Saddleback
Journal of Biology
Phytotelemata of Nepenthes major, a pitcher plant (see pg. 1)
Published by
Saddleback College Biological Society
Volume 5
Spring 2007
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Editors, Tony Huntley and Steve Teh
TABLE OF CONTENTS
Peer Reviewed Manuscripts from the Biology 3B Class
Author(s)
Giffany Deomampo and
Colleen Worne
Matin Khoshnevis and
Samuel Ollar
Erik Olmos and Avishan
Kolahdouz Nasiri
Mina Hamedanizadeh
and Megan Moshar
Omid Niavarani and
Ross Johnson
Jennifer Wilden and
Hasib Rahmani
Clay Jones and
Andrew Starsiak
Terrance M. Champieux
Amir Farsijani
Amantha Bagdon and
Nam Phuong Nguyen
Christine Nguyen and
Kevin Laitipaya
Kevin Mandala and
Greg Newkirk
Chris Pasvantis and
Callan Taylor
Nasrene Hosseini and
Nilou Moslehi
Dominique Alex and
Stephanie Olamendi
Lizbeth Barrera and
Felicia Dang
Jonathan Nadal
Isobel Santos and
Vyron Tayag
Title
The Existence of Bacteria in and the
Antibacterial Properties of the fluid
in Pitcher Plants, Nepenthes spp.
Testing for the Presence of Protein in Pitcher plant
(Nepenthes spp.) Phytotelmata
The Effect of Red Wine on the Growth of Oral Bacteria
Page
1
The Effect of Temperature on Light Emission by
Escherichia coli Transformed to Express the Vibrio
Luciferin-Luciferase System
Relationships among Maxillary Sinus Cavity Volume and
Various Maxillary Morphologies
Analysis of Lettuce Plants Grown in Escherichia coli
Contaminated Soil
The Effect of L-Arginine on Muscle Circumference and
Surface Temperature following Exercise in Recreational
Weight Lifters
A Genetic Analysis of the Urinary Odor Produced by
Asparagus in Humans
Effect of pH on the Growth of Aspergillus and Rhizopus
9
Effect of 5-Hydroxytryptophan on Weight in Mice (Mus
musculus)
Effect of Soil Inoculant on Growth of Phaseolus vulgaris L.
and Glycine max
The Effect of Monovalent, Divalent, and Trivalent Cations
on Transmembrane Plasmid Transport in Escherichia coli
The Effect of pH on the Speed of Closure of the Venus
Flytrap, Dionaea muscipula
Possible Isolation and Cultivation of Halophilic Bacteria
from Halite
Differences in Absorption Efficiency between Two Diets in
the Western Fence Lizard, Sceloporus occidentalis
Metabolic Rate of Melopsittacus undulatus Consuming
Dietary Omega-6 Fatty Acids
The Inhibitive Effect of Blue, Green and Red light on E. coli
growth
The Inhibitive Properties of Milk on Powdery Mildew
Sphaerotheca fuliginea
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Samir Al-Ayoubi and
Abdullah Ibish
Daniel Rounds*
Kelsey Quaranto*
Enas Abu Hammad*
The Effect of Various Brown Algae on Growth of Rhizopus
stolonifer
Contractile Vacuole Rates in Paramecia caudatum exposed
to varying Concentrations of Sucrose Solution
The Effect of Temperature on the Metabolic Rate of
Goldfish (Carassius auratus)
The Effect of Exercise on Carbon Dioxide Concentration
Levels in Exhaled in Humans
48
51
53
55
*indicates a Biology 20 Honors paper
Abstract Issue
Biology 3A abstracts for papers presented at the 4th Annual Biology 3A/3B/20 Honors
Scientific Meeting (Spring 2007)
The meeting organizers do not assume responsibility for any inconsistencies in quality or errors
in abstract information. Abstracts are in numerical order according to the abstract number
assigned to each presentation. Authorless abstracts appear at the end of all the abstracts,
including non-emailed abstracts. Abstracts begin on page 54.
Note: Author name(s) and abstract titles were printed directly from the abstract form without corrections. The
presentation order was determined by the order in which the emailed abstracts were received. All non-emailed
abstracts presentation order was also determined by the order in which the hardcopy was received.
TABLE OF CONTENTS
Biology 3A Abstracts
Fall 2006
Author(s)
Samir R. Al-Ayoubi
Omid Niavarani and
Ross Johnson
Courtney Alexander
and Colleen Worne
Hasib Rahmani and
Avishan Nasiri
Jennifer Wilden and
Kevin Laitipaya.
Jason Beltz
and
David Stapleton
Title
CAFFEINE’S AFFECT ON RESPIRATION DURING
EXERCISE
THE EFFECT OF PREMOLAR EXTRACTION ON
MAXILLARY WIDTH
GOLDFISH LONG TERM MEMORY
(Carassius auratus)
THE EFFECT OF FOOD ON METABOLIC RATE IN
GOLDFISH (Carassius auratus)
ARE OVERWEIGHT INDIVIDUALS MORE LIKELY
TO TEST POSITIVE FOR GLYCOSURIA?
PEDALING CADENCE EFFICIENCY AS MEASURED
BY DISTANCE TRAVELED VERSUS CARDIAC
OUTPUT
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Vol. 5, Spring 2007
Page
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57
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57
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Nicholas Davison
and
Erinn Deters
Mayra Mejia and
Annie John
Erik Olmos,
Isobel Santos and
Vyron Tayag
Amantha Bagdon
Blair Leaf,
Heidi Schaff and
Nasrene Hosseini
Dominique Alex and
Andrew Starsiak
April Jones
and
Stephanie Olamendi
Gimena Altamirano
and
David M. R. Quijano
Matin Khoshnevis and
Mortaza Aghazadah
Heather Rufino
and
Stephanie Reed
Author(s)
Tom Caldwell and
Mitra Foroutan
Jackie Olvera
Kristin Harm and Jana
Stverakova
Jonathan Castillo
and
Melissa Molina
COMPARATIVE VARIATION OF WATER LOSS IN
MALES AND FEMALES DURING INTENSE
PHYSICAL ACTIVITY
HOW EFFECTIVE ARE ANTI-BACTERIAL
CLEANERS AGAINST BACTERIAL GROWTH?
ENERGY PRODUCTION THROUGH MICROBIAL
OXIDATION-REDUCTION REACTION
COMING CLEAN: HAND SANITIZERS ARE MORE
EFFECTIVE AGAINST STAPHYLOCCOCUS AUREUS
THAN ANTIBACTERIAL SOAP
ANALYSIS OF THE ACCURACY OF SUGAR
CONCENTRATION IN SPORTS DRINKS THROUGH
FERMENTATION
MUSIC AFFECTS HUMAN HEART RATE
THE EFFECTS OF A HIGHER DOSE OF NITROGEN
VERSUS A HIGHER DOSE OF PHOSPHATE IN THE
MARIGOLD FLOWER, TAGETES PATULA
EFFECTS OF VARIOUS ENERGY DRINKS ON
ATHLETIC PERFORMANCE OF ADOLESCENTS
DUIRNG STRENUOUS SWIMMING ACTIVITIES
THE RATE OF OXYGEN CONSUMPTION OF
GOLDFISH BASED ON SIZE
EFFECTS OF CRYSTALLOID FLUID THERAPY ON
THE HEMATOCRIT OF HOSPITALIZED CANINE
PATIENTS
TABLE OF CONTENTS
Biology 3A Abstracts
Spring 2007
Title
THE EFFECTS OF pH ON BACTERIAL GROWTH
INHIBITION OF Staphylococcus aureus
EFFECTS OF ACID RAIN ON PLANT GROWTH AND
THE ENVIRONMENT
THE EFFECT OF ENVIRONMENTAL TEMPERATURE
ON EXERCISE EFFICIENCY
CAFFEINE STIMULATION AND EFFECTS ON
COGNITIVE MEMORY RECALL OF MICE
(Mus musculus)
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Page
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Cristine M. Kreitzer
and
Derrick Tran
Billy Belgen,
Jennifer Hipolite, and
Jerry Kephart
Jennifer Van Malsen
and
Kaitlyn Bake
Krystal N. Prince
and
Shaya Malekoshoarai
Kristopher Peterson &
John Lew
Milad Danesh
April Tegtmeier
and
Chris Glendinning
Stephanie Anstadt
and
Greg Nelson
Meilad J. Hojati
and
Taran S. Sondhu
Avelon Felsinger and
Zachary Beam
Matthew Kovach and
Chan Kim
Katie R. Rauschl
and
Jessica Lai
Cathy O’Connor and
Chris Yang
Maribeth Johnson and
Michelle Nazarifar
Lauren C. Ferris
and
Brook A. Thomas
THE EFFECT OF MICROWAVE (MW) RADIATED
FOOD ON THE OXYGEN CONSUMPTION OF MUS
MUSCULUS
THE EFFECT OF WATER TEMPERATURE ON THE
METABOLIC RATE OF GOLDFISH
62
GENDER DIFFERENCES IN DIRECTED ATTENTION
AND HEMISPHERIC BRAIN DOMINANCE IN
HUMANS
THE EFFECT OF RED AND BLUE LIGHT ON
CHLOROPHYLL CONCENTRATION IN JALAPENO
PLANTS CAPSICUM ANNUUM
THE EFFECT OF CARBONATED BEVERAGES ON A
TOOTH’S ENAMEL
THE EFFECTS OF CARBON DIOXIDE ON BIOMASS
DURING RADISH SEED GERMINATION (Raphanus
sativus
DNA QUANTITY OF SEEDED AND SEEDLESS RED
GRAPES AND THE POSITIVE EFFECTS ON
CELLULAR ACTIVITIES (Vitis spp.)
EFFECTS OF SUPER THRIVE GROWTH HORMONE
ON CO2 CONSUMPTION AND OVERALL GROWTH
AND DEVELOPMENT ON RED RADISHES (Raphanus
sativus)
THE EFFECTS OF SPORTS DRINKS ON HEART
RATE DURING EXERCISE COMPARED TO WATER
IN HUMANS
EFFECT OF TEMPERATURE ON OPERCULAR
PUMPING RATE OF SMALL AND LARGE GOLDFISH
COMPARISON OF THE METABOLIC RATES
BEFORE AND AFTER CONSUMPTION OF CAFFEINE
IN MUS MUSCULUS
COMPARING THE RESTING AND ACTIVE HEART
RATE AND CO2 PRODUCTION OF A NON-ATHLETE
TO AN ACTIVE ATHLETE
pH NEUTRALIZATION THROUGH SUGARLESS GUM
62
THE EFFECT OF BACTERIAL RESISTANCE TO A
DISANFECTANT
SHORT TERM EFFECTS OF CAFFEINE ON
METABOLIC RATE OF GOLDFISH
(CARASSIUS AURATUS)
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Ryan C. Clark
and
Josue J. Mandujano
Asami Uzawa
AJ Bezer and
Arash Shahrokhshahi
Payam & Sandra
Tony Shofer
and
Trevor Bodmer
Tiffanie L. Hand and
L. Jane Stewart
Michelle Chawner
THE COMPARISON OF CHLOROPHYLL IN SHADE
AND SUN LEAVES OF THE LEMONDADE BERRY
(RHUS INTEGRIGOLIA)
POOL TEMPERATURE AND ITS EFFECT ON
COMPETITIVE SWIMMERS’ PERFORMANCE
THE EFFECTS OF NICOTINE ON HEART RATE AND
BLOOD PRESSURE
THE EFFECTS OF ENERGY DRINKS ON OXYGEN
SATURATION
THE EFFECTS OF GATORADE VERSUS WATER ON
HUMAN HEART RATE DURING PROLONGED
EXERCISE
EFFECTS OF MODERATE DOSES OF CAFFEINE ON
COGNITIVE FUNCTION
THE EFFECTS OF DRINKING HOT AND COLD TEA
ON HUMAN BODY TEMPERATURE
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The Existence of Bacteria in and the Antibacterial Properties of the fluid in Pitcher Plants,
Nepenthes spp.
Giffany Deomampo and Colleen Worne
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Some carnivorous plants are known to have antibacterial properties, such as the
sundew, as well as bacteria that assist in digestion. The following tests were used to
determine whether the pitcher plant, Nepenthes spp., possesses these properties. For both
tests, four different pitcher plants were used. In testing for antibacterial properties, three
groups of eleven agar plates were spread with bacteria—E. coli, Staphyllococcus, and
Streptococcus. The plates were then incubated at 37ºC for 24 hours. Sterile paper discs
were then placed on each plate, and possible bacterial inhibition was studied. Then in
testing for bacterial presence, the four different pitcher plants’ fluid was incubated on agar
plates for 24 hours at 37ºC. The plates that were positive for bacterial growth underwent
Indole Production, Voges-Proskauer test, MR-VP test and Citrate test to discover if
Escherichia coli and Enterobacter-Klebsiella group (coliform bacilli) were present. The test
for antibacterial properties was negative for all three bacteria. The test for bacteria was
negative for all four pitchers.
Introduction
interactions between the pitcher plants and their
environment.
Nepenthes spp. (pitcher plants) are generally
found in acidic swamps, bogs and wet meadows.
They are carnivorous plants that grow leaves that are
filled with liquid, also known as pitfall traps. The
plant secretes more fluid only when food is trapped
inside the plant (Attenborough, 1926). Once the
insect is trapped, they drown, and the plant gradually
dissolves the body of the insect. This may occur by
bacterial action (the bacteria being washed into the
pitcher by rainfall) or by enzymes secreted by the
plant itself (Adams and Smith, 1977). As the prey
decomposes, the prey’s internal bacteria must also be
killed so as to not infect the plant. One such plant
that implements this procedure is the Drosera
rotundifolia L., the sundew. It has been found that
the sundew has spasmolytic, antibacterial and antiinflammatory properties (Kopp, 2006).
There has
been very little study on the digestive and microbial
activities in these plants and some researchers believe
there is a symbiotic relationship between the bacteria
and plant itself, such as E. coli in human intestines.
The objectives include determining if there are
any bacteria present, in particular E. coli and
Enterobater-Klebsiella, and possible antibacterial
qualities within the fluid of the pitcher plant. The
broader impacts of this experiment include possible
future antibacterial alternatives and discovering the
This first study examined the possible
antibacterial properties in the pitcher plant. The
cultivar Nepenthes spp. plant was obtained from T.
Huntley. A total of 36 Petri dishes were filled with
agar then split into three groups of 12. Each group
was spread with a different type of bacteria—E. coli,
Staphyllococcus, and Streptococcus. Four sterile
paper discs were soaked in the pitcher fluid and
placed in each agar plate, equidistant for each other.
There were three plates used as the control in which
no discs were placed. The fluid was derived from
four different pitcher plants. The plates were then
incubated for a period of 24 hours at 37ºC. After
incubation the plates were analyzed for any bacterial
inhibition.
The second study analyzed, bacterial existence in
four pitcher plants’ fluid. The four pitcher plants’
fluid were spread on eight agar plates with labels A,
B, C, and D, with two plates for each pitcher plant.
The plates were incubated for 48 hours at 37ºC. The
plates were checked to see the growth of bacteria.
Plates A and B had no growth of bacteria. Plates C
and D had growth of bacteria and were used to test
for the identity of the type of bacteria. The materials
that were used included nutrient broth cultures of
Escherichia coli and Enterobacter cloacae. Four
Materials and Methods
1
Saddleback Journal of Biology
Spring 2007
series of test were used to determine and identify
Gram-negative bacilli, particularly Escherichia coli
and the Enterobacter cloacae. For the first test, one
loop full of pitcher plant juice labeled (C) was
inoculated into one tube of tryptone broth and one
loop full of pitcher plant juice labeled (D) into
another tube. The tubes were incubated at 37ºC for 48
hours. The tubes were then added with a dropper full
of Kovac’s reagent to each culture and shaken from
side to side to determine the presence of indole. The
second and third test the Methyl Red Test and Voges
Proskauer Test were combined, which included
inoculating one tube of MR-VP broth with pitcher
plant juice labeled (C) and one with pitcher plant
juice labeled (D). The tubes were incubated at 37ºC
for 48 hours. A dropper full of methyl red indicator
was added to the tubes to test for a color change. The
fourth test included inoculating one Simmons citrate
agar slant with pitcher plant juice (C) and one slant
with pitcher plant juice (D). The slants were
incubated for 48 hours at 37ºC. The slants were
examined to see the color change.
Figure 1. A. E. coli, B. Staphococcus, and
Streptococcus. C. The final results in testing for
antibacterial properties in the pitcher plant fluid
gave a negative result for all three tested bacteria;
there was no inhibition.
Results
The group testing for antibacterial properties of the
pitcher plant fluid was negative for any result (Figure
1). When the experimental group and the control
group were compared, no obvious differences were
observed. The group testing for bacterial properties
of the pitcher fluid for the indole production ( first
test), was negative for both pitcher plants juice C and
D due to no color change in the tryptone broth
culture. The tubes were observed for 10 to 15
minutes until it was concluded that it was negative.
The second and third test, methyl red and Voges-
Proskauer Test were also negative; the tubes were
both yellow. The fourth test; citrate showed negative
due to no color change in the slants
Discussion
The study testing the antibacterial properties of
pitcher plant fluid was deemed negative. This could
be a result of being in a controlled environment.
Pitcher plants naturally growing in Southeast Asia
were used to treat infections, such as small pox
(Trimel, 2003).
When grown in controlled
environments, without naturally occurring prey, the
production of antibacterial properties may decrease.
As an insect decomposes its internal bacteria must
also be decomposed so as not to cause infection in
the plant. However, if no insect, or other prey, is
caught then the plant will not secret antibacterial
properties. Pharmaceutical drugs are now being
produced to treat infections, for example, some
strains of Staphylococcus (Trimel, 2003).
The study testing the presence of bacteria in the
pitcher plant fluid was also deemed negative. It has
been tested that E. coli is only sustained for up to five
days at 26ºC (Whitman, et al., 2005). With the
pitcher plants in the controlled environment, they
may not have been in the optimal temperature of
26ºC. It is also possible that the E. coli was no longer
available to be tested after five days.
A number of processes occurred during the indole
production. In a tryptone broth, indole is produced
through enzyme reactions. Bacteria used the amino
acid, tryptophan, found in tryptone as a source of
energy. As the bacteria fed, the tryptophan converted
into pyruvic acid and ammonia. In order for these
processes to be carried out properly, a certain
incubation period needs to be followed. If incubation
is not followed thoroughly, the indole may disappear
causing a negative result. The second and third test
used the methyl red and Voges-Proskauer test which
used the MR-VP broth. The products produced from
these tests are determined by peptone, dextrose
(glucose) and dipotassium phosphate. The E. coli
used the dextrose molecules for energy source. Their
by products raise the pH of the broth thus causing
Methyl Red indicator to turn yellow. The fourth test,
or the citrate test, includes an organic synthetic
medium, which contains mineral salts, thus providing
their source of carbon and energy. The Simmons
citrate test remained green constituting a negative
result (The IMVi C Tests ).
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Saddleback Journal of Biology
Spring 2007
Literature Cited
Adams, R. and Smith, G. An SEM Survey of the Five
Carnivorous Pitcher Plant Genera. Department of
Biological Sciences. 64(3): 265-72.
Attenborough, D. (1926). The Private life of Plants.
Princeton University Press. 320.
Kopp B, Wawrosch C, Buol I, Dorfer. T. (2006).
Efficient Production of Sundew ( Drosera
rotundifolia L.) in vitro using a temporary immersion
system. Planta Medica. 72.
Trimel, S. (2003) School of Continuing Education’s
Vezen Wu Helps develop New Public Health
Software. Columbia University Record. 2.
Whitman, R., Byers, S., Shively, D., Ferguson, D.,
Byappanahalli, M. (2005) Occurrence and growth
characteristics of Escherichia coli and enterococci
within the accumulated fluid of the northern pitcher
plant ( Sarracenia purpurea). Canadian Journal of
Microbiology.
51:12,
1027-37
.
Testing for the Presence of Protein in Pitcher plant (Nepenthes spp.) Phytotelmata
Matin Khoshnevis and Samuel Ollar
Department of Biological Science
Saddleback College
Mission Viejo Ca 92692
This study was designed to investigate the presence of protein in the phytotelmata of the
Nepenthes spp pitcher. This hypothesis was tested utilizing spectrophotometry, gel column
chromatography and gel electrophoresis. Comparisons of four phytotelmata samples from
the same plant were tested against serial dilutions of BSA from 50µg/mL to 5µg/mL using
the Bradford method and a Beckman DU730 Spectrophotometer. Protein concentrations
exceeded 5µg/mL in one sample but averaged <5µg/mL, a maximum absorbance of 0.222
and a mean absorbance of 0.212 at wavelength 590nm as compared to 5µg/mL of BSA
which gave an absorbance reading of 0.215 at 590nm. Gel filtration column
chromatography was used to purify samples and isolate proteins by size. Ten samples were
collected and re-tested at 590nm in order to determine which samples contained protein.
Finally, Gel electrophoresis was used to determine the molecular mass of the proteins. The
tests conducted confirmed the presence of at least two different proteins with a combined
concentration of around 5µg/mL in the pitcher fluid. Previous studies show the presence of
two proteinases in the phytotelmata of pitcher plants that function similar to pepsin, having
an optimum operating pH of 2 and temperature of about 40°C (Takahashi et al, 2005).
