesticides
 Pesticides include any chemical, antibacterial,
biological agent, or other similar substance used to
kill or repel unwanted species.
 Grouped in other categories, such as bactericides,
fungicides, herbicides, rodenticides, and insecticides.
 Insecticides are grouped under other titles such as
ovicides, larvicides, and adulticides.
Concern Insect Killing Soap
 Main ingredients include potassium salts of fatty
acids which pierce the cell membrane, causing the
contents to leak out and dehydrate.
 Works by penetrating and destroying the outer shells
of soft-bodied insect pests, resulting in dehydration
and death within hours of contact.
 Concentration of 1.03mL (3.5oz) per
gallon of water.
Ortho Rose Pride
 This insecticide uses acephate as its main active
ingredients.
 Acephate prevents the neurotransmitter
acetylcholine from being degraded and destroys the
nervous system.
Safer Brand Yard and Garden
 Contains pyrethrins and also potassium salts of fatty
acids.
 Pyrethrins attack the insect’s nervous system by
depolarizing a normally negatively charged cell, causing
the nerve cell to constantly trigger action potentials and
keep firing.
 Potassium salts weaken the insect’s protective outer
shell.
Non-Target Effects of Pesticides
 Potassium Salts of Fatty Acids:
 Slightly toxic through skin exposure, mildly toxic to plant leaves which
hold the soap to its surface.
 Highly toxic to aquatic invertebrates.
 Soil half-life of one day.
 Acephate:
 Very low toxicity to most animals, but extremely toxic to beneficial
insects (honey bees).
 Soil half-life of about half a day.
 Pyrethrins:
 Highly toxic to aquatic vertebrates that use skin as a primary means of
absorption.
 Soil half-life of 12 days.
Yeast
 Saccharomyces cerevisiae.
 Easy to manipulate in the laboratory.
 Most commonly studied cell.
 Very similar in structure and biochemistry to
more complex eukaryotes, including human
cells.
 Used as a eukaryotic cell model to mimic nontarget cells.
 Buds in single colonies, can be easily counted.
Purpose
 Determine if any of the pesticides will
have a significant effect on the
survivorship of yeast colonies.
Hypothesis
 Null-the pesticides used will have no
significant effect on yeast survivorship.
 Alternative-the pesticides will have a
significant effect on yeast survivorship.
Materials
 YEPD agar plates (1% yeast extract, 2% glucose, 1.5% agar)
 YEPD media (1% yeast extract, 2% peptone, 2% glucose)
 Sterile capped test tube sterile dilution fluid (SDF) (10 mM
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KH2PO4, 10 mM K2HPO4, 1 mM MgSO4, 0.1 mM CaCl2, 100 mM
NaCl)
Concern Insect Killing Soap
Ortho Rose Pride Insecticide
Safer Brand Yard and Garden Spray
Micropipette
Permanent marker
Plate spreader
Ethanol
Bunsen Burner
Klett Spectrophotometer
Incubator
Sidearm flask
Procedure (Continuous Contact Exposure)
1. S.c. yeast was grown overnight in sterile YEPD media.
2. A sample of the overnight culture was added to fresh media in a
sterile sidearm flask.
3. The culture was placed in a incubator (30 C) until a density of 50
Klett spectrophotometer units was reached. This represents a cell
density of approximately 107 cells/mL.
4. 0.1 mL of each variable at concentration of 1X (recommended
dosage) was pipetted onto five plates per variable.
5. The plates were labeled and the agar was allowed to absorb the
variable for thirty minutes.
6. The cell culture was diluted in sterile dilution fluid to a
concentration of approximately 103 cells/mL.
7. After vortexing to evenly suspend cells, 0.1mL aliquots were
removed from the tubes and spread on YEPD plates.
8. The plates were incubated at 30 C for 48 hours.
9. The resulting colonies were counted. Each colony is assumed to
have risen from one cell.