Introduction
Pitcher plants are carnivorous plants that derive
nutrients, but not energy, by trapping and consuming
animals, insects and protozoans. The prey-trapping
mechanism features a deep cavity filled with liquid
known as a pitfall trap. It has been widely assumed that
the various sorts of pitfall traps found in the Nepenthes
genus evolved from rolled leaves with selection
pressures favoring more deeply cupped leaves (Fish &
Hall, 1978). Insects and animals are attracted to the
pitcher by visual lures such as anthocyanin pigments in
the leaf, water-soluble vacuolar flavinoid pigments that
appear in colors ranging from red to blue depending on
pH and nectar which attracts pollinating animals
(Cipollini et al, 1994). The sides of the pitcher are
slippery and may be grooved in such a way so as to
ensure prey cannot climb out. The small body of liquid
contained within the pitcher, the phytotelmata (Greek
phyto: Plant & telma: pond) dissolves the prey allowing
for absorption of nitrates. The purpose of this project
was to test for the presence of protein in the
phytotelmata of the pitcher plant. Determining the
presence of protein produced by Nepenthes may
support the possibility that the species within the
phylum produce enzymes which aid in nitrate
absorption.
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Spring 2007
Materials and Methods
Results
Nepenthes spp. (Pitcher plant cultivar) was obtained
from T. Huntley. The plant was moved to the college
greenhouse and enclosed by two 15 gallon glass
aquariums. One of aquariums was filled with two to
three centimeters of deionized water and the second one
was set on top, in order to create a warm, humid
environment while preventing insects or other potential
contaminants from entering the phytotelmata of the
pitchers. The phytotelmata of four open pitchers were
collected and placed in sterile containers and labeled A,
B, C, and D, respectively. Samples were tested for pH
along with the presence and concentration of protein.
Using the Bradford Protein Micro Assay method, Serial
dilutions of BSA were used to create a standard dilution
curve, which was in tern used to create the formula y =
0.004x + 0.2007. Unknown samples were prepared by
combining 0.8mL of each sample with 0.2mL of G-250
dye in a 1mL cuvette. The cuvette was then inverted
several times to mix the solution and incubated at room
temperature for 5 minutes. The samples were measured
at an absorbance of 590nm in a Beckman DU730
Spectrophotometer (Spector, 1978). Five readings of
each sample were taken with the instrument being reblanked for accuracy.
Gel filtration column chromatography using
Sephadex G-100 was used to purify samples and isolate
the proteins in the fluid. As the sample moves through
the gel, larger particles will separate out faster while
smaller particles separate out later. By separating them
according to size we can purify out proteins into
separate samples for further testing. Two columns were
run, one with a 2mL sample dyed with Coomassie Blue
and the other run with 2mL of unaltered sample
(Sauterer et al 2000). The purpose of running one dyed
sample was to determine where the proteins were
separating out in the untreated sample. After draining
off 20 drops of buffer ten samples were taken, each
containing ten drops of purified phytotelemata. Once
the purified samples were collected, the untreated batch
was retested using the Bradford method and a Beckman
DU640 set at A590nm to determine which of the purified
samples contained protein.
Polyacrylamide gel electrophoresis was used to
measure the molecular mass of the purified protein
samples by placing them on a gel, in a physiological
buffer solution and passing 108 volts of current through
the solution and gel for one hour. The entire container
holding the gel and buffer were placed in an ice bath to
keep the temperature as close to 4°C as possible. The
gels were rinsed, and then stained with Coomassie Blue
dye for one hour. Finally the gel was de-stained by
agitation in deionized water for 24 hours.
All samples of phytotelemata had a pH of 2.5. The
average protein concentration of the phytotelemata was
calculated in table 1. The serial dilution of BSA
provided a trend line by which the formula y = 0.004x +
0.2007 was derived and is illustrated in figure 1. There
was an average concentration of <5µg/mL of protein in
the phytotelemata. The absorbance reading of each
sample can be seen in Table 1. Sample C was omitted
from the study because later investigation revealed a
partially dissolved sow bug which caused sample C to
give a calculated protein concentration of 12µg/mL;
more than twice the amount found in the other samples.
The averages of both the absorptions and the amount of
protein were recorded in table 1. After purifying sample
D, which contained the highest absorption reading, into
ten incremental samples of ten drops, the absorption
was recorded on a Beckman DU640. The purified
samples showed peaks in increments 1, 2 and 7 as
illustrated in figure 2. Peaks 1 and 2 could be and
probably are portions of the same protein thus, being
the largest particles of protein. The results of gel
electrophoresis were inconclusive as no protein was
Table1. The concentration of protein in solution
was determined by converting the absorption of
light at 590nm to µg/mL using the equation x = (y –
0.2207) /0 .004.
µg/mL
Phytotelemata
A590nm
Pitcher A
0.208
1.285
Pitcher B
0.206
1.325
Pitcher D
0.222
5.325
Average
0.212
2.825
identifiable after the staining process. This is probably
due to the extremely low volume of protein present in
the phytotelemata.
Discussion
This research was performed in order to determine
the presence of protein in the Nepenthes spp
phytotelmata. Based on the results it can be confidently
concluded that there are at least two different size
proteins present. It can be clearly seen that the protein
components found in the phytotelmata coincide with
the proteinases previously identified (Takahashi et al
2005). The fluid samples indicated a pH of 2.5 which
concur with earlier studies regarding proteinase. The
optimum pH of previously identified proteinases is at
approximately 2 and functions similar to pepsin, a
digestive enzyme secreted by chief cells in the lining of
the human stomach (Amagase 1971). This experiment
supports the theory that all species of Nepenthes
contain proteinases in their phytotelemata. Humans and
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Saddleback Journal of Biology
Spring 2007
0.45
0.4
Absorbance at 590nm
0.35
0.3
0.25
y = 0.004x + 0.2007
0.2
0.15
0.1
0.05
0
0
10
20
30
40
50
60
Protein Concentration µg/mL
Figure 1. A serial dilution of BSA was used to create the trend line from which the equation y = 0.004x + 0.2007
was created. This equation was used to find the specific concentration of the unknowns. A maximum absorbance
of 0.222 at 590nm, and a minimum absorbance of 0.206 for BSA were recorded.
0.1
0.08
Absorbance at 590nm
0.06
0.04
0.02
0
1
2
3
4
5
6
7
8
9
10
-0.02
-0.04
-0.06
Ten Drop Incremental Samples
Figure 2. The highest peaks of protein concentration were observed in first, second, and seventh samples. This
suggests the presence of at least two different size proteins, one larger and one smaller, in the Nepenthes spp
phytotelmata.
5
Saddleback Journal of Biology
Spring 2007
plants have no morphological and ecological
similarities except that they are both in domain
Eukarya. It is surprising that two such different
kingdoms of Eukarya have proteinases resembling each
other in such detail.
Saurterer, R.A., Jones J. 2000. A rapid, simple and
inexpensive
experiment
in
gel
filtration
chromatography. Am. Biology Teacher, 62(8): 602-607.
Literature Cited
Spector T. 1978. Refinement of the Coomassie blue
method of protein quantitation; A simple and linear
spectrophotometric assay for less than or equal to 0.5
to 50 micrograms of protein, Anal Biochem. 86: 142–
146.
Amagase, S. 1971. Digestive Enzymes in Insectivorous
Plants. J. Biochem. 72: 73-81.
Cipollini Jr D, Newell S, Nastase A. Apr 1994.
American Midland Naturalist. 131(2): 374-377.
Fish D, Hall D W. Jan 1978. American Midland
Naturalist. 99(1): 172-183.
Takahashi K. et al. Dec 2005. Nepenthesin, a Unique
Member of a Novel Subfamily of Aspartic Proteinases:
Enzymatic and Structural Characteristics. Current
Protein and Peptide Science. 6(6): 513-525.
The Effect of Red Wine on the Growth of Oral Bacteria
Erik Olmos and Avishan Kolahdouz Nasiri
Department of Biological Sciences
Saddleback College
Mission Viejo, California 92692
With multiple studies stating the various beneficial effects of red wine in different
species including Mus musculus immune cells and in the human body, the goal of this
experiment was to look at the effect red wine had on oral bacteria colonies. To determine if
the presence of ethanol in red wine was not the actual cause of inhibition, a 12.5%
concentration of ethanol was also tested as well as a control of sterilized water to determine
if filter paper didn’t cause the inhibition either. Colonies of oral bacteria were obtained
and grown in both broth and nutrient agar solutions. Once purified, 50μl of the bacterial
solution was smeared evenly onto 28 Petri dishes, and filter paper disks were submerged in
either the water, ethanol or the Merlot, a type of red wine and placed on top of the
bacteria-smeared agar. The formation of inhibition zones were looked for on the Petri
dishes after forty-eight hours of incubation at a temperature of 37°C. Only one winesoaked disk produced a small amount of inhibition while all the other disks had bacteria
growing on them. These results show that resveratrol does not work the same with humans
as it does in the M. musculus cells or else different types of red wine produce different
effects on the bacteria.
Introduction
In the past couple of years, there have been a
number of experiments in which the benefits of red
wine have been studied. In the majority of them, the
compound that is thought to produce the different
benefits has been resveratrol. Resveratrol is a
phytoalexin, a type of antibiotic produced by plants
as a defense system against invasions like fungus.
Although a common compound in a variety plants,
resveratrol is most prevalent in grape vines, roots,
seeds, stalks, and skin, where it has a concentration
of 50 to 100 µg per gram of grape skin.
The compound has already been shown to possess
beneficial health effects, such as cancer prevention,
6
Saddleback Journal of Biology
Spring 2007
neuro-protection, anti-aging, prolonging life, and as a
treatment of diseases in blood vessels, and the heart
and liver in non-human species. But now, a group of
scientists from Quebec have performed preliminary
studies demonstrating that resveratrol also inhibits
the growth of oral bacteria and has an antiinflammatory effect (Gaffney, 2006).
So far, the scientists have performed their
experiment only on Mus musculus immune system
cells
infected
by
Actinobacillus
actinomycetemcomitans
and
Fusobacterium
nucleatum. If left untreated, the bacteria can weaken
the jaw bones, break down gum tissue, and erode
enamel. They reported that even low doses of
resveratrol inhibited the growth of the bacteria and
that at a concentration of 100 micrograms per
milliliter it reduced one type of bacteria by 40 percent
and the other by 60 percent, compared with untreated
cell samples (Gaffney, 2006).
This is extremely useful since there are more than
400 different species of bacteria present in a human’s
oral cavity (Stevens, 1996). The most common
bacteria being Streptococcus pyogenes, which can
cause infections in the throat and skin; Streptococcus
mutans, a contributor to tooth decay (Dalwai 2006)
and Treponema denticola, a contributor to gum
disease (Genco, 2000). These results would be
helpful to the 80% of Americans that have some form
of gum disease. If the findings are true, it would
definitely decrease the number of individuals
suffering from gingivitis or even worse, periodontitis
which leads to bleeding, tooth decay and even tooth
loss. For this reason, the goal of this experiment is to
find out if red wine has the potential of inhibiting the
growth of bacteria in the human oral cavity.
Material and Methods
The agar was made by mixing 20.0g of nutrient
agar in a 1000 mL Erlenmeyer flask filled with 1000
mL deionized water and a magnetic stir bar. The
solution was then boiled to 100ºC until the nutrient
agar completely dissolved. The flask containing the
agar was afterwards placed in an autoclave so as to
sterilize the solution. After being sterilized, the agar
solution was poured evenly into thirty Petri dishes.
To help further firm the agar, the Petri dishes were
placed in a refrigerator.
The broth needed was made by mixing 4.0g of
nutrient broth in a 1000 mL Erlenmeyer flask filled
with 500 mL of deionized water and a magnetic stir
bar.
Once the broth solution dissolved at a
temperature of 100ºC, it was also placed in the
autoclave to sterilize it. The sterilized broth was then
poured into twelve test tubes.
The disks used to test for zones of inhibition were
made by punching holes in filter paper with a hole-
puncher. Once a sufficient amount of disks were
made, they were collected into a covered evaporating
dish and placed in the autoclave to kill any bacteria
that would have been present.
Oral bacteria samples were obtained by swabbing
between the teeth and cheeks of an individual with
sterile Q-tips. Afterwards, the Q-tips were stirred
into two of the broth test tubes to transfer bacteria.
The test tubes were then placed in an incubator at
37ºC. Once incubated for 48 hours, two Petri dishes
were inoculated with the cultured bacteria and again
incubated at the same temperature for 36 hours to let
the bacteria form colonies. After colonies formed 36
hours later, a single colony from each Petri dish was
transferred into two test tubes to culture a pure strain
of bacteria and placed in the incubator for 48 hours.
After the Petri dishes and the test tubes with the
purified bacteria strains were ready, 50 μL of the
bacterial solution was smeared evenly throughout the
agar of each of the 24 Petri dishes. Once smeared,
eight dishes were used for each of the three
substances being tested. The substances tested were
red wine with an alcohol concentration of 12.5%, a
12.5% ethanol solution, and sterilized water. Disks
were immersed in 10 ml beakers filled with either of
the substances. Four of the soaked disks were placed
in each of the eight Petri dishes assigned to each of
the liquids. Finally, the Petri dishes were placed in
the incubator for a period of 48 hours at 37°C to
culture the bacteria.
Once cultured, the dishes were placed in a
refrigerator until they were examined for the presence
of an inhibition zone around the filter paper disks.
All data were graphed using Microsoft Excel®.
After the examination for inhibition, a gram stain was
performed to determine what type of bacteria was
being tested.
Results
When examined, the Petri dishes showed no
significant inhibition from the red wine. All the
dishes looked alike with the bacteria growing
throughout the agar in tiny colonies (Figure 1). Only
a single wine-soaked filter paper disk had a thin line
of inhibition around it, while the three other disks on
the same dish showed no inhibition. The water and
alcohol dishes even had bacteria growing beneath the
disks on the plate.
The results of the gram stain showed that there
was a combination of both gram-negative and grampositive cocci present in the Petri dishes.
7
Saddleback Journal of Biology
Spring 2007
Number of Disks without Inhibition
known wines used in similar research include
Cabernet Sauvignon and Chianti.
35
30
25
Literature Cited
20
Cotter P, and Hill C. (2003). Surviving the Acid Test:
Responses of Gram-Positive Bacteria to Low pH.
Microbiology and Molecular Biology Reviews, 67(3):
429–453.
15
10
5
0
Red Wine
Ethanol 12.5%
Water
Samples Tested
Figure 1. The difference in the amount of disks
without inhibition between the various solutions
tested. There was no significant difference between
the groups since only one disk soaked in wine showed
some inhibition.
Discussion
One explanation for the absence of inhibition
might be that there are various types of red wine, and
they might have had different concentrations of
resveratrol. Thus, resulting in there being varieties of
red wine which produce inhibition of oral bacteria
due to their high concentration of the phytoalexin.
Since the gram stain resulted in different bacteria
being present in the Petri dishes during the
experiment, there is a possibility that the red wine did
in fact inhibit bacteria but the other bacteria masked
the inhibition by growing in its place.
The researchers in Canada also performed their
experiments on bacteria infecting the immune cells of
Mus musculus while this experiment obtained the
samples from a human’s oral cavity (Gaffney, 2006).
This means that there could have been a difference in
the genetic makeup of the different bacteria that
made those infecting the immune cells susceptible to
the pytoalexin compound.
Future experiments could be made using the
distinct concentrations of resveratrol found in
different red wines. While Merlot was used, other
Dalwai F, Spratt D, and Pratten J. (2006). Modeling
Shifts in Microbial Populations Associated with
Health or Disease. Applied and Environmental
Microbiology, 72(5): 3678–3684.
Gaffney J. Red-Wine Compound May Be Good for
the Gums, Study Finds. Wine Spectator. 22 May
2006.
Genco R. Frank A, and Harold C. (2000). Oral
Reports. Sciences. 40(6): 25.
King S. and Beelman R. (1986). Metabolic
Interactions Between Saccharomyces cerevisiae and
Leuconostoc oenos in a Model Grape Juice/Wine
System. American Journal of Enology and
Viticulture. 37(1): 53-60.
Munro C, and Grap M. (2004). Oral Health and Care
in the Intensive Care Unit: State of the Science.
American Journal of Critical Care. 13: 25-34.
Stevens J. (1996). It’s a Jungle out There. Bioscience.
46(5): 314.
Wang W, Lai H, Hsueh P, Chiou R, Lin S, and Liaw
S. (2006). Inhibition of swarming and virulence
factor expression in Proteus mirabilis by resveratrol.
Journal Medical Microbiology 55: 1313-1321.
8
Saddleback Journal of Biology
Spring 2007
The Effect of Temperature on Light Emission by Escherichia coli Transformed to Express
the Vibrio Luciferin-Luciferase System
Mina Hamedanizadeh and Megan Moshar
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Bioluminescence is a biological transduction of energy and an offshoot from the general
metabolic scheme of respiration. It results from the conversion of chemical to radiant
energy. As long as ATP and O2 are present, luciferin can continue to generate light in the
presence of luciferase. Temperature, which affects all enzyme mediated reactions, affects
the rate of the luciferin-luciferase reaction, and the general rule is that for every 10°C
change in temperature, reaction rates will change. This experiment determined the amount
of light emitted by pLUX in Escherichia coli while placed in various temperatures. Five
experimental conditions were met throughout this experiment (-15.5°C, 3.5°C, 21.5°C,
30.8°C, and 37.0°C) . The light intensity showed a maximum at 37.0°C and minimal at 15.5°C. There was an overall significant difference between all five temperatures (p=
0.001).
Introduction
Bioluminescence is an emission of light as a result
of a chemical reaction in living organisms. It results
from the conversion of chemical energy into radiant
energy. The amount of luminescence is measured by
the reaction of ATP with luciferin plus luciferase,
ATP + luciferin + luciferase Æ light (McGowen.,
Detwiler, 1985). It is assumed that the basic reactions
of all bioluminescence involve an enzymatic reaction
between luciferin and its complimentary enzyme
luciferase. Heat production is significant to the
amount of luminescence produced in the oxidation of
the luciferin (Harvey, 1919). It is said that for every
ten degrees Celsius increase in temperature, the
reaction rate, also, increases. It has proven that
activity increases due to a rise in temperature
between 6 and 25 degrees Celsius (Dickinson, et. al,
1993). In previous experiments conducted on
fireflies, the optimal temperature for maximum light
emission was 22.5°C (Ueda, et. al, 1994). In a
previous experiment the effects of increasing the
optimum temperature showed a detrimental effect on
the pH (Johnson, et. al, 1945). No major research has
been conducted to isolate only temperature as a
single factor the enzymatic reaction between luciferin
luciferase. The purpose of this experiment is to
determine if a change in temperature would effect the
emission of light given off by the luciferin enzyme. It
is expected that the luciferin luciferase reaction will
emit the most light at a higher temperature, 37.0°C,
in Escherichia coli.
Materials and Methods
To carry out the experiment, first the E. coli was
transformed by the following process. First, 250 µL
of ice cold calcium chloride were added to four tubes,
along with ten µL of the plasmid pLUX. This
plasmid is a recombinant plasmid that contains genes
from Vibrio fischeri that are involved in the process
of bioluminescence. Isolated colonies of E. coli were
inoculated and placed into each tube. Next, the tubes
were placed on ice for 15 minutes. Immediately after
the 15 minute incubation on ice, the tubes were
placed into a 42°C water bath for 90 seconds. After
the heat shock, 250 µL of Lurid broth were added to
each tube using a sterile pipette. The tubes were
placed in a rack for ten minutes to allow for a
recovery time. After the recovery, 100 µL of the
transformed plasmid were added to eight Lurid
broth/Ampicillin (LB/AMP) plates. The plates were
placed upside down in room temperature for a 24
hour period.
Four of the 1/1000 plates were placed in five
different temperatures (-15.5°C, 3.5°C, 21.5°C,
30.8°C, and 37.0°C) to determine the intensity of
luminescence given off at each temperature. All four
plates were placed in each temperature for 15 minute
intervals. To determine the intensity of light given
off, pictures were taken of each plate. A Canon D3
camera was used to take the pictures. The pictures
were taken in a dark room over a one minute time
period. With the use of Photoshop, the brightness of
9
Saddleback Journal of Biology
Spring 2007
each picture was determined. The brightness of each
picture was determined as a percentage. At room
temperature, 21.5°C, the percentage was 100. A
change in percentage above or below 100 indicated a
change in light intensity.
The intensity of luminescence was analyzed using
a repeated-measures one-way analysis of variance
(ANOVA). A Bonferroni correction was ran to avoid
a type 1 error. All the data was expressed as a mean
plus or minus the S.E.M.
Results
Average Brightness (%)
The temperature scan in the luciferin luciferase
reaction showed that the maximum light intensity
was observed at 37.0°C. No statistical difference was
observed between 21.5°C (98.2 %) and the following
three temperatures, 3.5°C (56.8%), 30.8°C (123%)
and 37.0°C (134%). There was a statistical difference
between 21.5°C and -15.5°C (34.5%). A statistical
difference was observed at 3.5°C and all the
temperatures, except at -15.5°C. There was a
significant change in brightness at -15.5°C in
comparison to 30.8°C and 37.0°C. No significant
difference was observed between 30.8°C and 37.0°C
(Figure 1).
150
100
light expressed was temperature sensitive. The
plasmid, pLUX, was sensitive due to the presence of
the Vibrio fischeri which plays a key role in
bioluminescence. The LUX operon of Vibrio fischeri
is responsible for the expression of the protein that is
involved in the luminescence process. In this
experiment it was crucial that the bacteria that
contained the pLUX plasmid was inoculated at room
temperature, otherwise no fluoresce would occur due
to the denaturing of the proteins.
In a previous experiment the effects of
temperature upon the uninhibited enzyme was
determined and found that activity increased with a
rise in temperature from 6°C to 25°C (Dickinson, et.
al, 1993). This experiment supports Dickinson
because there was a statistical difference between 15.5°C and the higher temperatures, 21.5°C, 30.8°C
and 37.0°C. Another experiment suggests that the
luminescence intensity was greater at low
temperatures and decreased as the temperature was
raised towards the optimum temperature (Johnson, et.
al, 1945). In this experiment light intensity was
greater at higher temperatures, therefore it does not
support the claim from Johnson that the luminescence
decreased as it reached the optimum temperature,
21.5°C. Thus these results are consistent with, but
certainly do not prove, the idea that when pLUX
transforms non-bioluminescent bacteria, such as
Escherichia coli, temperature may be the only factor
in the process of bioluminescence.
50
Literature Cited
0
-15.5
3.5
21.5
30.8
37
Tem perature (°C)
Figure 1. Temperature dependence of the luciferin
luciferase reaction in transformed E. coli. Each
group was subjected to five different temperatures, 15.5°C, 3.5°C, 21.5°C, 30.8°C, and 37.0°C, over
fifteen minute intervals. There a statistical difference
among all five temperatures (p= 0.001). At 3.5°C a
statistical difference was shown with all temperatures
except at -15.5°C. At -15.5°C a statistical difference
was shown with 21.5°C, 30.8°C and 37.0°C.
Discussion
This experiment suggests that the enzyme
luciferase favors higher temperatures, 37.0°C, due to
the amount of light emitted. Comparison between the
lower temperatures, -15.5°C and 3.5°C, did not show
a significant difference. When 3.5°C was compared
to 37.0°C and 30.8°C, a significant difference was
found. The significant difference in the amount of
Dickinson R., Franks N.P., Lieb W.R., 1993.
Thermodynamics of anesthetic/protein interactions:
Temperature studies on firefly luciferase. Biophysical
Journal 64: 1264-1271.
Harvey E., 1919. Studies on bioluminescence: Heat
production during luminescence of Cypridina
Luciferin. Nela Research Laboratory.
Johnson F., Eyring H., Steblay R., Chaplin H., Huber
C., Gherardi G., 1945. The nature and control of
reactions in bioluminescence: With special reference
to the mechanism of reversible and irreversible
inhibitions by hydrogen and hydroxyl ions,
temperature, pressure, alcohol, urethane, and
sulfanilamide in bacteria. The Journal of General
Physiology 28:463-537.
McGowan E., Detwiler T., 1985. Modified platelet
responses to thrombin: Evidence for two types of
receptors or coupling mechanisms. The Journal of
Biological Chemistry 261(2):739-746.
10
Saddleback Journal of Biology
Spring 2007
Ueda I, Shinoda F, Kamaya H., 1994. Temperaturedependent effects of high pressure on the
bioluminescence of firefly luciferase. Biophysical
Journal 66(6):2107-10.
Relationships among Maxillary Sinus Cavity Volume and Various Maxillary Morphologies
Omid Niavarani and Ross Johnson
Department of Dental Surgery
Saddleback College
Mission Viejo, CA 92692
Altering maxillary morphologies is common place in orthodontics. This is done in order
to achieve proper teeth alignments or more aesthetically pleasing smiles. The purpose of
this study was to investigate the relationship(s) various maxillary morphologies had on
maxillary sinus cavity volume. The hypothesis stated that there was a correlation between
maxillary morphologies and maxillary sinus cavity volume. The skulls that were measured
(n=12) were supplied by the Saddleback Biological Sciences Department and consisted of
varying ethnic backgrounds. Analysis showed that the mean maxillary sinus cavity volume
was 20.44 ml ± 4.88 ml (± se). The maxillary morphologies of width, exterior arch length,
and height showed mean values of 46.17 mm ± 2.41 mm (± se), 82.42 mm ± 6.04 mm (± se),
and 11.42mm ± 2.94 mm (± se), respectively. The calculated correlation coefficients (r) of
the measurements against maxillary sinus cavity volume are as follows: with respect to
width, r = 0.63; with respect to exterior arch length, r = 0.61; with respect to height, r =
0.92. These values support the hypothesis and indicate that there is a correlation between
the measured maxillary morphologies and maxillary sinus cavity volume.
Introduction
There are various maxillary morphologies
present in the current population. The difference in
morphologies is related to ancestry, ethnicity, and
tooth alignment. This variation in the shape of the
maxillary also alters cranial morphologies:
A
widening of the maxillary arch results in a
broadening of the midface (Hoffer and Walters,
1975). It is the purpose of this study to investigate the
relationship(s) that various maxillary morphologies
will have with maxillary sinus cavity volume. The
hypothesis will assert that there is a correlation
between maxillary morphologies and maxillary sinus
cavity volume.
Because one can alter the structures and bones
that comprise the facial skeleton, it is of great
importance to understand the consequences of
various alterations (Meikle, 2007). Understanding the
implications of various orthodontic treatments is
paramount in the field of orthodontics where it is
common practice to manipulate teeth relationships.
Literature on this subject has indicated that an
expansion of the maxillary arch has increased the size
of the nasal chambers of the individuals studied
(Wertz, 1968). There is also evidence to suggest that
an expansion of the maxillary reduces nasal airway
restriction, which would indicate an increase in nasal
cavity volume (Doruk et al, 2004). However, much of
the literature fails to touch on the subject of different
maxillary morphologies and resultant maxillary sinus
cavity volumes. Hence, this study will investigate the
relationship between the respective sinus cavity
volumes and that of various maxillary morphologies.
Materials and Methods
The subjects consisted of 12 human skulls of
various ethnic backgrounds. The maxillary
morphologies were measured in millimeters (mm)
and the maxillary sinus cavity volume was measured
in milliliters (ml). Three different measurements were
made: 1) the maxillary palatal exterior arch length of
the skulls, which was measured from the lateral side
of the second molar to the lateral side of the
corresponding second molar; 2) the maxillary width,
which was measured from the buccal pit of the
second molar to the buccal pit of the corresponding
11
Saddleback Journal of Biology
Spring 2007
maxillary sinus cavity
volume (ml)
Results
The data indicated that the mean maxillary sinus
cavity volume was 20.44 mL ± 4.88 mL (± se). The
maxillary morphologies of width, exterior arch
length, and height showed mean values of 46.17 mm
± 2.41 mm (± se), 82.42 mm ± 6.04 mm (± se), and
11.42 mm ± 2.94 mm (± se), respectively. Correlation
tests between maxillary sinus cavity volume and the
investigated morphologies yield r values that present
the interdependence of each of the morphology
quantities with maxillary sinus cavity volume: The
relationship between width and sinus volume was
significant (Figure 1); the correlation with respect to
exterior arch length was significant (Figure 2); the
relationship with respect to height was significant
(Figure 3).
30
25
20
15
10
5
0
y = 1.2272x - 36.119
0
20
40
60
width (mm)
Figure 1. Maxillary width has a significant
correlation with maxillary sinus cavity volume (r =
0.63). The mean maxillary sinus cavity volume and
maxillary width are 20.44 mL ± 4.88 mL (± se) and
46.17 mm ± 2.41 mm (± se), respectively.
maxillary sinus cavity
volume (ml)
30
25
20
15
10
5
0
y = 0.4682x - 18.081
0
20
40
60
80
100
length (mm)
Figure 2. Maxillary arch length has a significant
correlation with maxillary sinus cavity volume (r =
0.61). The mean maxillary sinus cavity volume and
maxillary length are 20.44 mL ± 4.88 mL (± se) and
82.42 mm ± 6.04 mm (± se), respectively.
maxillary sinus cavity
volume (ml)
second molar; 3) the arch height which was measured
from the roof of the palate, at the mid-palatal suture,
to the bone-line of the respective individual. A string
was employed for measurements and then applied to
a ruler to determine the units of length. Maxillary
sinus cavity volume was measured by packing the
cavity with fine sand. The volume of sand used to fill
the cavity was measured by dividing its mass by its
density.
The data analyses were then restricted to finding
relationships between maxillary width, exterior arch
length, height and corresponding maxillary sinus
cavity volume. The statistical methods employed for
a quantitative report of the data include
measurements of the mean and Pearson product
moment correlation coefficient (r) values to show if
there is a correlation between the two anatomical
structures that are being investigated. Significance
was determined by applying the critical value (CV =
0.55), obtained from the calculated degrees of
freedom (d.f. = 11) and a significance value of 95%
(α = 0.05), to the measured correlation coefficients.
30
25
20
15
10
5
0
y = 1.4543x + 3.7851
0
5
10
15
20
height (mm)
Figure 3. Maxillary height has a significant
correlation with maxillary sinus cavity volume (r =
0.92). The mean maxillary sinus cavity volume and
maxillary height are 20.44 mL ± 4.88 mL (± se) and
11.42 mm ± 2.94 mm (± se), respectively.
Discussion
The relationship between maxillary sinus cavity
volume and all measured maxillary morphologies
were significant. Thus, the data supports the
hypothesis that there is indeed a relationship between
maxillary morphologies and maxillary sinus cavity
volume. This implies that alteration of maxillary
morphologies cannot be undertaken with the
assumption that cranial morphologies (maxillary
sinuses specifically) will not be affected. Instead, one
must focus on the maxillary as part of a whole.
Further investigation may be appropriate to ascertain
the effects of a reduction in maxillary sinus cavity
volume with respect to drainage capabilities and
infection rates of the sinus cavities.
References
Doruk, C., Sokucu, O., Sezer, H. and Canbay, E.
(2004). Evaluation of Nasal Airway Resistance
During Rapid Maxillary Expansion Using Acoustic
Rhinometry. European Journal of Orthodontics. 25:
397-40
12
Saddleback Journal of Biology
Spring 2007
Hoffer, F. and Walters, R. (1975). Adaptive Changes
in the Face of the Macaca Mulatta Monkey
Following Orthopedic Opening of the Mid-palatal
Suture. Angle Orthodontist. 45(4): 282-290.
Meikle, M. (2007). Remodeling the Dentofacial
Skeleton: The Biological Basis of Orthopedics and
Dentofacial Orthopedics.
Research. 86(1): 12-24.
Journal
of
Dental
Wertz, R. (1968). Changes in Nasal Airflow Incident
to Rapid Maxillary Expansion. Angle Orthodontist.
38(1): 1-11.
Analysis of Lettuce Plants Grown in Escherichia coli Contaminated Soil
Jennifer Wilden and Hasib Rahmani
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Leaf vegetables may become infected with Escherichia coli when exposed to contaminated
water. This study investigated the ability of E. coli to colonize lettuce plant tissue internally
through its root system. Iceberg and Romaine lettuce plants were grown in plastic pots that
were placed in water inoculated with E. coli transformed to express green fluorescent
protein (3.9 x 105 CFU · mL –1). Lettuce samples were plated on Luria-Bertani agar that
contained ampicillin, and analysis of E. coli was demonstrated with fluorescent UV light.
The results showed that E. coli did not migrate through the roots and become entrapped
within the lettuce plant tissue. The results of this study indicated that lettuce plants were
unsusceptible to internal infection when grown in soil contaminated with low
concentrations of E. coli. However, further research on the various routes of E. coli
contamination in produce is necessary for the prevention of foodborne infections.
Introduction
The prevention of foodborne disease associated with
pathogenic Escherichia coli, a causative agent of
hemorrhagic colitis and hemolytic uremic syndrome, is
of necessity (Kudva et al., 1998). Recent outbreaks
linked to infected produce, specifically leaf vegetables,
have triggered a greater public outcry for increased
food safety measures (Ingham et al., 2004; Johannessen
et al., 2005). However, epidemiological data on the
various methods of E. coli contamination in vegetables
are insufficient.
Recent studies indicate that contaminated lettuce
may be a mechanism for the transmission of human
enteropathogens in some cases of foodborne illnesses
(Franz et al., 2005; Johannessen et al., 2005; Solomon
et al., 2001). One possibility is that the lettuce plant
may become infected with E. coli when exposed to
contaminated irrigation water or manure that is used as
fertilizer (Franz et al., 2005; Johannessen et al., 2005;
Solomon et al., 2002).
The aim of this study was to determine if E. coli was
able to colonize the lettuce plant internally through its
root system. E. coli transformed to express green
fluorescent protein was used to study the uptake of E.
coli within the plant tissue (A. Huntley, pers. comm.,
30 Jan 2007).
Materials and Methods
The present study was conducted between April 12 and
April 20, 2007 at Saddleback College Laboratory. A
total of 10 lettuce plants (4 Romaine and 6 Iceberg)
were grown indoors and contained in plastic pots with
drainage holes (Green Thumb International, Lake
Forest, Calif.). All ten lettuce plants were placed inside
a bucket that contained water inoculated with E. coli
transformed to express green fluorescent protein for 48
hours at 23°C (A. Huntley, pers. comm., 30 Jan 2007).
The collected lettuce samples were cut 2 cm above the
soil surface with a sterile surgical knife and washed in
deionized water for one minute (Johannessen et al.,
2004; Solomon et al. 2001). Each lettuce sample was
13
Saddleback Journal of Biology
Spring 2007
blended with Luria-Bertani broth supplemented with
ampicillin in a sterile mason jar for one minute using
an Osterizer™ blender (A. Huntley, pers. comm., 30
Jan 2007). Ten samples (50 μL) were then individually
plated on Luria-Bertani agar that contained ampicillin
(1 mL/100mL of medium). After the plated samples
were incubated at 37°C for 24 hours they were
examined for E. coli transformed to express green
fluorescent protein with fluorescent UV light at 365 nm
(Franz et al., 2005).
Luria-Bertani broth was inoculated with E. coli
transformed to express green fluorescent protein, and a
serial dilution was conducted to determine the final
concentration of E. coli as 3.9 x 105 CFU · mL–1 (A.
Huntley, pers. comm., 20 Feb 2007; Solomon et al.,
2002).
Results
Analysis of the samples with fluorescent UV light
determined that the lettuce plants (n=10) were negative
for contamination of E. coli transformed to express
green fluorescent protein (Table 1).
TABLE 1. Number of positive/total samples based on
soil concentration (3.9 x 105 CFU · mL–1) of E. coli
Plants Examined
Positive Samples/Total
Romaine Lettuce
0/4
Iceberg Lettuce
0/6
Discussion
Recent studies have implicated animal waste as the
primary vehicle for the dissemination of pathogenic E.
coli to vegetable plants (Franz et al., 2005; Ingham et
al., 2004; Johannessen et al., 2005; Kudva et al., 1998;
Solomon et al., 2001; Solomon et al., 2002). A study
conducted by Ingham et al. (2005), for example,
suggested that E. coli might recur in manure-fertilized
soil from the excrement of birds or mammals traveling
through agricultural fields. Although lettuce leaves are
sanitized with chlorine as part of common
manufacturing practice, foodborne infections persist
and research is needed to elucidate all routes of crop
contamination (Solomon et al., 2001; Solomon et al.,
2002). Thus, E. coli infection within lettuce plant tissue
was examined in this study.
Solomon et al. (2002) found that E. coli was able to
enter the roots and become entrapped within the tissue
of mature lettuce plants by means of contaminated soil.
Alternatively, the results of this study showed that E.
coli did not travel through the root system and colonize
the lettuce plant internally. The present study had a soil
concentration of E. coli at 3.9 x 105 CFU · mL-1,
whereas Solomon et al. (2002) reported using a higher
soil concentration of E. coli at 7.5 x 107 CFU · mL-1.
This is an indication that other environmental factors
may have influenced the plants ability to uptake E. coli.
It was interesting to note that the lettuce seedlings in
this study were grown in low concentrations of
contaminated soil and were not infected with E. coli,
which coincides with Johannessen et al. (2005) data.
Moreover, Johannessen et al. (2005) suggested that the
infection of vegetables might be related to the growth
stage of the plant along with the bacterial concentration
of the fertilized soil.
Soil microorganisms may have played an influential
role in the results of this study. Pseudomonads, which
are commonly found in soil, are more plentiful and
acclimated to their environment and may have
inhibited the newly introduced E. coli from penetrating
the lettuce roots (Johannessen et al., 2005). It might be
beneficial to contaminate the soil with different
concentrations of E. coli in future research.
Soil temperature may have also been a significant
determinant in the results of this study. Kudva et al.
(1998) found E. coli to have greater survival rates in
fertilized soil at temperatures below 23°C as compared
to decreased survival rates at temperatures above 37°C.
It might be conducive to place the lettuce plants at
various temperatures, and to study the lettuce at
germination and vegetative growth stages in future
research as well.
In conclusion, the uptake of E. coli was not observed
in the lettuce plant tissue. The results of this study
suggest that lettuce plants are unsusceptible to internal
infection when grown in soil with low concentrations
of E. coli. Additional research would be necessary in
order to ameliorate further crop contamination, and as a
result, aid in the prevention of foodborne infections.
Acknowledgments
The authors acknowledge Dr. Anthony Huntley and
Steve Teh for their assistance during the study.
Literature Cited
Franz, E., van Diepeningen, A.D., de Vos, O.J., van
Bruggen, A.H.C. 2005. Effects of Cattle Feeding
Regimen and Soil Management Type on the Fate of
Escherichia coli O157:H7 and Salmonella enterica
Serovar Typhimurium in Manure, Manure-Amended
Soil, and Lettuce. Applied and Environmental
Microbiology; 71: 6165-6174.
Ingham, S.C., Losinski, J.A., Andrews, M.P., Breuer,
J.E., Breuer, J.R., Wood, T.M., and Wright, T.H. 2004.
Escherichia coli Contamination of Vegetables Grown
in Soils Fertilized with Noncomposted Bovine Manure:
Garden-Scale Studies. Applied and Environmental
Microbiology; 70(11): 6420–6427.
14
Saddleback Journal of Biology
Spring 2007
Johannessen, G.S., Bengtsson, G.B., Heier, B.T.,
Bredholt, S., Wasteson, Y., Rorvik, L.M. 2005.
Potential Uptake of Escherichia coli O157:H7 from
Organic Manure into Crisphead Lettuce. Applied and
Environmental Microbiology; 71: 2221-2225.
Kudva, I.T., Blanch, K., and Hovde, C.J. 1998.
Analysis of Escherichia coli O157:H7 Survival in
Ovine or Bovine Manure and Manure Slurry. Applied
and Environmental Microbiology; 64 (9): 3166-3174.
Solomon, E.B., Potenski, C.J., Matthews, K.R. 2001.
Effect of Irrigation Method on Transmission to and
Persistence of Escherichia coli O157:H7 on Lettuce.
Journal of Food Protection; 65: 673-676.
Solomon, E.B., Yaron, S., Matthews, K.R. 2002.
Transmission of Escherichia coli O157:H7 from
Contaminated Manure and Irrigation Water to Lettuce
Plant Tissue and Its Subsequent Internalization.
Applied and Environmental Microbiology; 68: 397400.
The Effect of L-Arginine on Muscle Circumference and Surface Temperature following
Exercise in Recreational Weight Lifters
Clay Jones and Andrew Starsiak
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Temporarily increasing blood flow in the biceps and triceps during and after exercise is
usually associated with vasodilation. As a precursor to the gas nitric oxide, ingesting the
amino acid L-Arginine prior to exercise will increase the effect of vasodilation by further
relaxing the smooth muscle in blood vessel walls. L-Arginine fuels nitric oxide production,
and since there is evidence of increased blood flow during physical activity, it was predicted
that ingesting seven grams of L-Arginine prior to a moderate arm workout would
temporarily increase blood flow in the biceps and triceps by means of vasodilation. Fifteen
athletic males ages 19-23 who have consistently used L-Arginine for at least one year
performed two arm exercises. The surface temperature and circumference of the biceps
and triceps were measured pre and post exercise. The following day, subjects ingested
seven grams of powdered L-Arginine with 12 fluid ounces of water on an empty stomach 45
minutes prior to exercise, and performed the same two exercises. Measurements of the
biceps and triceps were recorded immediately before and after exercise. The results
showed that ingesting seven grams of L-Arginine prior to exercise had no significant effect
on increasing blood flow after exercise. Without seven grams of L-Arginine, test subjects
averaged a 1.9 cm increase in biceps and triceps circumference as well as a 1.4 °C in
surface temperature post exercise. With seven grams of L-Arginine, test subjects averaged
a 2.1 cm increase in circumference as well as a 1.5 °C in surface temperature post exercise,
indicating an increase in circumference by 3.7 % (p=0.184). These differences were
reflected by biceps and triceps circumference and surface temperature with and without LArginine. Though not statistically significant, a possible explanation for the increased
surface temperature and circumference measurements when ingesting L-Arginine prior to
exercise could be that the L-Arginine increased the production of nitric oxide in the blood
vessels. Thus higher levels of nitric oxide temporarily allow the blood vessels to dilate
leading to an in increase blood flow.