Procedure (Pulse Exposure)
1. S.c. yeast was grown overnight in sterile YEPD media.
2. A sample of the overnight culture was added to fresh media in a
sterile sidearm flask.
3. The culture was placed in a incubator (30 C) until a density of 50
Klett spectrophotometer units was reached. This represents a cell
density of approximately 107 cells/mL.
4. The cell culture was diluted in sterile dilution fluid to a
concentration of approximately 103 cells/mL.
5. Each of the variables was pipetted into the SDF tube to reach the
concentration of 1X, where it remained for thirty minutes.
6. 0.1 mL of the yeast/variable concentration was pipetted onto
five plates per variable.
7. The plates were incubated at 30 C for 48 hours.
8. The resulting colonies were counted. Each colony is assumed to
have risen from one cell.
Yeast Survivorship After Continuous Contact
Exposure
Survivorship After Indirect Exposure
300
250
p=7.65 ^-10
200
Resulting
Colonies 150
Survivorship After
Indirect Exposure
100
50
0
Control Concern
Soap
Ortho
Rose
Pride
Safer
Brand
Groups of Variables
alpha: p = .05
Yeast Survivorship After Pulse Exposure
Survivorship After Direct Exposure
300
250
Resulting
Colonies
p=3.39 ^-11
200
150
Survivorship After
Direct Exposure
100
50
0
Control Concern
Soap
Ortho
Rose
Pride
Safer
Brand
Groups of Variables
alpha: p = .05
Dunnett’s Test
 If t-value is > than the t-critical (3.29), significant
variation.
Dunnett’s Tests Results for Continuous
Contact Exposure
Variables Used
Results
Concern Soap
t-value(9.25)>3.48
(Significant)
Ortho Rose Pride
t-value(14.32)>3.48
(Significant)
Safer Brand
t-value(3.49)>3.48
(Significant)
Not significant if cut-off of .01 is used.
(t-critical of 4.98)
Dunnett’s Tests Results for Pulse Exposure
Variables Used
Results
Concern Soap
t-value(13.14)>3.48
(Significant)
Ortho Rose Pride
t-value(18.44)>3.48
(Significant)
Safer Brand
t-value(9.58)>3.48
(Significant)
Variance Between Similar Variables
Pulse Exposure
Continuous Contact Exposure
Concern Soap- Average: 173.4
Concern Soap-Average: 204.8
Ortho Rose Pride-Average: 130.8
Ortho Rose Pride-Average: 164.2
Safer Brand-Average: 202
Safer Brand-Average: 251
Single Factor ANOVA Results
Concern Soap: p value = .008846 (significant)
Ortho Rose Pride: p value= .008151 (significant)
Safer Brand: p value= .000129 (significant)
Interpretation
 Pulse exposure appeared to have a more significant
effect on yeast survivorship when compared to the
continuous contact exposure based upon the
statistical analysis.
 The continuous contact exposure of Safer Brand
resulted in a significant t-value when the t-critical in
the Dunnett’s test was 3.48, but not 5.22. These tcritical values represent .05 and the more stringent
alpha of .01, respectively.
Conclusions
 Null hypothesis rejected for all trials in both the
pulse and continuous contact exposures.
 However, the result found in the Safer Brand
continuous contact trial, although significant when
compared to the standard cut-off of .05, the result
would not be significant with the more rigid value of
.01.
Limitations and Inconsistencies
 Although no visible contaminants, environmental
contamination could have been a factor.
 Other methods of destruction from the pesticides
could have effected the yeast growth aside of the
main active ingredients.
 Plating was not exactly synchronized, which could
possibly result in varying colony counts.
Continuations
 Use a wider range of pesticides in addition to
insecticides.
 Test the specific ingredients of the pesticide instead of
the entire product.
 Test different concentrations of each variable instead of
the listed recommended concentration.
 A combination of pesticides could be used
simultaneously (synergetic exposures).
Works Cited
 www.planetnatural.com
 www.saferbrand.com
 www.npic.orst.edu
 www.epa.gov