15
Saddleback Journal of Biology
Spring 2007
Introduction
Materials and Methods
The experiment involved fifteen male test subjects
who had already been consistently taking L-Arginine
for at least one year and lift weights recreationally 3-4
days a week. The tests were preformed over a twoday time span, March fourth and fifth of 2007. The
first day involved testing and measuring the subjects
without supplemental L-Arginine. The following day
the subjects performed the exact same workout but
this time with the L-Arginine supplement. Subjects
were given seven grams of powdered L-Arginine with
12 fluid ounces of water on an empty stomach 45
minutes prior to exercise. The results were obtained at
a 24 Hour Fitness center in Irvine. The subjects
performed the two arm workouts at a level of eight
out of ten difficulty scale, ten being the most difficult.
After a 20-minute warm up on a stationary bicycle,
the first exercise was three sets of light weight barbell
curls involving 15 repetitions with 60-second breaks
between each set. After performing bicep curls,
subjects then performed three sets of cable triceps
extensions for 15 repetitions with 60 second breaks
between each set. Before and after performing both of
the exercises, subjects were asked to flex their biceps
and triceps (Figure 1). Right and left arm apex
circumferences were measured with a standard
measuring tape. Surface temperature in the bicep
region were measured as well with an Orion®
infrared thermometer gun. The data were next
statistically analyzed with Paired T-test in Microsoft’s
Excel® to check for statistical discrepancies.
Figure 1. Example of a subject being measured
properly.
Results
The mean circumference for both right and left
arms after exercise, and without the supplemental LArginine achieved a mean increase of 1.9 cm; they
also produced an average bicep temperature of 1.4 Co.
After supplemental L-Arginine the test subjects
achieved 2.1 cm mean increase for their
circumferences, and biceps temperatures of 1.5 Co
means (Figure 2). This was a 3.7% increase in size
and 3.5% in temperature compared to the
supplement’s absence. Even though there was an
increase, there was no statistical difference in arm
circumference and surface temperature between the
use and the absence of supplemental L-Arginine,
therefore disproving our hypothesis.
2.5
2
Mean Increase in cm and Degrees C
Most recreational weight lifters show interest in
legal supplements that may help with skeletal muscle
hypertrophy and skeletal muscle definition. In the
past, there have been numerous studies on L-Arginine
uptake affects for nitric oxide production and blood
flow for aerobic exercise, but not anaerobic exercise.
Numerous studies have been concentrated on the
affects of L-Arginine and blood flow during aerobic
exercise in rats (Deng, et. al., 1996; D’Amours, et. al.
2001). The objective of this study was to examine the
effects of supplemental L-Arginine on the human
skeletal muscle and amount of blood flow to the
biceps and triceps muscles post work out. Previous
studies on humans have mainly been concentrated on
the increase of blood flow in skeletal muscle for
aerobic exercise, but few touch on anaerobic exercise
(Ehsani, et. al. 1997; Ambring, et. al. 1997). The
study was performed to determine if there is any
increase in blood flow for anaerobic exercise, and to
test the validity of supplemental L-Arginine for
recreational weight lifters interested in skeletal
muscle hypertrophy.
1.5
1
Before
After
0.5
0
arm
temp
Measuring Mode
Figure 2. Although there were increases in size and
temperature, there was no statistical difference
between the use and absence of L-Arginine (p=
0.184) for the arm circumference, and (p=0.186) for
surface temperature.
16
Saddleback Journal of Biology
Spring 2007
Discussion
Results showed that L-Arginine did not
significantly increase blood flow in athletic males
during anaerobic exercise. There may be several
reasons why L-Arginine did not significantly affect
the subjects in this study. One reason is the means of
ingestion. As the powdered L-Arginine is orally
ingested, the supplement is digested in the stomach
then absorbed in the small intestine and next the liver,
followed by the tissues of the body. The low pH of
the stomach acid could have decreased the
effectiveness of the supplement, preventing the full
dose of L-Arginine from entering the endothelial cells
for synthesis of nitric oxide (Anderson, 2000).
Enteric-coated L-Arginine capsules may be more
effective for this purpose.
Another reason why L-Arginine did not
significantly affect the subjects is that the production
of nitric oxide was possibly reduced due to a highly
oxidative environment (Galland, 1998). Cigarette
smoke, alcohol use, radiation, cleaning fluids, and
processed foods contribute to oxidative stress. Boger
and others in 1998 speculated that during bouts of
oxidative stress on endothelial cells, production of
nitric oxide would decrease due to lowered activity of
nitric oxide synthesizing enzymes. Though results
were not statistically significant, test subjects
exhibited a slight increase in blood flow during
anaerobic exercise.
Other reasons why this experiment might not have
preformed as expected could be due to the duration of
the exercise. By increasing the volume of repetitions
or sets, blood flow could have increased to that area.
With the additional blood flow to the arms, greater
gains in surface temperature and circumference could
have been obtained. These greater gains could have
adjusted our data to statistically significant increases
in our results.
Literature Cited
Ambring A, Jungersten L, Wall B, Wennmalm A.
1997. Both physical fitness and acute exercise
regulate nitric oxide formation in healthy humans. J.
Appl. Physiol. 82(3): 760-764.
Anderson J. 2000. A role for nitric oxide in muscle
repair: nitric oxide-mediated activation of muscle
satellite cells. J. Cell Sci. 11(5): 1859-1874.
Boger R, Bode M, Thiele W. 1998. Restoring
vascular nitric oxide formation by L-arginine
improves the symptoms of intermittent claudication in
patients with peripheral arterial occlusive disease. J.
Am. Coll. Cardiol. 32:1336–1344.
D'Amours M, Larivière R, Lebel M, Yannick D.
2001. Supplementation with a low dose
of L-arginine reduces blood pressure and endothelin-1
production in hypertensive uraemic rats. Nephrol.
Dial Transplant. 16: 746-754.
Deng L, Deschepper C, Li J, Grove K, Schiffrin E.
1996. Comparison of effect of
endothelin antagonism and angiotensin-converting
enzyme inhibition on blood pressure and vascular
structure in spontaneously hypertensive rats treated
with
NW-Nitro-L-Arginine
methyl
ester.
Hypertension. 28: 188-195.
Ehsani A, Fisher J, Hickner R, Kohrt W. 1997. Role
of nitric oxide in skeletal muscle
blood flow at rest and during dynamic exercise in
humans. Am. J. Physiol. 273(1): 405-410.
Galland A. 1998. L-arginine-induced vasodilation in
healthy humans: pharmacokinetic-pharmacodynamic
relationship. Br. J. Clin. Pharmacol. 46:489-497.
Hiramori K, Kanaya Y, Kobayashi N, Nakamura M.
1999. Effects of l-arginine on lower limb vasodilator
reserve and exercise capacity in patients with chronic
heart failure. Heart. 81: 512-517.
Maxwell A, Schauble E, Bernstein D, Cooke J. 1998.
Limb blood flow during exercise is dependent on
nitric oxide. Circulation. 98: 369-374.
17
Saddleback Journal of Biology
Spring 2007
A Genetic Analysis of the Urinary Odor Produced by Asparagus in Humans
Terrance M. Champieux
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Malodorous urine following the ingestion of asparagus is a well known phenomenon.
This study assumed that the odor is an inheritable genetic trait and looked at the
distribution of the gene that allows some people to be positive for the trait, then applied the
results of a survey to a standard Hardy-Weinberg model. The hypothesis that there are
more people who are positive for the smelly urine than people who are negative was
suggested. One hundred people were asked, “Do you have an unusual odor in your urine
after you eat asparagus?” Of the hundred individuals, 45 had an unusual odor after
consuming asparagus, and 55 did not have an unusual odor. Using a chi-squared analysis a
p-value of 0.3173 was calculated showing no significant difference from the expected to the
actual. These results did not support the hypothesis. Further analysis provided a possible
genotypic frequency for the genotypes. Of the 100 individuals a frequency of 0.0666 for
homozygous dominant (AA), 0.383 for heterozygous (Aa), and 0.55 for homozygous
recessive (aa) was found. AA and Aa being positive for the trait and aa being negative.
Introduction
There is an interesting phenomenon that occurs
when some people eat asparagus. After eating
asparagus they smell an odor in their urine that is
unlike the normal urinary smell. Some individuals are
positive for this phenomenon and some are negative.
For about half the population, asparagus produces the
characteristic odor in their urine (Gearhart and Pierce
1977).
Asparagus contains certain compounds that may
produce a mixture of sulfur containing chemicals that
have a strong, distinctive odor when metabolized and
excreted by some humans (Wolke 1999). The
question from this observation is do only some people
produce the chemicals in their urine or do all people
produce the chemicals but only some people are able
to smell them. Lison et al. suggests in their study that
every person emits the odorous urine and there is a
gene that allows some to smell it and some to not.
There is an agreement among scientists that what
determines which people experience this phenomenon
and which do not is in their genes. However, there is
disagreement on whether or not people have a gene
for producing the pungent smell or they have a gene
for smelling pungent chemicals (Mitchell 2001).
This study assumed that the odor is an inheritable
genetic trait and looked at the distribution of the gene
that allows some people to be positive for the trait,
then applied the results of a survey to a standard
Hardy-Weinberg model. Further study will be
necessary to identify whether it is the gene for
producing the pungent smell or the gene for smelling
pungent chemicals. The question of whether the gene
would be expressed more often than not was
explored. The hypothesis is that there are more people
who are positive for the smelly urine than people who
are negative.
Materials and Methods
For this study one hundred individuals were
randomly selected and interviewed as to whether they
were positive or negative for the distinctive odor
produced after eating asparagus in their own urine.
The question asked to the individuals was, “Does
your urine have an unusual odor after consuming
asparagus?” The sex of each individual and their
answer was noted. If the answer was yes, no further
questions were asked. If the answer was no or that
they were not sure, the individual was asked to verify
their answer by sampling two spears of asparagus and
reporting on whether or not their urine did have an
unusual odor. The totals were added up and, using a
chi-squared
analysis
on
graphpad.com/quickcalcs/chisquared1.cfm, the pvalue and significance were found, using the expected
amount of fifty individuals that would be positive and
the expected amount of fifty individuals that would be
negative. Using the Hardy-Weinberg equilibrium
18
Saddleback Journal of Biology
Spring 2007
equation the genotypic frequency was calculated,
assuming that the gene that produces a positive result
is dominant (AA or Aa) as a previous study found
evidence to support (White 1975).
for heterozygous (Aa), and 0.55 for homozygous
recessive (aa). AA and Aa being positive and aa being
negative.
Results
The results show no significant difference from the
expected amount to the actual amount of individuals
that were positive for the chemicals in their urine and
individuals that were negative for the chemicals in
their urine. This does not support the hypothesis that
there are more people who are positive for the smelly
urine than people who are negative. The HardyWeinberg equation is useful in that it shows the
effects of selection, non random mating, or the factors
that influence the genetic make up of a population. As
the odor of urine probably is not known by
prospective mates, it should not effect selection,
mating, or other factors. The only things that would
affect Hardy-Weinberg equilibrium would be that the
population size sampled was small and there was
gene flow in and out of the population.
In conclusion a larger population sample would
give more accurate results and further study is
required to identify whether it is the gene for
producing the pungent smell or the gene for smelling
pungent chemicals that gives malodorous urine to
humans following the ingestion of asparagus.
Discussion
There were 45 individuals who said their urine was
positive for the unusual odor after consuming
asparagus, and there were 55 individuals who said
their urine was negative for the unusual odor. A chisquared test showed a two-tailed p-value that equals
0.3173
which shows that this difference is not considered to
be statistically significant. (Figure 1).
Using
p2+2pq+q2=1, where p equals the frequency of the
dominant allele (A) and q equals the frequency of the
recessive allele (a), a frequency of 0.0666 was
calculated for the homozygous dominant positive
genotype (AA), 0.383 was calculated for the
heterozygous positive genotype (Aa), and 0.55 was
calculated for the homozygous recessive negative
genotype (aa) (Figure 2).
60
Frequency
50
40
30
Literature Cited
20
Gearhart H, Pierce S. 1977. Volatile organic
components in human urine after ingestion of
asparagus. Clinical Chemistry (23):1941.
10
0
Odor
None
Figure 1. The frequency of individuals that were
positive for an unusual odor in their urine after
eating asparagus was 45 and the frequency of
individuals that were negative was 55 (p=0.317, twotailed).
Genotypic Frequency
0.6
AA
0.5
Aa
aa
0.4
Lison M, Blondheim S, Melmed R. 1980. A
polymorphism of the ability to smell urinary
metabolites of asparagus. British Medical Journal
(281): 1676-1678.
Mitchell S. 2001. Food idiosyncrasies: beetroot and
asparagus. Drug Metabolism and Disposition (29):
539-543.
White R. 1975. Occurrence of S-methyl thioesters in
urines of humans after they have eaten asparagus.
Science (189): 810-811.
0.3
Wolke R. 1999. There’s something about asparagus.
The Washington Post (5/19/99): pg. F.03.
0.2
0.1
0
Genotype
Figure 2. The genotypic frequency of the positive
asparagus/ urine gene in 100 individuals was shown
to be 0.0666 for homozygous dominant (AA), 0.383
19
Saddleback Journal of Biology
Spring 2007
Effect of pH on the Growth of Aspergillus and Rhizopus
Amir Farsijani
Department of Biological Sciences
Saddleback College
Mission Viejo, California USA
Studies have shown that fungi are beneficial to plants and the environment. Some fungi
that colonize the root of the plant and its nearby soil can be helpful for plant growth and
other fungi suck up nutrients for the plant, and as in effect, the plant’s resistance to disease
increases. The impact that acid rain could have on fungi in nature is crucial to some
organisms and the environment. It was hypothesized that different pH levels would affect
fungal growth. Also in this study, the effect of different acidity levels of acid rain on fungi
growth is determined. By growing different fungi in different pH levels, we could
determine how much rain acidity affects living organisms. There were two common fungi
used in this project: Rhizopus and Aspergillus. These two fungi were grown at different pH
levels (5.5, 4.0 and 2.0) in potato dextrose agar and were kept at room temperature for five
days. There were different results for different pH levels. For Rhizopus fungi, at pH level
5.5, out of five Petri dishes, there was growth in all of the Petri dishes. At pH level 4.0 and
2.0, four had grown fungi. For Aspergillus, at pH 5.5, out of 5 Petri dishes, there was
growth in all of the Petri dishes. At pH level 4.0 and 2.0, four had grown fungi.
Introduction
Since Acid Rain negatively influences the plant
kingdom, to what extent does the same acid rain
affect how prosperous the growths of Rhizopus and
Aspergillus are? Ashenden (1989) showed that the
growth of plants is severely inhibited when subjected
to acidity as opposed to soil with an average pH of
5.6. Acid rain has a pH of around 4 and plants can not
survive in that environment. Certain fungi release
base substances, such as ammonia, to counteract the
acidity of the environment that they live in. Naofumi
et al. (2004) showed that microbial cells are capable
of emitting basic substances such as ammonia into
acidic soil to ensure survival. This research considers
that fungi are capable of releasing base substances to
raise the pH of the immediate soil, counteracting
acidity and helping the soil surrounding the plant, to
increase the pH and have a chance for growing and
living. In this study, it was hypothesized that different
pH levels will affect fungi growth. Mushrooms grow
more when it is damp; however, some years have
more mushrooms than others. Is this because of the
acidity of the rain? Many species are dependent upon
fungi for survival. According to Gerardo et al.
(2006), ants cultivate food and allow parasitic micro
fungi to attack it, thus breaking it down. Fungi have
other beneficial applications as well. For instance
fungi, according to Gerardo et al. (2006), could be
used to replace the artificial fertilizers that farmers
use to improve crop yields; therefore, farmers save
money. The expected significance from this study is
to understand how mushroom survival impacts their
environment, including potential symbionts.
Materials and Methods
19.5 grams of potato dextrose was measured with
measuring devices. One liter of water was added to an
Erlenmeyer flask and the potato dextrose was added to the
water. The Erlenmeyer flask was then heated for 15
minutes and Nitric acid was then added. By adding Nitric
acid, the pH of the solution came out to be 5.5. The
solution was then divided into three volumes of 333
Milliliters (ml). One of them remained untouched
(control); two drops of sulfuric acid were then added to
the second volume (to make a pH of 4.0) and 5 drops was
added to the third volume (to give the solution a pH of
2.0.)
After pouring the potato dextrose solutions with
different pH levels into Petri dishes, the Petri dishes with
the pH of 4 and 2 did not get solid. It was decided to put
more potato dextrose solution with a higher volume of
water for the acidic pHs. After putting 6 more grams of
potato dextrose agar to another 200 millimeters (ml) of
water and heating it for 10 minutes, it was poured into the
Petri dishes for the second time; after 15 minutes at room
temperature, the solution became solid and the two
different fungi, Rhizopus and Aspergillus, were added to
the Petri dishes for measuring how acidity affects fungi
20
Saddleback Journal of Biology
Spring 2007
growth and life (Previous grown Rhizopus and
Aspergillus in Bio 3B were used to represent different
fungi in this study.)
After five days the amount of fungi growth was
observed in each Petri dish and the data was recorded in
the lab notebook. Also, Microsoft Office Excel was used
to draw a graph of different pHs and their corresponding
fungi growth.
Results
The Petri dishes containing Rhizopus grew very well in
every condition. All Petri dishes (Rhizopus) with a 5.5
pH, had a substantial growth. Petri dishes (Rhizopus) with
a 4.0 pH and 2.0 pH also had growth. Four of the Petri
dishes in each pH level grew very well and the last saw no
growth. The Petri dishes containing Aspergillus grew very
well in every condition as well. Petri dishes (Aspergillus)
with a 5.5 pH, had a substantial growth. Petri dishes
(Aspergillus) with a 4.0 pH and 2.0 pH, also had growth.
Four of them had significant growth and the last saw no
growth.
Num ber of petri dishes that fungi
was observed
6
pH= 5.5
pH= 4.0
pH= 2.0
5
4
Rhizopus had more fungi growing on the agars compared
to the Aspergillus agars. Figure 1 illustrates different pHs
and their corresponding fungi growth. It was hypothesized
that different pH levels would affect the growth of the
bacteria, and in the effect, it would affect the growth of the
plant. Although that there are different amounts of fungi
and mushroom in every season of the year, it would be
reasonable to say that in rain (acid rain) seasons, low fungi
growth affects the plants with decreased growth. After
executing the study for five days at room temperature, a
significant amount of fungi growth was observed in each
of pH levels. Therefore, there is no conclusive result to
protect the hypothesis. Also, the results can not explain
why mushrooms grow more in a particular season or how
mushroom survival impacts their environment. There may
have been minor fluctuations in the study that may have
influenced the results. The agar that was used in this study
was potato dextrose agar, and it is not the best agar to be
used for this study and also minor changes in temperature
(in the bio 3B lab) might have affected the fungi growth
on the agars where fungi growth was limited.
Literature Cited
Ashenden TW, Bell SA. 1987. The effects of simulated
acid rain on the growth of three herbaceous species grown
on a range of British soils. Environ Pollut. 48 (4): 295310.
3
Ashenden TW, Bell SA. 1989. Growth responses of
three legume species exposed to simulated acid rain.
Environ Pollut. 62 (1): 21-29.
2
1
0
Rhizopus
Fungi
Aspergillus
Figure 1.two fungi and their corresponding growth, out of 5
petri dishes containing different acidity levels in potato
dextrose agar
Discussion
Generally, different pH levels have major impacts on
different living organisms. 72 percent of the destruction in
the forests is caused by acid rain (Ashenden, 1987). Fungi
grew in every condition that is possible (acidity wise);
different pH levels did not affect the growth of fungi; thus,
we can infer that different pH levels would not affect
fungi living in nature and around living organisms. In both
of the fungi (Rhizopus and Aspergillus), the results of
how much the fungi grew, were slightly different as the
Gerardo NM, Mueller UG, Currie CR. 2006. Complex
host-pathogen co-evolution in the Apterostigma fungusgrowing ant-microbe symbiosis. BMC Evol Biol. 3 (6):
88.
Naofumi S, Takako Y, Yoku I, Noriko K, Saori I,
Tomohisa K, Shigeo K. 2004. Characteristics of
Neutralization of Acids by Newly Isolated Fungal Cells.
Journal of Bioscience and Bioengineering. 97 (1): 54-58.
21
Saddleback Journal of Biology
Spring 2007
Effect of 5-Hydroxytryptophan on Weight in Mice (Mus musculus)
Amantha Bagdon and Nam Phuong Nguyen
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
The purpose of this experiment was to examine the effect of a serotonin supplement, 5Hydroxytryptophan, or 5-HTP, on the metabolism of mice (Mus musculus) and to determine
the effectiveness of 5-HTP as a beneficial dietary supplement. The study was conducted
using 20 mice, ten mice were the controls and their water source did not contain any 5-HTP.
The remaining ten out of the 20 mice subjects had 5-HTP introduced into their diets by
dissolving the supplement into their source of water. The amount of water and food intake
was monitored and recorded every other day and the weight of the mice was recorded once
a week throughout the course of the experiment. At the end of the experiment, the weights
of the mice were compared. The results demonstrate that 5-HTP was effective as a
beneficial dietary supplement. The mice receiving 5-HTP in their diets gained an average
weight of 3.00 g by the end of the experiment, while the mice that received no 5-HTP in their
diets gained an average weight of 6.40 g, 3.40 g more than the mice that were taking 5-HTP.
The success of 5-HTP as a weight gain inhibitor may lead to an increased use of 5-HTP as a
productive dietary supplement by consumers seeking to lose weight.
Introduction
The experiment was conducted using 5Hydroxytryptophan, or 5-HTP, a serotonin
supplement extracted from the seeds of Griffonia
simplicifolia, a West African medicinal plant.
Serotonin is a neurotransmitter that plays in an
important role in the regulation of mood, sleep, and
appetite. High levels of 5-HTP are believed to cause
the serotonin-producing neurons in the brain to
increase production. Increased serotonin production
leads directly to an increased serotonin release. Low
levels of serotonin in the brain has been affiliated
with several disorders including anxiety disorders,
clinical depression, obsessive-compulsive disorder,
and severe migraines (Fernstrom, et. al., 2000).
Although 5-HTP has been marketed by health food
companies as an alternative solution to prescription
medications for depression and other mood disorders,
the studies to date regarding the use of 5-HTP as an
effective alternative are incomplete and require
further trials before a firm conclusion can be formed
(Antonucci, et. al., 1992; Cairella, et. al., 1991).
Health food companies have also marketed
5-HTP as a solution for weight control. Studies
suggest that 5-HTP decreases carbohydrate and fat
consumption in individuals by stimulation a sense of
satiety, which results in a decreased food intake
(Angelico, et. al., 1998; Cooper, et. al., 2000; Cowen
and Smith, 1999). The supplement also works by
curbing cravings for sugars and carbohydrates
(Cairella, et. al., 1989; Elte-de Wever, et. al., 1996).
The project studied the effectiveness of 5-HTP on
weight control in Mus musculus.
Materials and Methods
The experiment included 20 mice at the starting
age of three weeks old. The mice were divided into
two groups and each group was kept in a glass
terrarium. The first group of mice was the control
group, consisting of ten mice raised under normal
conditions: they were kept at room temperature and
received a regular diet of mouse food and water that
was free of 5-HTP. The second group of mice was the
variable group; these ten mice were raised under
similar conditions with the exception that 5-HTP was
added into their source of water. Fifty mg of the
supplement was dissolved in 2.5 L of water to make
the concentration of 5-HTP in the water 1mg/50mL.
At the beginning of the experiment, the initial weights
of the three-week-old mice were recorded. After that,
the weights of the mice were recorded once a week to
monitor the significance of change in the weight. The
completion of the experiment came at the end of a
four-week period. All data were then transferred to
22
Saddleback Journal of Biology
Spring 2007
MS Excel (Microsoft Corporation, Redmond,
Washington) where all further statistical formulations,
such as a two-tailed, unpaired t-test, were performed
and descriptive statistics were determined.
Results
The average overall weight gained (Figure 1) by
the control mice (6.40 + 0.61 g; n=10) averaged 3.40
g greater than the average overall weight gained by
the mice receiving 5-HTP (3.00 + 0.38 g; n=10) in
this study (two-sample t-test, p=0.01). The frequency
of mice that gained more than 3.00 g (Figure 2) is
greater for the control mice than for the mice
receiving the 5-HTP.
8
6
6
5
5
4
Confidence
Level = + 0.38 g
4
Frequency
Average Overall
Weight Gained (g)
7
Confidence
Level = + 1.39 g
located in the hypothalamus of the brain, which then
releases a hormone that acts on the MC4R to reduce
appetite (Alexander and Leibowitz, 1998). Serotonin
also blocks other neurons that inhibit the activity of
MC4Rs that cause an increase in appetite. Blocking
this inhibitory action prevents the occurrence of an
increase in appetite.
The average overall weight gained by the
control mice was 6.40 g, which is 3.40 g greater than
that of the variable mice, which gained an overall
weight of 3.00 g. These findings agree with previous
studies regarding the effectiveness of 5-HTP on
weight control. The high efficacy of 5-HTP in
reducing weight gain suggests that 5-HTP should be
marketed to consumers as an effective and beneficiary
dietary supplement.
3
3
2
2
1
1
0
2
0
Control
1
Discussion
The
results
demonstrated
that
5Hydroxytryptophan was effective in treating weight
control. The effectiveness of 5-HTP is due to the fact
that it increases the amount of serotonin in the brain.
Serotonin activates melanocortin-4 receptors, or
MC4Rs, which studies have shown that mutations in
this gene are related to morbid obesity. Drug-induced
serotonin also activates pro-opiomelanocortin
neurons, or POMC, in the arcuate nucleus, or ARC,
4
5
6
7
Average Weight Gained (g)
5-HTP
5-HTP
Figure 1. Mean overall weight gained by 10 mice that
did not receive 5-HTP in their diets and 10 mice that
did receive 5-HTP in their diets. Mean overall weight
gained by the control mice (6.40 + 0.61 g; n=10) was
greater than the mean overall weight gained by the
variable mice (3.00 + 0.38 g; n=10). Mean overall
weight gained by the control mice is significantly
different from mean overall weight gained by the
variable mice (p=0.01, two-tailed, unpaired t-test).
Error bars show 95% confidence interval.
3
8
9
Control
Figure 2. Frequency distribution of average
weight gained by 10 mice that did receive 5-HTP
in their diets and 10 mice that did not receive 5HTP in their diets for this study (p=0.01).
Literature Cited
Alexander, J.T. and Leibowitz, S.F. (1998).
Hypothalamic serotonin in control of eating behavior,
meal size, and body weight. Biological Psychiatry
44(9): 851-64.
Angelico, F., Cangiano, C., Cascino, A., Del Ben, M.,
Laviano, A., Preziosa, I., and Rossi-Fanelli, F. (1998).
Effects of oral 5-hydroxy-tryptophan on energy intake
and macronutrient selection in non-insulin dependent
diabetic patients. International Journal of Obesity
and Related Metabolic Disorders 22(7): 648-54.
Antonucci, F., Cangiano, C., Cascino, A., Ceci, F.,
Del Ben, M., Laviano, A., Muscaritoli, M., and RossiFanelli, F. (1992). Eating behavior and adherence to
23
Saddleback Journal of Biology
Spring 2007
dietary prescriptions in obese adult subjects treated
with 5-hydroxytryptophan. American Journal of
Clinical Nutrition 56(5): 863-7.
Cairella, M., Cangiano, C., Cascino, A., Ceci, F., Del
Ben, M., Muscaritoli, M., Rossi-Fanelli, F., and
Sibilia, L. (1989). The effects of oral 5hydroxytryptophan administration on feeding
behavior in obese adult female subjects. Journal of
Neural Transmission 76(2): 109-17.
Cairella, M., Cangiano, C., Cascino, A., Ceci, F., Del
Ben, M., Muscaritoli, M., and Rossi-Fanelli, F.
(1991). Effects of 5-hydroxytryptophan on eating
behavior and adherence to dietary prescriptions in
obese adult subjects. Advances in Experimental
Medical Biology 294: 591-3.
Serotonin function following remission from bulimia
nervosa. Neuropsychopharmacology 22(3): 257-63.
Cowen, P.J., and Smith, K.A. (1999). Serotonin,
dieting, and bulimia nervosa. Advances in
Experimental Medical Biology 467: 101-4.
Elte-de Wever, B.M., Hopman, E., Meinders, A.E.,
Pijl, H., and Toornvliet, A.C. (1996). Serotoninergic
drug-induced weight loss in carbohydrate craving
people. International Journal of Obesity and Related
Metabolic Disorders 20(10): 917-20.
Fernstrom, J.D., Fernstrom, M.H., Gendall, K.A.,
Kaye, W.H., McConahan, C.W., and Weltzin, T.E.
(2000). Effects of acute tryptophan depletion on
mood in bulimia nervosa. Biological Psychiatry
47(2): 151-7.
Cooper, T.B., Finkelstein, D.M., Jimerson, D.C.,
Levine, J.M., Metzger, E.D., and Wolfe, B.E. (2000).
Effect of Soil Inoculant on Growth of Phaseolus vulgaris L. and Glycine max
Christine Nguyen and Kevin Laitipaya
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Plants and bacteria have evolved together to perfect and mold a symbiotic partnership
that has greatly advanced their ability to survive. Beneficial bacteria in Dr. Earth’s Super
Active soil and seed inoculant, which includes nitrogen fixing bacteria, was used to
investigate whether plants can be over nourished by this partnership and hinder growth.
Separate studies were conducted on the garden variety plants Phaseolus vulgaris L. (pea)
and Glycine max (soybean) to test different concentrations of beneficial bacteria including
Bradyrhizobium japonicum and its correlation to stem to root biomass and the formation of
lateral roots (LR), respectively. The study conducted on Phaseolus vulgaris L. yielded
results showing no significant difference when comparing the stem to root biomass ratio of
low concentration (of inoculant) to high concentration (P=0.143, two-tailed test assuming
unequal variances). In the study of G. max and the effect of differing concentrations of
inoculant, a significant difference was found (P<0.001, ANOVA). Further analysis
(ANOVA Post-Hoc) revealed a significant difference (P< 0.05) when comparing
concentrations below recommended concentration (CBRC) or recommended concentration
(RC) to concentration above recommended concentration (CARC). However, no significant
difference was seen when comparing recommended concentration (RC) to CBRC (P> 0.05).
The data obtained from the pea plants supports the hypothesis. In addition the data
obtained from the soybean seedling shows that there is no benefit in adding more inoculant
(bacteria) than is recommended, since at CARC LR formation diminish, from those seen at
RC and CBRC.
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Saddleback Journal of Biology
Spring 2007
Introduction
The symbiotic partnership between nitrogen-fixing
bacteria (Rhizobium) and plants provides great
beneficial advancements for the growth of plants
(Sachs, 2004). This partnership allows the availability
of usable nitrates fixed by the bacteria that is
necessary for plant growth. It is formed from the
invasion of nitrogen-fixing bacteria onto the roots of
the legumes. Root nodules are formed by the plant
upon infection of the root hairs and lateral roots,
which acts as a new organ in the plant that functions
solely for nitrogen fixation (Mathesius, 2000). It is
shown that nodulation will only occur at the sites of
mature root hairs and lateral roots due to a surplus of
plant flavonoids to induce the bacterial genes required
for nodulation (Mathesius, 2000). Thus, soil
inoculants that supply nitrogen-fixing bacteria will
also supply other beneficial bacteria to promote root
growth to increase the occurrence of nodulation.
These nodules then can take the natural N2 from the
atmosphere and convert it into NH3 thus allowing the
plant to break down the nitrogenous compound so
that it may get its needed nitrogen (Kaminski et al.,
1998; Makarova, 2004; Spaink, 2000). However, can
plants be over invaded with nitrogen-fixing bacteria
to cause too many nodules to grow and produce a
lethal amount of nitrates? Findings have shown that
plants are capable of responding to their environment,
thus not allowing over invasions of bacteria to occur
(Gage, 2004).
In order to investigate whether high concentrations
of beneficial bacteria can hinder the growth of plants,
a study was performed using varying concentrations
of beneficial bacteria and looking at its effects on the
plant. In this study we conducted two experiments
that test different concentrations of beneficial bacteria
along with Bradyrhizobium japonicum and its
correlation to biomass and the formation of lateral
roots (LR). Since the amount of lateral roots will
increase the amount of root hair present, this leads to
bacterial invasion and nodule formation (Cardenas,
2006). In doing so, data gathered from this study will
broaden the understanding of plants and how they
interact with bacteria.
Materials and Methods
Inoculant concentrations and inoculation methods
of Phaseolus vulgaris L.
Phaseolus vulgaris L. (beans) were grown in
different concentrations of Dr. Earth’s Super Active
soil and seed inoculant at Saddleback College
greenhouse. Five varying concentrations of the
following amount of inoculant were mixed with 1.00
L of DI water: 6.00, 3.00, 1.50, 0.75, 0.00g of
inoculant. Test groups were watered twice with
inoculant solution. First inoculation occurred one day
after the seeds were sowed and the second inoculation
occurred one month after. Each container in a test
group was watered with 0.100 L inoculant solution.
Prior to the planting of seeds and inoculation, the
soil of each test group were baked for a 24 hour
period at 90°C to ensure that any pre-existing bacteria
would not affect the results of the study once the seed
is planted. Soil was sterilized collectively. Soil was
sterilized on February 16, 2007 and was sown the
following day. Each group consisted of 10 plants and
each container housing the plants held 200 mL to 300
mL of sterilized soil. Sixty-seven days after sowing
(DAS), plants were taken out of the soil and the roots
were cleaned of soil by means of washing through
water. The number to lateral roots formed were tailed
and recorded. Plants were sectioned into roots (cut at
first lateral roots and included all mass of taproot only
below cut), stem (section above root and below
cotyledon), and other (cotyledon and up), and were
dried in a drying oven at 15.6°C for 24 hours.
Dried stem and root masses were then entered into
Microsoft Excel® (Microsoft Corporation, Redmond,
Washington) under their specified concentration
groups. Data were then further analyzed by separating
the data into low concentration and high
concentration group. A two-tailed t-test (assuming
unequal variances) was then ran on the two sets of
data
Inoculant concentrations and inoculation methods
of Glycine max
A total of 90 Glycine max (soybean), a commercial
variety from Botanical Interests ™, were used in this
study. A dilution was performed to obtain the
following six concentrations of Dr. Earth’s Super
Active soil and seed inoculant: 4x, 1x (recommended
concentration RC, control group), 1/4x, 1/8x, 1/32x,
1/64x, and no inoculant. Five Petri dishes were used
with each concentration. Each Petri dish included
three seeds and filter paper. Then 3.00 mL of
inoculant was pipeted into each Petri dish.
Petri dishes were then covered and placed in a
dark environment at room temperature (23°C) to
induce germination for 15 days. Throughout the
experiment the seeds were kept moist by adding
1.00mL of DI H2O into each Petri dish every two
days to ensure seed growth.
Lateral roots were tallied and then entered into
Microsoft Excel® (Microsoft Corporation, Redmond,
Washington). The data was categorized into three
groups: CBRC, CARC, and RC. Statistical analysis
(ANOVA) was performed on the three groups.
25
Saddleback Journal of Biology
Spring 2007
Results
Phaseolus vulgaris L. Data
Dry weight data was obtained from 42 plants.
Mean root mass and stem mass values were
calculated from each group (Figure 1).
0.3
An ANOVA statistical test was performed on the
three groups that showed a significant difference (P<
0.001). An ANOVA Post-Hoc was then computed.
Results show when the RC was compared to CBRC
there is no significant difference (P>0.05). However,
when comparing the RC to the CARC and the CBRC
to the CARC there was a significant difference
(P<0.05).
0.25
30
0.15
0.1
0.05
0
0
0.75
1.5
3
6
Lateral Roots
Mass (g)
0.2
25
20
15
10
Inoculant (g)
5
0
Figure 1. Mean of root mass (■) in 0 (n=9), 0.75
(n=9), 1.5 (n=6), 3(n=9), and 6g (n=7) of inoculant
were 0.033, 0.027, 0.038, 0.032, and 0.028g
respectively. Mean of stem mass (□) in 0 (n=9), 0.75
(n=9), 1.5 (n=8), 3 (n=9), and 6g (n=7) of inoculant
were 0.18, 0.20, 0.22, 0.25, and 0.20g respectively.
Stem to root mass ratios were then calculated and
ran through a statistical two-tailed t-test (assuming
unequal variances). Stem to root mass ratios were
then organized into two groups; low concentration (0,
0.75, and 1.50g) and high concentration groups (3.0
and 6.0g) (Table 1). Statistical analysis of the grouped
data showed that there were no significant differences
between the data from low to high concentrations
(P=0.143).
Table 1. Stem mass to Root mass ratios for each
concentration group shown under its specific
concentration group (ie. Low or High).
Low
High
Concentration
Concentration
Concentration(g) 0.00 0.75 1.50 3.00
6.00
Stem mass (g)
5.49 7.31 5.76 7.67
7.16
Root mass(g)
Glycine max Data
LR was tallied from a total of 64 G. max seedlings.
The concentration groups studied were 0 (n=8), 1/64
(n=7), 1/32 (n=6), 1/8 (n=10), 1/4 (n=11), 1 (n=4),
and 4 x RC (n=12). The distribution of mean LR
formed for each group is shown in Figure 2. The
highest mean of LR were seen in the RC group (28.5
LR), and the lowest mean of LR were seen in the 4x
RC group (6 LR).
0.00
0.02
0.03
0.13
0.20
1.00
4.00
Inoculant Concentration (x RC)
Figure 2. Mean of LR formed on Glycine max
seedlings under differing inoculant concentrations: 0,
0.02, 0.03, 0.13, 0.25, 1, and 4 x RC; with means of
25.6, 27.4, 20.7, 20.4, 23.5, 28.5, and 6 respectively.
Discussion
Inoculant
The soil inoculant by Dr. Earth was used for this
study because it contained Bradyrhizobium
japonicum which was a bacterium which causes root
nodulation on a specific host plant. In the case of B.
japonicum the host plant is Glycine max.
The soil inoculant also contained other bacteria
which included: Bacillus subtilis, Bacillus cereus,
Bacillus
megaterium,
Azobacter
vinelandi,
Lactobacillus acidophilus, and Asperigillus oryzae.
Majority of these bacteria are found in the soil,
however; usually not at the concentrations found in
this soil inoculant (Bai et al., 2001, 2002).
Most of the bacteria (if not all) help the plant with
degradation of other plant material that can be used as
a chemical resource (such as N) necessary for growth.
Azobacter vinelandii helps with the fixation of N2
(Ehaliotis et al., 1999; Moreno et al., 1990).
Lactobacillus acidophilus helps in the breakdown of
lignin and cellulose. Aspergillus oryzae is a fungus
which helps with the separation of soil and
degradation of other plant materials.
Other than Bradyrhizobium japonicum the only
other bacteria of importance in the inoculant is
Bacillus subtilis, cereus, and megaterium. These
bacteria have profound effects on not only the upkeep
26
Saddleback Journal of Biology
Spring 2007
of soil but also the health of the plant (Bai et al. 2001,
2002; Bernhard et al., 1978).
Effect of soil inoculation on Phaseolus vulgaris L.
In this study pea seedlings were sowed and then
inoculated twice to ensure that all bacteria were
present in solution and that runoff did not occur after
first inoculation. Peas were inoculated with varying
concentrations of inoculant in order to determine if
there was a significant difference between groups
which had low concentrations of inoculant vs. those
with high concentrations. Previous studies have found
that by introducing bacteria such as Bacillus subtilis
along with other “good” bacteria a significant
difference should be seen in almost every aspect of
the plant, such as shoot mass, root mass, and nodule
mass (Bai et al., 2001, 2002).
Results indicate that statistically there was no
significant difference between the data (P=0.143). In
other words, this data indicates that plant growth at
higher concentrations shows more growth than seen
in lower concentrations; however, this growth is not
significant. This may indicate the plants’ ability to
regulate intake nutrients from the soil. This p value
was obtained by running a two-tailed t-test (assuming
unequal variances) on the stem mass to root mass
ratio. It is therefore possible that differing results may
be obtained of t-test were ran on other values. It is
also important to note that once the pea plants grew
large enough, they were pinched back in order to
stimulate nodulation of root hairs. Therefore if this
experiment were to be performed again, it is advisable
that terminating pea seedlings be used in order to
allow for growth which terminates after the peas have
fruited. This would allow for cleaner data and would
allow for the researchers to have a better view of the
effects of soil inoculants on stem mass and root mass.
Another symbiosis of importance between pea
plants and bacteria is of the one seen with Rhizobium.
The Phaseolus vulgaris L. specific Rhizobium strain
is Rhizobium leguminosarum (Schultze and
Kondorosi, 1998; Berkum et al., 1996; HernandezLucas et al., 1995; Albrecht et al., 1999; Bai et al.,
2002; Makarova et al., 2004). Most recent research in
terms has been geared towards Rhizobium due to its
ability to fix nitrogen to the plants that it infects. This
symbiosis was not studied because the researchers
were unable to attain R. leguminosarum.
Effect of soil inoculation on Glycine max
In this study, varying soil inoculant concentrations
were added to G. max seedlings prior to germination
in order to determine its effects on LR formation.
Previous studies have correlated LR formation with
root nodulation, due to the increased amount of root
hairs found on the lateral roots (Hoffman).
It was hypothesized by the researchers that at high
concentrations no significant difference would be
seen because the host organism (G. max) would be
able to regulate the effects of the bacteria found in the
inoculant. In addition, the LR formation would not
differ significantly from what was seen in the lower
concentration groups. However, this was not the case.
According to the results there was a significant
difference seen between RC or CBRC to CARC (P<
0.05). It is also important to note that as the
concentration of inoculant increased past the RC, LR
count decreased. This decrease in the LR formation
could be due to high concentration of bacteria which
at such high levels could have become detrimental to
the plant. However due to amount of bacteria in the
inoculant it is difficult to say which bacteria is
responsible.
It is important to note that the highest
concentration group (4x) rotting of the seedlings
occurred 14 days after inoculation. This could be due
to one of the many bacteria found in the soil
inoculant. There was no available test to run (at the
disposal of the researchers) to determine to source of
the rotting.
Of more importance is that no nodulation
occurred. However this might be expected due to the
amount of time seeds were left to germinate.
Conclusion
The results have shown that at higher
concentrations of inoculant no significant growth was
seen in either of the experiments. This leads the
researchers to conclude that plants that have evolved
over times with bacteria, such as Glycine max with B.
japonicum have managed to self regulate the amount
of bacteria that infects it (in the case of Rhizobium).
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Bai Y, Zhou X, Smith DL. 2002. Enhanced Soybean
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27
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Spring 2007
Berkum P, Beyene D, Eardly BD. 1996. Phylogenetic
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Makarova L, Sokolova M, Akimova GP, Luzova GB,
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leguminosarum. Agrokhimiia; 12: 29-35.
Ehaliotis C, Papadopoulou K, Kotsou M, Mari I,
Balis C. 1999. Adaptation and Population Dynamics
of Azotobacter vinelandii During Aerobic Biological
Treatment of Olive-Mill Wastewater. FEMS
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MA. 2000. Rhizobia Can Induce Nodules in White
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Gage DJ. 2004. Infection and Invasion of Roots by
Symbiotic, Nitrogen-Fixing Rhizobia During
Nodulation of Temperate Legumes. Microbiology and
Molecular Biology Reviews; 68(2): 280-300.
Moreno J, De La Rubia T, Ramos-Cormenzana A,
Vela GR. 1990. Growth and Nitrogenase Activity of
Azotobacter vinelandii On Soil Phenolic Acids.
Journal of Applied Bacteriology; 69(6): 850-855.
Hernandez-Lucas I, Segovia L, Martinez-Romero E,
Pueppke SG. 1995. Phylogenetic Relationships and
Host Range of Rhizobium spp. That nodulate
Phaseolus vulgaris L.. Applied and Environmental
Microbiology; 61(7): 2775-2779.
Sachs JL, Mueller UG, Bull JJ. 2004. The Evolution
of Cooperation. The Quarterly Review of Biology;
79(2): 135-160.
Hoffman F, Akashi R, Hoffman-Tsay S. Nodulation
in Legume Root Cultures: micrografted or
Schultze M, Kondorosi A. 1998. Regulation of
symbiotic root nodule development. Annual Review
of Genetics; 32(2): 33.
28
Saddleback Journal of Biology
Spring 2007
The Effect of Monovalent, Divalent, and Trivalent Cations on
Transmembrane Plasmid Transport in Escherichia coli
Kevin Mandala and Greg Newkirk
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
During plasmid transformation, calcium-like cations bond to Poly-BHydroxybutyrate/Calcium Polyphosphate membrane complexes that are formed in
cytoplasmic membranes of Escherichia coli. In this experiment, cations were tested to see
whether cations with different geometric configurations than calcium would be able to
complex with the Poly-B-Hydroxybutyrate and facilitate transformation of an ampicillinresistant plasmid. Solutions of BaCl2, Fe(NO3)3, KCl, and CaCl2 were made, applied to
competent cells in an attempt to make them permeable, spread, and then grown for 24
hours. The results showed that only cations with geometric configurations similar to
calcium were able to facilitate transformation; cations with altered geometric
configurations were unable to facilitate the complex for transformation of the ampicillinresistant plasmid.
Introduction
Reproduction is a staple of biological life forms.
Allowing organisms to transfer DNA from one to
another, reproduction allows the progression of strong
DNA through kin. There are various means as to how
cells may acquire new DNA: conjugation,
transfection, and transformation. Viruses called
bacteriophages inject their DNA into the host in a
process known as transfection. Conjugation of DNA
happens when two cells mate and transfer DNA. And
while rare in nature, transformation occurs when the
cell membrane becomes permeable, and accepts DNA
through the cell membrane. Even though cell
transformation accounts for a very small percentage
of DNA swapping in nature, it has become an
invaluable tool in biochemistry.
Cell transformation allows the insertion of
plasmids, containing DNA, into cells. Recently, E.
coli has been injected with DNA to express
recombinant proteins, such as insulin for humans
(Huang and Reusch, 1996). Bacteria, laced with
injected DNA to help them survive the harshest of
environments, are found in bioremediation to help
clean up toxic materials, such as oil spills (Reusch
and Sadoff, 1988). With the importance of
bioengineering, much emphasis has been put on the
expression of important traits in the laboratory.
While the cell membrane’s affinity to calcium is
well documented, not much has been known about the
actual mechanism that allows plasmid DNA to enter
into the cell (Jones, et. al., 2003). However, recently,
complexes of Poly-B-Hydroxybutyrate (PHB) were
found to have formed in membranes of E. coli that
had been made competent by cold calcium buffers;
these complexes facilitated the transport of DNA
through the cell (Reusch, 1999).
The main aim of the hypothesis is to test various
cations, Ba2+, Fe3+, K+, and Ca2+, and to see whether
the cations will be able to facilitate transformation of
an ampicillin-resistant plasmid (pAMP). If the cell
transformation does not occur, we can infer that the
PHB membrane complexes failed to form.
Materials and Methods
In preparation of the experiment, Luria-Bertani
(LB) broth was made from 10.0g of triptone, 5.00g of
yeast, 10.0g of NaCl, and 18.0g of agar. With this LB
broth, 48 plates were made; 24 plates with ampicillin
and 24 without. Next, 50.00 mM solutions of calcium
chloride (CaCl2), potassium chloride (KCl), barium
chloride (BaCl2) and iron (III) nitrate (Fe(NO3)3)
were added to Erlenmeyer flasks. The CaCl2, BaCl2
and the KCl solutions were buffered to a pH of 7,
while the Fe(NO3)3 solution was buffered to a pH of
5; due to iron’s insolubility in water, the most
applicable pH to buffer the solution to was a pH of 5.
While the pH of iron varied from the others, a pH of 5
is still scientifically accurate because it is the same
pH of tap water. A starter plate was made by plating
cultures of E. coli onto standard agar plates and left to
grow for two days in an incubator.
Sixteen transformation tubes were marked with
their according cation solution; four for each cation.
29
Saddleback Journal of Biology
Spring 2007
Next, 250.0 µl of each solution was pippetted into the
tube, and put on ice. Using a sterile inoculating loop,
colonies of E. coli were transferred from the starter
plate into the solution of cations. Immediately
following, the competent cells were suspended with a
micropipette. 10.00 µl of 0.005 µg/µl plasmid (m288)
was placed directly into the solution, and then put on
ice. The tubes were then left to set for a fifteen minute
incubation period on ice. All tubes were then heat
shocked in a 42.0 ºC water bath for 90 seconds, and
immediately placed onto a room temperature test tube
rack. 250.0µl of LB broth was then placed into each
test tube. For spreading, 100.0 µl of solution was
placed onto the according plate: LB agar plate with
ampicillin resistance plasmid, LB agar plate without
ampicillin resistance plasmid, LB agar plate with
ampicillin and no ampicillin resistance plasmid, and
LB agar plates with ampicillin and ampicillin resistant
plasmids. Sterilizing between each application, the
solutions were spread on plates, and then put into a
37.0°C incubator for 24 hours.
Results
The results of the experiment were distinct and
discriminate. All positive controls, LB agar plates
with and without plasmids for resistance, grew lawn
bacteria; this shows that while transformation of
critical DNA was not critical, all DNA were able to
replicate on their own in a stable environment. If
these controls did not grow, then it would show the
bacteria were unable to replicate to begin with, so
transformation would be impossible. The negative
control, LB agar plate with ampicillin but no
ampicillin resistant plasmid, all failed to grow (Table
1). Negative controls allowed us to see that without
the ampicillin resistant plasmid, the bacteria would
indeed die. Our controls showed clearly that not only
were the cells able to reproduce, but none of them had
natural resistance to the ampicillin.
The experimental plates also had clear results
between the different cations. K+ and Fe3+ were both
unable to make the cell permeable enough to transfer
the DNA through the cell membrane. However, the
plates with Ca2+ and Ba2+ clearly were able to survive,
which was obvious by the presence of lawn bacteria
(Table 1). In our results, it was clear that Ca2+ and
Ba2+ were the only cations that allowed any kind of
transformation of plasmids through the cell
membrane.
Table 1. K+ and Fe3+ lawn growth of E. coli observed
on plates with and without ampicillin resistance.
Cells were transformed with an ampicillin-resistance
plasmid (pAMP). The negative control consisted of
the LB/amp plate with nontransformed cells, and the
positive controls were LB broth plates with and
without transformed cells.
Broth
Transformed
Nontransformed
LB
Growth
Growth
LB/amp
No growth
No growth
Table 2. E. coli growth observed after the cells were
transformed with Ca2+ and Ba2+ and an ampicillinresistance plasmid (pAMP). All plates, of Ca2+ and
Ba2+ cations, with the pAMP grew E. coli.
Broth
LB
LB/amp
Transformed
Growth
Growth
Nontransformed
Growth
No growth
Discussion
CaCl2 and BaCl2 were the only solutions that
were capable of making the cells uptake the
ampicillin resistant plasmid. Ca2+ and Ba2+, the
cations in CaCl2 and BaCl2, showed that there was a
distinct difference in the cations capable of causing
cell transformation from those that were not. These
results show that cell transformation is ion specific
based on cation shape, and cannot be achieved by any
cation.
With the addition of Ba2+ to the environment, the
cell was able to transport the DNA across the
membrane; the common shape of Ba2+ to Ca2+ played
a crucial role in the formation of PHB/Ca2+/polyP
(Pavlov, et. al., 2005). Since K+ and Fe3+ failed to
facilitate transformation, we can infer that these two
cations failed to form proper channels to transport
DNA into the cell. If PHB/Ca2+/polyP, the complex
responsible for channeling plasmids through the cell
membrane, was not cation shape dependant, these
cations would have been able to transform DNA and
become resistant to the ampicillin.
In 1995, Reusch and Huang discovered that the
selectivity of Ca2+ extends passed the cation charge.
They found that Zn2+, a cation with nearly the same
shape and charge as Ca2+, fails to facilitate any kind
of transformation. From that study, Reusch and
Huang concluded that the cation binding sites of PHB
must contain seven or eight oxygen ligands in
irregular geometry. While this model for the ligands
of the complex holds up to earlier work done by
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Saddleback Journal of Biology
Spring 2007
Reusch, it is still not known how the PHB complex
differentiates between Ca2+and Zn2+ cations.
While the experiment suggests that cations with
similar geometric configurations will complex with
the PHB to facilitate transformation, further research
is required to determine whether all cations with
similar shapes will have the same affects as Ca2+ and
Ba2+. Also, research could be done to look at the
efficiency of transformation if PHB were added to a
solution, rather than having the cell manufacture it.
PHB is known to be required in cell transformation,
but at what stage the PHB is needed is not entirely
known (Reusch, et. al., 1988). Research into
facilitation efficiency of certain plasmids could help
science get a better understanding of how to induce
and speed up transformation.
Literature Cited
Huang, R., & Reusch, R.N. (1996). Poly(3hydroxybutyrate) is associated with specific proteins
in the cytoplasm and membranes of Escherichia coli.
The Journal of biological chemistry, 271(36), 22196202.
Jones, H.E., Holland, I.B., Jacq, A., Wall, T.,
Campbell, A.K.(2003). Escherichia coli lacking the
AcrAB multidrug efflux pump also lacks
nonproteinaceous,
PHB-polyphosphate
Ca2+
channels in the membrane. Biochimica et biophysica
acta, 1612(1), 90-7.
Reusch, R.N., Hunag, R. (1995). Genetic Competence
in
Escherichia
coli
Requires
Poly-BHydroxybutyrate/Calcium Polyphosphate Membrane
Complexes and Certain Divalent Cations. Journal of
Bacteriology, 486-490.
Reusch, R.N., Hunag, R. (1995). Genetic Competence
in
Escherichia
coli
Requires
Poly-BHydroxybutyrate/Calcium Polyphosphate Membrane
Complexes and Certain Divalent Cations. Journal of
Bacteriology, 486-490.
Reusch, R.N., Huang, R., & Bramble, L.L. (1995).
Poly-3-hydroxybutyrate/polyphosphate
complexes
form voltage-activated Ca2+ channels in the plasma
membranes of Escherichia coli. Biophysical journal,
69(3), 754-66.
Reusch R.N., H.L. Sadoff. (1988). Function of a polB-hydroxybutyrate/calcium polyphosphate channel in
bacterial plasma membranes. Proc. Natl. Acad. Sci.
USA 85:4176-4180
Reusch, R.N., Hunag, R. (1995). Genetic Competence
in
Escherichia
coli
Requires
Poly-BHydroxybutyrate/Calcium Polyphosphate Membrane
Complexes and Certain Divalent Cations. Journal of
Bacteriolog, 486-490
Pavlov, E., Grimbly, C., Diao, C.T.M., & French, R.J.
(2005). A high-conductance mode of a poly-3hydroxybutyrate/calcium/polyphosphate
channel
isolated from competent Escherichia coli cells. FEBS
letters, 579(23), 5187-92.
Reusch, R.N. (1999). Polyphosphate/poly-(R)-3hydroxybutyrate) ion channels in cell membranes.
Progress in molecular and subcellular biology, 23,
151-82.
31
Saddleback Journal of Biology
Spring 2007
The Effect of pH on the Speed of Closure of the Venus Flytrap, Dionaea muscipula
Chris Pasvantis and Callan Taylor
Department of Biological Science
Saddleback College
Mission Viejo, CA 92692
The Venus flytrap (Dionaea muscipula) is a carnivorous plant that uses acid growth to
catch its prey. Once caught, Dionaea muscipula digests and obtains nutrients not available
in their habitat. The ideal habitat for a Venus flytrap is a bog with a pH range between 4
and 5. It is believed that the lower the pH of the Venus flytrap, the faster the rate of
closure of the “trap”. In this experiment, the effects of pH on the speed of closure of Venus
flytraps were studied. The stems of the Venus flytraps were placed in floral tubes and left
to absorb three different pH solutions of 3, 5, and 7 for one hour. After absorption was
complete, the traps were closed and captured with a video camera capturing at sixty
frames per second. The videos were analyzed and the results demonstrated that pH did
play a role in the speed of closure of the traps. The lower the pH of the solution, the
greater the frequency and speed of the reaction. This observation is interpreted as a result
of increased hydrogen ions (lower pH) in the cell, therefore allowing the trap to be less
restricted and close at a quicker speed. Thus, increased acidity directly affects the speed of
trap closure.
Introduction
The Venus flytrap (Dionaea muscipula) is a
carnivorous plant that catches and digests insects.
The “trap” of a Venus flytrap is a modified leaf and
the edges of Venus flytrap are surrounded by trigger
hairs. The red pigment inside the trap attracts insects.
As the insect lands on the pigment, hairs are
stimulated causing the trap to close (Dumais, et. al. ,
2005). It is believed the Venus flytrap uses the “acid
growth hypothesis” for closure. The acid growth
hypothesis suggests that increased acidity in the walls
of certain cells increases their flexibility and
expandability, so that more water can diffuse into the
cells and cause cell elongation. This occurs as
hydrogen ions enter the cell, increasing cell acidity,
causing the microfibrils holding the cells together to
weaken, and allowing them to grow to their
maximum potential (Summers, 2006). Once the traps
have clamped down on the prey, the trap digests the
insect. Digestion usually takes one to two weeks to
complete. After digestion is complete, the trap
reopens and is ready to catch new prey (Robins,
1980). During its lifespan a single trap usually closes
a maximum of three times. Each trap can grow from
a range of three to seven centimeters (Hodick and
Sievers, 1989).
The ideal habitat for a Venus flytrap is a nitrogen
poor environment. The poor condition of the soil,
usually ranging from a pH of 4 to 5, causes the traps
to gain nutrients from elsewhere, thus the formation
of the trap. Insects they trap and digest provide
nitrogen and protein. The speed of closure for a trap
varies on humidity, light, and growing conditions.
Also, the speed of closure can be used as an indicator
of the plant’s health. Tap water can be fatal to Venus
flytraps because salts in the water can kill the traps.
Soft water, distilled water, or clean rain water is ideal
(Kudlinski and Wexler 1998).
The purpose of this experiment was to determine
the effect of pH in the speed of closure of a Venus
flytrap (Dionaea muscipula). It is believed that due
to acid growth, the Venus flytraps will close at a
much faster rate at a lower pH.
Materials and Methods
Three pH solutions were created from a 0.50M
HCl solution. Forty milliliters of deionized water was
measured three times. To form a solution with a pH
of 3 solutions, 0.75 milliliters of HCl were
micropipetted into forty milliliters of water. For the
pH of five solutions, 0.20 milliliters of HCl was
micropipetted into forty milliliters of water and for
the solution with the pH of 7, it was determined the
pH of the deionized water from the tap was optimal.
The habitat for the flytraps was then prepared.
Floral clay was used to fasten floral tubes in place and
7.5 milliliters of each solution (pH 3, pH 5, and pH 7)
were poured into five respective floral tubes.
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Saddleback Journal of Biology
Spring 2007
6
5
Amount of Traps Closed
Therefore, a total of fifteen separate habitats were
created, five for each pH value. Flytrap heads were
chosen based on similar color, size, and status of
closure. It was determined the flytraps with a pink
mouth, and at least two centimeters in width, were the
healthiest and most mature.
Flytrap stems, with attached trap, were cut at 3.5
centimeters in length and placed in the floral tubes.
They were then left to absorb the solutions for one
hour and were then tested. The closure of the plant
was captured using the Fast Speed Capture setting on
a Canon digital camera recording at a speed of sixty
frames per second. Flytraps were closed one by one,
using a probe to stimulate and cause closing.
Adobe Premiere Elements 3.0 was used to
calculate frames per second of the closing. The times
were taken and converted into decimal form dividing
1/60th of a second into 1/100th of a second for easy
reference and charting. After the full spectrum of
data was entered into an Excel database, an ANOVA
and post-hoc test was used to analyze the data.
4
3
2
1
0
3
5
7
pH
Figure 1. The number of traps that closed vs. the
pH of the solutions.
4.5
4
Results
As the acidity increased, the frequency of closure
of the traps increased (Figure 1). The solution with
the pH of 3 yielded the best results, with five out of
five possible closures. As the acidity decreased, so
did the frequency of closure. The solution with the
pH of 7 yielded three out of five possible closures.
In addition to increased frequency of closure, the
increased acidity was directly related to an increased
speed of flytrap closure (Figure 2). The traps exposed
to a solution with the pH of 3 responded twice as
quickly as those with the pH of 5. Likewise, the
traps exposed to the solution with the pH of 7 showed
the slowest closures of the three solutions with a
speed of 3.6 seconds.
Statistical analysis was performed with ANOVA
utilizing a 95% confidence interval; the test yielded a
p-value of 0.003. A post hoc test was performed since
the p-values for the ANOVA test was significant.
The post-hoc test showed the relationships between
the pH’s of 3 and 5, and 3 and 7 were significant, but
the relationship between 5 and 7 was not significant.
Discussion
Dionaea muscipula was affected by the pH of the
solution. First, the amount of traps that closed
increased as the acidity of the solution increased.
Second, the test showed a significant difference in the
rate of closure between the pH’s of 3 and 5, and 3 and
7, but the relationship between 5 and 7 was not
significant. Therefore, it is believed that the pH did
alter the closing speed of the Venus flytrap.
Time (sec)
3.5
3
2.5
2
1.5
1
0.5
0
3
5
7
pH
Figure 2. The time (sec) for the traps to close vs.
the pH of the solutions. Statistical significance
between 3 and 5 pH. Statistical significance
between 3 and 7 pH.
This experiment supports the “acid growth
hypothesis”. The hypothesis stated that increased
acidity in the walls of certain cells increases their
flexibility and expandability. More water can diffuse
into the cells and cause cell elongation, allowing the
traps of the Venus flytrap to close (Rayle and
Cleland, 1992). As the pH of the solution lowered,
the response was quicker and more traps responded.
It is believed a significant increase in the H+
concentration allowed this to happen. Any other
reason would be unknown at this point without
further experimentation.
Further research could be done comparing trap
closures for plants that are domesticated or in the
wild. The Venus flytrap closure is one of the fastest
movements in the plant kingdom with rates of closure
at one hundred milliseconds (Dumais, et. al. , 2005).
The fastest closure in this experiment was 0.720
seconds, with the slowest closure being 3.70 seconds.
This is by far slower than the average Venus flytrap.
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Saddleback Journal of Biology
Spring 2007
This could be attributed to poor Venus flytrap health
and an improper environment for growth and
maturation. Another theory is that the flytrap may
have been domesticated and therefore lost the need to
react quickly to capture its own prey. Natural
selection was no longer present
Further research is required to determine if acid
growth is truly responsible for the closing of a Venus
flytrap.
Hodick D, Sievers A. (1989). The action potential of
Dionaea muscipula Ellis. Planta. 174:8-18.
Literature Cited
Robins R.J., Juniper B.E. (1980). The secretory cycle
of Dionaea muscipula Ellis. I. The fine structure and
the effect of stimulation on the fine structure of the
digestive gland cells. New Phytologist. 86:279-296.
Dumais J, Forterre Y, Mahadvan L, Skotheim JM.
(2005). How the Venus Flytrap Snaps. 433:421-425.
Hodick D, Sievers A. (1989). On the mechanism of
closure of Venus flytrap. Planta. 174:8-18.
Kudlinski K, Wexler J. (1998). Venus Flytraps.
Minneapolis: Lerner Publishing. 48 p.
Rayle DL, Cleland RE. (1992). The acid growth
theory of auxin-induced cell elongation is alive and
well. Plant Physiology. 99:1271-1274.
Summers A. (2006). Biomechanics.
Museum of Natural History.
American
Possible Isolation and Cultivation of Halophilic Bacteria from Halite
Nasrene Hosseini and Nilou Moslehi
Department of Biologcal Sciences
Saddleback College
Mission Viejo, CA 92692
Halophilic bacteria can be found in hypersaline environments with salt concentrations
exceeding those of sea water. These unique organisms exhibit a form of tolerance that
researchers are continuing to explore. Through the study of these organisms insights can
be gained regarding external life on other planets. In this experiment the possible growth
of halophilic bacteria on cultured media is considered. Ten halite crystals were used as the
potential sources for obtaining halophilic organisms with the hopes of cultivation. Fluid
inclusions extracted from these halite crystals were cultured on a Salt Tryptose Glucose
Yeast Extract agar medium at various concentrations and incubated at 37ºC. Bacterial
growth was observed on four of ten plates containing 10 g/L of NaCl, three of ten plates
containing 30 g/L, three of ten plates containing 60 g/L, and none of the ten plates
containing 120 g/L of NaCl. These results provide support that there is a threshold for
halophilic bacterial growth and that the particular bacteria isolated and cultivated in this
experiment thrive on agar containing 10 g/L of NaCl.
Introduction
Some of the most tolerant organisms inhabiting
the planet are halophilic extremophiles, archaea
bacteria that can withstand highly saline
environments such as salty lakes and in subterranean
crystals (DasSarma, 2006). During the process of salt
crystal formation, halophiles that are trapped in fluid
inclusions utilize the brine and nitrogen where they
may thrive for years at a time (Day, 2004).
Characteristic metabolic functions, acidic proteins,
and salt-dependent enzymes are what enable
halophilic organisms to survive in such environments
(DasSarma, 2006; Fendrihan, et. al. 2006).
It is the ability of halophilic organisms to
experience periods of dormancy that contributes to
one of the most fascinating aspects of halophile
physiology (DasSarma, 2006).
By lowering
metabolic activities and minimally exploiting energy
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Saddleback Journal of Biology
Spring 2007
resources halophiles can live for millions of years,
and the potential exists to isolate halophilic organisms
dating back to 250 million years ago (Chu and Sheng;
DasSarma, 2006).
The study of these microorganisms is compelling
since information about their tolerance to highly
saline environments and other stressful factors such as
radiation and temperature, hold much promise in the
area of exobiology—the survival of organisms on
other planets, such as Mars (DasSarma, 2006). By
understanding halophile physiology, information can
be provided regarding the requirements to survive in
places once believed to be uninhabitable (DasSarma,
2006).
In this study, salt crystals obtained from Detroit,
Michigan were selected for fluid inclusions
containing brine and potentially harboring halophiles.
The extracted fluid containing organisms was
cultured under sterile conditions in order to minimize
contamination. In order to determine the limit to
which halophiles can survive in saline environments
culture mediums of various NaCl concentrations were
used. Ultimately the objective was to produce
bacterial growth on the agar medium and determine
the threshold of bacterial tolerance to NaCl.
Materials and Methods
Ten halite crystals selected for considerable fluid
inclusions were submerged in isopropyl alcohol for
superficial sterilization. A wire drill was used as a
means of penetrating the halite in order to extract the
fluid. After the halite was drilled, individual sterile
syringes were used for extraction of brine from each
crystal (Powers et. al, 2000). The extracted fluid
from each of the ten crystals was placed into separate
test tubes containing 5 mL of broth. ATCC broth was
prepared in 1 L of water with 97g NaCl, 8g MgCl2,
12g MgSO4, 0.5g CaCl2, 0.1g NaHCO3, 2.5g KCl,
0.25g NaBr, 0.25g Yeast Extract for bacterial
magnification. Salt Tryptose Glucose Yeast Extract
Agar used for plating was prepared with 5.0g
Tryptone, 2.5 g yeast extract, 1.0g glucose, 10 g
NaCl, in 1 L of distilled water. Agar was prepared
with four different salt concentrations: 10, 30, 60, and
120 g/L in order to determine the bacterial tolerance
to salinity. Broth containing brine extractions were
placed in an incubator at 37ºC for a period of three
days. Four plates of each salt concentration were
inoculated with broth from the same test tube and the
process was performed for all ten test tubes on a total
of forty plates. The plates were then placed in the
incubator at 37ºC for five days. Bacterial growth was
removed from plates and placed back into broth for
further magnification over a period of six days under
the same temperature conditions. Plates with no
obvious initial growth were reinoculated with the
broth and incubated for 10 days (Chu and Sheng).
Plates were examined for bacterial growth and data
was collected by determining the number of plates
from each salt concentration that produced growth.
Results
The Salt Tryptose Glucose Yeast Extract agar
plating produced bacterial growth on a total ten of
the forty plates inoculated with ATCC broth
containing fluid inclusions. Bacterial growth was
observed on four of the plates containing 10 g/L of
NaCl, on three plates containing 30 g/L, on three of
the plates containing 60 g/L, and on none of the plates
containing 120 g/L of NaCl. The number of plates
containing bacterial growth leveled off at 30 g/L and
then decreased at 120 g/L to zero plates. Bacterial
growth appeared to display colonial morphology on
all of the ten plates as well as a similar ivory color.
No further testing was performed for bacterial
identification.
Discussion
These results support the hypothesis that
halophilic bacteria existing in halite crystals can be
cultured. Additionally, results indicate that halophilic
bacteria have a threshold for which they can tolerate
NaCl. Although this particular threshold is not
known, Chu and Sheng’s study provided that
culturing halophiles on a 25% agar solution has
minimal feasibility due to the crystallization of salt on
the agar plate.
In this study, the concentration of NaCl directly
affected the amount of plates with growth. The
number of plates with bacterial growth decreased as
the concentration of NaCl increased in the agar. Asha
et. al’s (2005) study indicated similar results,
although different agar media were used. Powers et
al, (2000) did not do a serial dilution, however
bacterial growth was found only on casein-derived
amino acid (CAS) agar medium rather than other
media, showing bacteria to have preference to certain
media.
The validity of previous studies in which
halophilic bacteria was cultivated and isolated has
been questioned due to the fact that contamination of
the source of bacteria may lend false information
regarding the presence of authentic ancient bacteria
(Powers et. al, 2000). Thus, sterile procedures are
particularly relevant in the conduct of this
experiment. Contamination of culture media and
other components may have rendered false results.
This is unlikely, however, as most other bacteria do
not grow on such media due to the high salt
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Saddleback Journal of Biology
Spring 2007
concentration (T. Huntley, personal communication,
10 April 2007).
More specific analyses of the bacteria can be
performed in order to determine how bacteria are
affected by other conditions such as temperature and
pH. Bacteria can be microscopically analyzed to
determine the appearance of an individual halophilic
organism. Chu and Sheng found that cultured
bacteria were gram positive cocci shaped halophiles.
Day (2004) identified a series of halophilic
morphotypes to be cocci, bacilli, square, triangle, and
bent or curved. Further studies may reveal other
potential morphotypes and species of halophiles.
Acknowledgements
The authors wish to acknowledge the support of the
Geology Department of Saddleback College,
especially Dr. James Repka and Mr. John Robinson
for their help with this project.
Literature Cited
Asha K., Vinitha D., Kiran, S., Manjusha W.,
Sukamaran N., Selvin J. 2005. Isolation and
Cultivation of Halophilic Archaea from Solar Salterns
Located in Peninsular Coast of India. Inter. J.
Microbiology. 1(2).
Chu H., Sheng W.. Exobiology: The Survival Ability
of Halophiles Under Martian Conditions.
DasSarma S.. 2006. Tolerance of extremes in salinity,
radiation, and temperature may permit halophiles to
survive elsewhere in the universe. American Society
For Microbiology 1(3): 120-127.
Day J.. 2004. Revival of Halophiles from Recently
Formed Great Salt Lake Salt Crystals. Myriad.
Fendrihan S., Legat A., Pfaffenhuemer M., Gruber C.,
Weidler G., Gerbl F., Stan-Lotter H.. 2006. Extremely
Halophilic Archaea and the Issue of Long-term
Microbial Survival. Springer Science.
Powers D.W., Rosenzweig W.D., Vreeland R.H..
2000. Isolation of a 250 million-year-old halotolerant
bacterium. Nature 407: 897-900.
Differences in Absorption Efficiency between Two Diets in the Western Fence
Lizard, Sceloporus occidentalis
Dominique Alex and Stephanie Olamendi
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
The difference in assimilation efficiencies between two diets was tested in the
western fence lizard, Sceloporus occidentalis. The study focused on measuring the
amount of calories absorbed from the consumption of a cricket diet versus a
mealworm diet. Ten lizards (N=10) were caught and housed in an incubator set at
their optimum temperature (31 °C) for proper digestion. Once captured, the lizards
were starved to assure consistent post-absorptive states. The lizards were fed both
diets with a two day period of starvation in between. The feces collected from each
diet were individually collected, dried, pulverized and pressed into approximately
one-gram pellets. The caloric value was determined using an oxygen bomb
calorimeter. The assimilation efficiencies were calculated by subtracting the energy
excreted from the energy consumed. The absorption efficiencies were calculated to
be 89.8% in the cricket diet and 85.4% in the mealworm diet. This supported the
tested hypothesis that there is a difference in the absorption efficiency when
comparing a cricket diet and a mealworm diet in the lizard S. occidentalis.
36
Saddleback Journal of Biology
Spring 2007
Introduction
There have been many studies that focus on
the assimilation efficiencies of various lizards
(Ruppert, 1980; Johnson and Lillywhite, 1979;
Muller, 1970; Slade, et al., 1944). Furthermore,
these studies support the hypothesis that there is
a difference in the absorption efficiency in two
diets consumed by the lizard S. occidentalis.
When comparing the two experimental diets
(mealworm and cricket), the evolution and
energetics of lizards suggest that a mealworm
diet would be more beneficial. For a lizard, the
Gryllodes sigillatus cricket diet requires a higher
energy expenditure when compared to a
mealworm diet. A study stated that Tenebrio
larvae, commonly known as mealworm,
contained an average of 61.0% water, with a
caloric value of 5707cal/g (Avery, 1971).
Another study used this data and compared it to
a cricket diet. This study calculated that crickets
contained an average of 73.0% water with a
caloric value of 5556 cal/g (Ruppert, 1980). This
raised an evolutionary inquiry further questioned
by Mueller (1970).
Materials and Methods
This study measured the absorption efficiency
of a cricket diet compared to a mealworm diet.
Ten lizards (N=10) labeled A through J, were
used as the experimental group, consuming both
the mealworm and the cricket diet. For more
accurate results in comparing absorption
efficiency, no control group was used. Each
lizard was captured in March at Saddleback
College, Mission Viejo. The captured lizards
were transported to the laboratory where they
were housed in individual glass jars, modified to
provide the lizards with oxygen. All lizards were
held at optimum digestive temperature (31°C)
and no water was provided due to a high waterbased diet, (Ruppert, 1980; Avery, 1971).
Different diets
The first diet tested was an exclusively
mealworm diet. The lizards were fed the
mealworm diet for eight days, often eating up to
three times a day. Following the mealworm diet,
the lizards were starved for two days prior to
feeding to ensure consistent post-absorptive
states. Subsequent to the mealworm diet, the
lizards were exposed to a solely cricket based
diet. The lizards consumed this diet for about six
days, often eating up to three times a day.
Temperature relations
Because most reptiles regulate their body
temperatures
behaviorally
by
exploiting
environmental heat, the body temperature that
lizards maintain through thermoregulation
greatly reflects a compromise of optimal body
temperatures for proper digestion (Du et al.,
2000). To allow proper digestion, the lizards
were kept in a VWR Incubator provided by
Saddleback College. According to a study, the
body temperature of a lizard has a significant
impact on its feeding behavior (Avery, 1971).
The incubator was set to 31.0°C, the optimum
temperature for digestion of S. occidentalis. This
temperature was agreed upon based on previous
studies stating the thermal dependence of food
assimilation and locomotion performance in
ectoderms (Angilletta, 2001). It has been further
shown that the digestion time decreases with an
increase in total body temperature within the
range from 24°C to 34°C, however this will
increase at higher body temperatures (Zhang and
Ji, 2004).
Experimental procedure
After the lizards were exposed to a specific
diet, the feces were collected and placed in
plastic vials labeled A through J. Lizards were
fed 4 days without collection of feces. This
allowed time for physiological adjustment
required by the lizards (Ruppert, 1980). All the
feces and nitrogenous waste from each diet was
pooled together in separate containers.
According to Mueller (1970), no correction for
the bacterial content of the fecal matter or the
effect of bacteria on the feces was necessary.
This error would prove no or an insignificant
difference in this study. The fecal samples were
placed in a Lindberg/Blue drying oven, set at a
constant temperature of 60°C. The samples of
consumed crickets and mealworms were left to
dry until the dry weight appeared constant,
indicating a complete loss of moisture in the
fecal sample. The dried, pooled samples were
placed in a pulverizing machine, provided by
Saddleback College. Once dried and grinded the
samples were pressed into approximately 1.00g
pellets. Calorimetry was performed using a Parr
Oxygen Bomb Calorimeter. After the sample had
been disintegrated, bomb washings were titrated
against Na2CO3. This allowed for proper acid
corrections.
37
Saddleback Journal of Biology
Spring 2007
Table 1. Symbols and Definitions used to
Calculate Assimilation Efficiencies. Modified
from Johnson and Lillywhite (1979).
C
E
C-E
(C – E) / C
x 100
Definition
calories consumed
calories of both fecal and urinary
wastes
8000
7000
6000
5000
4000
3000
2000
1000
0
Cricket
Cricket Feces
Mealworm
Mealworm Feces
Samples
Fig. 1. Comparison the caloric value of calories
ingested versus calories excreted for both diets.
The cricket diet contained 5530 cal/g, and
excreted a mean of 2620 cal/g, whereas the
mealworm diet contained 6519 cal/g, and
excreted a mean of 2591 cal/g.
useable calories retained by animal
useable calories retained by animal (%)
This procedure was altered for the cricket
diet. Due to time constraints, the total amount of
cricket feces could not be collected. A study by
Johnson and Lillywhite (1979) provided the
necessary values to draw an educated guess to
calculate the assimilation efficiency and calories
absorbed in a cricket diet. By following the same
procedure for the mealworm diet, the percent
assimilation efficiency was estimated for the
cricket diet.
Results
Mean caloric value of a cricket diet was
calculated to be 5530 cal/g. The mean caloric
value of a mealworm diet was calculated to be
Assimilation Efficiency (AE)
Percentage
Symbol
6519 cal/g. The collected feces samples from
mealworm averaged 2591 cal/g while the
collected feces samples from the crickets
averaged 2620 cal/g. These data are
demonstrated in Figure 1. The data were utilized
in obtaining the total calories absorbed from each
diet. Between the two diets, the lizard absorbed
89.8% of the cricket’s caloric value, using data
provided by Johnson and Lillywhite (1979),
whereas it only absorbed 85.4% of the
mealworm’s caloric value (Figure 2). The lizards
absorbed a total of 17930 calories from the
cricket diet, using the information provided by
Johnson and Lillywhite (1979), and a total of
55100 calories from the mealworm diet (Figure
3).
Calorie Content (cal/g)
Calculations
Absorption efficiency was estimated through
a series of calculations. For the first diet, the
ratio of dry to wet weight of the mealworms
digested was used to calculate the percentage of
dry weight consumed. The total number of grams
consumed was multiplied by the ratio of dry to
wet weight (measured in grams) of the
mealworms consumed. This was used to get the
dry weight consumed. The dry weight consumed
was multiplied by the caloric value of the
mealworms to get the total energy ingested
(measured in calories). To measure the total
calories excreted, the total grams of pulverized
mealworms was multiplied by the caloric value
of the feces from mealworms (measured cal/g).
The amount of energy consumed was subtracted
from the energy excreted to calculate the total
calories absorbed by the lizards. Furthermore,
this number was divided by the amount of
energy in taken and multiplied by 100 to get the
percent assimilation efficiency. No statistical
analysis was needed to estimate assimilation
efficiency percentage. Calculations provided in
Table 1 display the equation used for calculating
the absorbance efficiency for each diet.
100
90
80
70
60
50
Mealworm Diet
Cricket Diet
Diets
Fig. 2. Absorption efficiency as a percent of a
cricket and a mealworm diet. The lizard
absorbed 85.4% of the mealworm’s caloric
value, whereas it absorbed 89.8% of the
cricket’s caloric value. This support the
hypothesis tested that there is a difference in
absorption efficiency between the two diets.
38
Saddleback Journal of Biology
Spring 2007
Calories (cal)
60000
50000
40000
30000
20000
10000
0
Mealworm
Cricket
Diets
Fig. 3. Caloric absorption from the cricket diet
equaled 17930 calories, and 55100 calories from
the mealworm diet.
Discussion
The caloric value absorbed by the lizard S.
occidentalis varies depending on the diet. When
exposed to a high caloric diet such as a
mealworm diet, the caloric value of the
mealworms consumed was calculated to be 6519
cal/g with an absorption efficiency of 85.4%.
When compared to a lesser caloric diet, such as a
cricket diet, the absorption efficiency rose to
89.8 %, using data provided by Johnson and
Lillywhite (1979). This proves the tested
hypothesis that there is a difference in absorption
efficiency between the two diets. The caloric
value of crickets calculated from this study was
averaged to be 5530 cal/g. This number can be
compared to the study by Ruppert (1970), who
calculated a consistent caloric value of 5556
cal/g for crickets. The similarity of the results
gives our data a specific accuracy. Within each
diet, the amount of calories fed to each lizard
was recorded and totaled. When feeding the
lizards, the optimum temperature was highly
accounted for. Furthermore, because the
experimental lizards were exposed to a constant
temperature and a strict diet, the finding of this
study may be significantly different when
compared to free-living lizards.
Lizards die if they are kept at a constant high
temperature for a long period of time, due to
high calorie expenditure and little calorie
consumption (T. Huntley, pers. comm.). In this
study, exposing the three smallest lizards to the
constant optimum temperature, lead them to
expire, due to their high surface to volume ratios.
The smaller lizards continued to be in a digestive
state, however, they did not consume any energy,
for unknown reasons. This assumption was
determined by weighing the body mass of the
lizards. Weighing the lizards was done by
massaging the lizards on the ventral surface until
enough lactic acid was produced (T. Huntley,
pers. comm.). The lactic acid build up paralyzed
the lizards, allowing them to be easily placed on
the measuring scale.
When considering the two diets, nutritional
factors may explain why the lizards S.
occidentalis, prefer a specific diet. A study
showed that many lizards ingest a considerable
percentage of calories in the form of cellulose
(Johnson and Lillywhite, 1979). Nagy (1977)
examined different concentrations of nutrients
absorbed and not absorbed by different digestive
organs, data that gave a better understanding of
the lizard’s digestive system and nutritional
needs. Further studies may research the
nutritional contents, such as protein and
cellulose, between a mealworm diet and a cricket
diet. Foraging strategies, exposing whether
lizards prefer one diet over another in their
natural habitat, could be a further study. This
could show whether lizards have a natural
instinct to hunt the prey that would provide more
caloric absorption and less caloric expenditure
when hunting.
Acknowledgements
We would like to thank Dr. Tony Huntley for
the time and knowledge he provided for this
study. Further more, we would like to thank Dr.
M. Ruppert for providing us with his research
paper on comparative assimilation efficiencies of
two lizards.
Literature Cited
Angilletta, M. J. (2001). Variation in Metabolic
Rate between Populations of Geographically
Widespread
Lizards.
Physiological
and
Biochemical Zoology. 74(1): 11-21.
Avery, A. R. (1971). Estimates of food
consumption by the lizard Lacerta vivipara
(Jacquin). Journal of Animal Ecology. 40: 35165.
Du, W.G., Yan S.J., AND X. Ji. (2000). Selected
body temperature, thermal tolerance and thermal
dependence of food assimilation and locomotor
performance in adult blue-tailed skinks, Eumeces
elegans. Journal of Thermal Biology. 25: 197202.
Johnson, N.R. AND Lillywhite B.H. (1979).
Digestive Efficiency of the Omnivorous Lizard
Klauberina riversiana. Copeia.3: 431-437.
Mueller, F.C. (1970). Energy utilization in the
lizards Scleoporus graciosus and S. occidentalis.
Journal of Herptetology. 4: 131-34.
39
Saddleback Journal of Biology
Spring 2007
Nagy, K.A. (1977). Cellulose Digestion
andNutrient Assimilation in Sauromalus obesus,
a Plant-Eating Lizard. Copeia. 2: 355-62.
Ruppert,
M.R.
(1980).
Comparative
assimilationefficiencies
of
two
lizards.
Comp.Biochem. Physiol. 67A: 491-96.
Zhang, Y.P., AND X. Ji. (2004). The Thermal
dependence of food assimilation and locomotor
performance in southern grass lizards,
Takydromus sexlineatus (Lacertidae). Journal of
Thermal Biology. 29: 45-53.
Slade, J.H., Arnold, B.W., AND V.M. Plummer.
(1994). Efficiencies of Digestion and
Assimilation in the gecko Hemidactylus turcicus.
Journal of Herpetology. 28: 513-14.
Metabolic Rate of Melopsittacus undulatus Consuming Dietary Omega-6 Fatty
Acids
Lizbeth Barrera and Felicia Dang
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92691
Birds have an alarming metabolic rate; the Melopsittacus undulatus or parakeet is no
exception. Parakeets are a medium sized bird that can eat up to 3 tablespoons of food in 20
small meals daily. They need a constant well balanced diet with all essential amino acids
and omega fatty acids. Parakeets are diet sensitive and so a change in their established diet
can alter the bird’s metabolism drastically. When feeding the experimental birds, omega 6
fatty oil seed (eg. sunflower seeds, hemp seed, rape seed ect.) the mean consumption of
oxygen was 30.47 ± .05 SE (n=8). The omega 6 group (p > 0.004631) changed drastically in
weight and as well in metabolic rate from the control group. The more a bird eats the
slower its metabolism becomes because the caloric intake is surpassing the capability of an
avian body daily function usage. Therefore, their bodies store the excess food as fatty
chains in adipose tissue. From this study the one thing that should be highly noted is that
aves are synonymous to mammals and the obesity in aves can help further understand
other obesity problems.
Introduction
Food is an essential necessity that takes up a large
majority of time finding – that is if one is an animal
or as in this instance a bird. Birds are known to have
fast metabolisms: dependent on species and size,
some metabolisms are faster than others.
For
example, the hummingbird, Allen’s hummingbird,
Selasphorus sasin, has a metabolism where it has to
feed every 10 to 15 minutes. Hummingbirds on
average have to consume around 3.14 to 7.16 calories
a day. To put in prospective -- a human with this type
of metabolism would have to eat 155,000 calories a
day! However, birds that are domesticated have no
need to scavenge or forage for food. Therefore,
domesticated birds require fewer calories; they are on
average fed daily with 2 tablespoons (per bird) of new
fresh seed each day.
The seed choice is vast for the household bird;
different species of birds require different varieties of
nutrients. There are oily seed which are high fatty
acid chains, high in energy content but in excess can
cause obesity (Hillgartner and Charron, 1998). There
is also regulated seed that has extra vitamins, (Earle,
K.E., Clarke, N.R., 1991), flaxseed (Omega-3), and
extra fat (Omega-6). Birds that get a highcarbohydrate mash diets tend to develop avian fatty
40
Saddleback Journal of Biology
Spring 2007
liver disease, usually seen in goslings, chicks and
ducklings (Back et al., 1986). The omega 6 fatty
acid, the bad fat, can in the long run cause bodily
harm to an avian body. One way omega 6 is
introduced into diet is through seed with glucose.
Glucose is used as an immediate energy source, and
in moderation is helpful in an avian body. Glucose is
also good in avian diet because it helps regulate
(speed up or slow down) the transcription of fatty acid
synthase and malic enzyme; both of which helps in
storage of excess fat in the diet (Hillgartner FB and
Charron T, 1998). However, parakeets, being a
domesticated breed, can be over fed or have the
wrong seed diet. What would happen if the seed was
one with too much omega 6 would their metabolism
slow down?
Materials and Methods
A total of 8 store bought (Pet co.,
Westminster) parakeets were used in the experiment.
The control group, 4 birds, was kept on a regular diet.
The regular diet consisted of a 2:1 ratio (omega 3
fatty seed to omega 6 fatty seed) and then they were
also given fresh greens every 3 days.
The
experimental group, 4 birds were given a “junk diet.”
The diet was composed of a 1:3 ratio (omega 3 to
omega 6). The experimental group was on the diet
for 2 weeks. They were weighed at the beginning of
the experiment using a triple beam balance (OHAUS,
Florida Park, NJ)
After the 2-week period both groups of birds
were brought into lab. At which time they were reweighed using a triple beam balance and it was
recorded. Then each bird had 3 tests run, in the fox
box (Sable Systems International). Also Drierite
(Xenia, Ohio) and Ascarite (Swedesboro, NJ) were
added in a small test tube to take out moisture, which
could alter the reading. The device was opened for 2
minutes to allow the birds to stabilize again. All data
was stored then finally purged on to a lab top. Where
further statistical analysis using Microsoft excel cBirds
ould
be evaluated. The weight specific metabolic rate was
Experime
calculated:
MR=
20.95cc x
t
60sec x
min
60 min x
hr
P1 V 1 = P 2 V 2
T1
T2
Where P1 is the room barometric pressure (in mm
Hg), V1 is the gas volume (i.e. the MR), T1 is the
temperature of the respirometer in °K, P2 is standard
pressure (760 mm Hg), T2 is 273 °K, and V2 is the
adjusted volume (STP).
The birds were also examined for any
change in behavior or in daily activity in a biology
notebook, used throughout the experiment.
Results
The body weight of before and after the
experiment is given for each group of birds in table 1
below. The birds ended up gaining weight, slightly.
The test was given at ambient temperature (Ta)
(22oC) and barometric pressure of 1atm. Vapor
pressure was not included in the calculation because
of the drierite and ascarite test tube took out moisture
from the apparatus.
The parakeets should an
immense decrease in metabolism after being fed on
the omega 6 seed as seen in figure 1.
A big observation was also on the bird activity.
The birds on the healthier diet were much more
attentive and energetic. They also were friendlier,
more tamed, the birds overall fared better. The
omega 6 group tended to be quieter and sleepier by
the end of the project the birds had changed
immensely since the commencement of dietary
change.
Table 1. The body weight of parakeets before and
after the experiment. The control group stayed
relatively constant.
On the other hand the
experimental group did get bigger.
Weight (g)
Experimental
Initial
Final
Weight (g)
Control
Initial
Final
1
24.3
26.2
23.5
23.6
2
1
3
wt
34.42
37.46
26.95
27.01
26.95
29.95
35.45
35.45
4
24.1
25.5
25.02
25.2
20 .95 cc was the volume used as the starting point in
the chamber, t was the time to return to 20.95, and wt
was the weight of the individual parakeet. After that
was calculated, using Boyle’s law, one was able to
obtain adjusted volumes at STP.
41
Saddleback Journal of Biology
Spring 2007
Figure 1. Mean metabolic rate of birds fed on omega
6 seed vs. birds fed on a balanced feed. The birds
(n= 8) on omega 6 diet had an overall lower
metabolic rate (p > 0.004631) which is highly
significant. Then mean of the experimental group
was 30.47 mLO2/gm/hr, STPD (± .05 SD). While the
control group had a mean metabolic rate of 51.6
food supply, if only the trainers would pay attention
to diet.
Other studies have provided evidence for other
diseases in birds which begin at transcription phases
(Back et al., 1986) do to diet. If fatty acid chains can
affect a system all the way back down to the
transcription phase, tests could be done on how that
effects egg development would the egg contain more
or less of its essentials and would liver disease be
found a chick, this would be interesting to see if it
passed on the excess fatty acid (Back et al., 1986).
Furthermore, tests could be done on birds to find
out where most of the fat deposits on the body and the
correlation to heart disease or liver problems -- if any.
Finding out where fat deposits (Crespo, and EsteveGarcia, 2002) allows for further understanding as to
what gene or predisposition a species must have in
order to obtain that disease. What ever the case diet
does have a significant affect on avian metabolism
and also on behavior.
Literature Cited
Angel R and Ballam G. 1999. Dietary Protein Effect
on Parakeet Reproduction, Growth, and Plasma Uric
Acid. Purina Mills Inc., St. Louis, Missouri.
mLO2/gm/hr, STPD (± .05 SD).
Conclusion
The parakeet diet had a huge significance in both
metabolic rates and also on behavioral factors. The
omega 6 fatty seed significantly slowed the rate at
which a bird metabolizes daily food. Consequently,
there is support that dietary stresses on animals, aves,
can alter their state of being. An example is a once
docile bird can become much more aggressive
(Gonzalez-Esquerra and Leeson, 2000), especially
around matting season. Males during mating season
were found to have increased aggressiveness. The
dietary change is big on animals that normally
foraged for food. Which begs the question does food
shortage affects the aggressiveness of caged birds.
Birds that were fed the omega 6 fatty diet ended up
becoming slightly larger and fatter than the control
group. The weight gain on the birds had no
immediate health affect; however, if the birds were
allowed to continue the diet, it can alter regulation of
fatty acid synthesis which is associated with several
disease states such as obesity, diabetes, and
atherosclerosis (Hillgartner FB and Charron T, 1998).
This study has helped understand a little more on
avian obesity. Birds should be monitored and
provided a healthy well balanced diet. After all
domesticated birds have an advantage to have a ready
Back DW, Goldman MJ, Fisch JE, Ochs RS and
Goodridge AG. 1986. The fatty acid synthase gene
in avian liver. Two mRNAs are expressed and
regulated in parallel by feeding, primarily at the level
of transcription. J. Biol. Chem; 261: 4190 – 4197.
Crespo N and Esteve-Garcia E. 2002. Nutrient and
fatty acid deposition in broilers fed different dietary
fatty acid profiles. Poultry Science, Vol 81, Issue 10,
1533-1542.
Earle K.E.,and Clarke N.R. 1991. The nutrition of the
budgerigar (Melopsittacus undulatus). Journal of
nutrition 121: 5186-5192.
Gonzalez-Esquerra R and Leeson S. 2000. Studies
on the metabolizable energy content of ground full-fat
flaxseed fed in mash, pellet, and crumbled diets
assayed with birds of different ages. Poultry Science,
Vol 79, Issue 11, 1603-1607.
Hillgartner FB and Charron T. 1998. Glucose
stimulates transcription of fatty acid synthase and
malic enzyme in avian hepatocytes. Am J Physiol
Endocrinol Metab 274: E493-E501.
42
Saddleback Journal of Biology
Spring 2007
The Inhibitive Effect of Blue, Green and Red light on E. coli growth
Jonathan Nadal
Department of Biological Sciences
Saddleback College, California, USA
The management of septic water for sewage treatment plants is never a simple process.
One must always take into account the many pathological organisms present in waste water
(coliform bacteria for example). The exact procedure for eliminating these organisms
(bacteria) is time-consuming and costly. It would be interesting and quite useful if simple
methods such as the exposure to green, red or blue (colored) light would inhibit E. coli
(coliform bacteria) growth at least to some degree. Forty Petri dishes were cultured with E.
coli and exposed to red, green and blue light, experimental, and white light and dark,
control, with eight dishes for each. An ANOVA test yielded a p-value of 0.002 which
prompted a Bonferroni connection test with results that showed a significant difference for
red and blue light against no light with t values of 4.02 and 3.04 respectively. It should be
noted that temperature possibly skewed the data in favor of the hypothesis (inhibition of
growth).
investigation of this hypothesis would consider it a
possible avenue for future use.
Introduction
The objective of the experiment is to investigate
the inhibitive effect of different spectrums of visible
light (specifically; blue, red and green) on the growth
of coliform bacteria (E. coli in this case). Pathological
bacteria (especially coliform bacteria from fecal
matter) have always been a problem for water
treatment facilities. Treatment plants never clean
sewage water directly to a potable status; water must
be disinfected before release into the environment.
Coliform bacteria and other contaminants located in
sewage water have been treated biologically,
chemically and physically. The process is a lengthy
procedure and there is the possibility the water will be
contaminated from biological sources (animal wastes)
during the process. Others have investigated
sanitizing techniques using ultraviolet light (Fiksdal
and Tryland, 1999), solar radiation coupled with
chemicals (Acher and Juven, 1977) or just sunlight
(Fuijoka et. al., 1981) to sanitize septic or sewage
water. The methods stated earlier use high energy
waves (the wavelength is short and frequency long,
such as UV light) for decontamination of waste water.
It is curious if the wavelengths red, blue and green;
i.e., visible colored light, will have an inhibiting
effect on bacterial growth (considering these colors of
light in itself don’t contain as much energy as UV
light does) compared to growth simply in the dark or
white light. Mostly likely this method will not stop
growth of E. coli on nutrient agar entirely. Any
technique to further help the reduction of dangerous
bacteria in waste water would be appreciated and the
Methods and Materials
The investigator would need to culture coliform
bacteria (E. coli) in a broth then apply it to a petridish
(Nutrient Agar) and shine red, blue, green, white light
on the respective petridishes. This is essentially three
steps: broth inoculation, petridish preparation, and the
exposure. The first step would include the growth of
E. coli in four broth media containing 20mL of
distilled water. Petridish preparation would include
two mL of each broth tube mixed with 200ml of
distilled water. A portion of this mixed solution will
be applied on the petridish. Petridish exposure would
include exposing the E. coli under one of its
prescribed eleven watt lights for 48 hours (with eight
petridishes with no light). The two controls would be
the same cultured bacteria sample but have a white
light and a dark light exposed to it for the same
amount of time. Sample number for the control will
include three categories: red, blue and green each
with eight petridishes and the control will have two
categories: white light and no light. Quantification of
results was measured in number of colonies grown on
each petridish and added up for each category.
Results
The data seems to confirm the hypothesis of blue, red
and green light having a inhibiting effect on coliform
bacteria growth (E.coli). Figure 2 shows the total
number of colonies from the control (n=16) and
experimental (n=24) groups. An ANOVA was used
43
Saddleback Journal of Biology
Spring 2007
Discussion
7
# of colonies
6
5
4
3
2
1
0
control
experimental
Figure 1. Mean number of colonies per Petri dish for
white and dark control (n=16 total) and green, blue
and red experimental (n=24 total) groups (n=40)
120
# of colonies
100
80
60
40
20
0
control
experimental
Figure 2. Number of colonies total for white and dark
control (n=16) and red, blue and green experimental
(n=24) groups (n=40)
16
14
# of colonies
12
10
8
6
4
2
0
red
blue
green
dark
white
Figure 3. Colonies of colonies on each petridish
versus light exposure (dark being no light) with
green, red and blue being the experimental group and
dark and white being the control
for the data Figure 3 (individual bacterial colony
number for each petridish). ANOVA results in a pvalue of 0.002, prompting a Bonferonni connection
test. This statistical test yields a significant difference
between red (t-value 4.02) and blue (t-value 3.04)
light against no light. It should be noted that
temperature could have skewed the data because the
Petri dishes were left in the incubator for ninety hours
instead of forty-eight due to human error.
The results from the experiment show that there is
a significant difference between control and
experimental groups with a p-value 0.002 form
ANOVA and a Bonferroni connection test indicating
a significant difference between red and blue light
against no light. This outcome lends support to the
hypothesis that at least red and blue light will have an
inhibitive effect on coliform bacterial growth (E.
coli). It is an interesting conclusion considering that
the small variation from the colors of the visible light
spectrum would have a hindering effect on E. coli
compared to normal light conditions. Though this
outcome wasn’t anticipated, it wasn’t a far stretch
compared to other studies in this field where results
showed that certain wavelengths do inhibit coliform
growth (Meckes, 1982) and (Meyer and Reed, 2001).
Unfortunately, due to human error, the petri dishes
were left in an incubator that did not adjust for
temperature variation for ninety hours instead of
forty-eight hours. The deviation caused by an increase
in temperature could have been a factor skewing the
data off.
Although there is the possibility of data variation it
should not impede future studies in this area. Simple
and innovative methods to exterminating coliform
bacteria, a scourge of potable water, should be
encouraged, such as investigating if slight variations
in the environment will adversely effect bacterial
growth (Barcina et. al., 1986). Coliform organisms in
drinking water are particularly prevalent in flooded
areas. This is usually due to the fact that people
relieve themselves in this excess water (it’s
everywhere) while the water to begin with probably
mixed with contaminated sources. If there ever was a
incentive to use a cost effective method of
decontaminating septic water it would be here; sadly
because this problem generally happens in third world
countries were water sanitation usually is unreliable
to begin with. If simple procedures such as applying
colored light on septic water could in fact kill
coliform bacteria (through experimentation) it would
be a great assistance to not only vulnerable disaster
victims or areas; but could be used to lower the cost
of sanitizing waste water (at least for release into the
environment).
Acknowledgements
I would like to thank Dr. Huntley and Nam Vu for
their help, expertise and permission to use incubator
space, Petri dish plates and nutrient agar.
44
Saddleback Journal of Biology
Spring 2007
Literature Cited
Acher A. J., and Juven B. J. 1977. Destruction of
coliforms in water and sewage water by dyesensitized photooxidation. Appl. Environ. Microbiol.
33: 1019-1022
Fuijoka R. S., H. H. Hashimoto, E. B. Siwak, and H.
F. Reginald. 1981. Effect of sunlight on survival of
indicator bacteria in seawater. Appl. Environ.
Microbiol. 41: 690-696
Barcina, I., I. Arana, J. Iriberri, and L. Egea. 1986.
Factors affecting the survival of E. coli in a river.
Hydrobiologia. 141: 249-253.
Meckes, M. C. 1982. Effect of UV light disinfection
on antibiotic-resistant coliforms in wastewater
effluents. Appl. Environ.Microbiol. 43:371-377.
Fiksdal, L., and I. Tryland. 1999. Effect of UV light
irradiation, starvation and heat on Escherichia coli ßD-galactosidase activity and other potential viability
parameters. J. Appl. Microbiol. 87:62-71
Meyer V & Reed R. H. 2001. SOLAIR disinfection of
coliform bacteria in hand-drawn drinking water.
Journal of Tropical Medicine & Hygiene 87(2): 53-9
The Inhibitive Properties of Milk on Powdery Mildew Sphaerotheca fuliginea
Isobel Santos and Vyron Tayag
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
This experiment investigated the effectiveness of milk in fungal inhibition. A
comparative evaluation was done based on milk concentrations and the variable fat
percentages in milk that exhibits the most anti-fungal properties. The main objective of the
study was to find a fungicide that would be practical under standard conditions, costeffective and would make the less environmental impact. In the laboratory, cultures of
powdery mildew were grown on potato agar at relatively controllable settings. In an
environmental setting zucchini squash plants (Cuccurbita peppo) were used which was
sprayed with the milk solution. Rough visual evaluations were done to measure the density
of mildew on the leaves before and after the treatment. Milk inhibited mildew growth at
all concentrations on both the agar plates and the Zucchini squash plants. The results
showed a positive outcome 48 hours after spraying it with the milk solution. There has
been research on milk’s germicidal and antifungal properties that may have been
responsible for fungal inhibition. Compounds present in milk may be held accountable for
the milk’s inhibiting properties. In this study, milk exhibited a high potential in inhibiting
the growth of powdery mildew in plants. In the future, milk may perhaps provide an
essential resource in agriculture owing to its tremendous inhibiting properties and may in
fact create a relatively negligible detriment to both the environment and the economy.
Introduction
Sphaerotheca fuliginea, which is a type of powdery
mildew, is one of the many widespread fungal
diseases in conventional plantations. This type of
mildew is controlled by chemical fungicides like
sulfur and silicates that could potentially harm the
environment. An alternative that came up in the year
1999 was the use of milk as a fungicide for powdery
mildew. Research was done to prove the efficacy of
milk in inhibiting the growth of the mildew. There is
evidence that the milk has a direct effect on powdery
mildew.
The factors include the germicidal
45
Saddleback Journal of Biology
Spring 2007
properties of raw milk, the production of free radicals
upon exposure to sunlight and the antifungal action
of the fatty acids (Crisp 2006). There is an apparent
production of methionine (a chelating agent) and
riboflavin.
Milk components like lactoferrin, an
iron-bonding glycoprotein which is known for its
antifungal properties and lactoperoxidase which is an
antimicrobial reagent help to inhibit fungal growth.
(Boas et al 2002). The lactoferrin is a strong ironbonding agent which means that it has a strong
affinity for iron in the ferric state. This glycoprotein
depletes the pathogenic fungus and or bacteria of iron
which serves as an essential element for growth
leaving them incapable of surviving. There are a
number of advantages in using milk as a fungicide:
The milk provides a fungal inhibiting effect that does
not strain the plant itself, it is relatively inexpensive
and accessible, there is less or at most no possibility
for the fungi to evolve a resistance in milk therefore
decreasing the probability of an arms race between
the fungi and the fungicide, and milk fungicide is
probably the only fungicide that can produce the least
environmental impact.
Materials and Methods
The agar plate was made by mixing 39 grams of
potato dextrose agar into a 1000 mL of boiling water
in an Erlenmeyer flask until it reached a uniform
solution. It was then placed into the autoclave at
121°C for 15 minutes. Samples of the fungi were
collected from a native plant which was infested with
powdery mildew along the slopes of Saddleback
College in Mission Viejo California. The inoculum
was made by gently swabbing the surface of the
leaves with sterile cotton swabs and then smearing
the sample on the agar plates. It was then left to
proliferate for 48 hours. The powdery mildew was
then isolated from the rest of the colonies present in
the sample to avoid contamination and inhibition of
powdery mildew growth itself. The mildew was
smeared onto twenty, sterile potato agar plates. After
48 hours, the milk solutions were made. One solution
consisted of a dilution of 30% milk and 70% water.
The other solution however has a dilution ratio of
10% milk and 90% water. The control group
however, consisted mainly of water. Eighty 5mm
disk were then cut out of filter paper and were dipped
into their respective milk solutions. In each agar
plate, a total of four paper disks were equidistantly
positioned between each other to test for fungal
inhibition. The agar plates were then set in room
temperature with moderate sunlight. The plates were
visually evaluated every twelve hours for four days.
The plant experiments were done under an
uncontrolled setting that was made to reproduce the
plant’s normal habitat in the presence of the variables
and factors that exists in a natural situation. This
was done to test the maximum capability of milk and
its effects in a standard condition. Zucchini squash
(Cuccurbita peppo) was used in this experiment. The
plant was infected with the cultivated inoculum by
smearing the mildew on the leaves with a cotton
swab. The plant was then placed under direct
sunlight to initiate mildew growth and it was then
transferred to a shaded area. The milk solutions
were sprayed over the plants twice a day for three
days. It was watered everyday avoiding water
contact with the leaves. After 72 hours, the plants
were visually evaluated and were checked for any
pronounced differences between the effects of the
milk solutions and between the individual infected
leaves. The results that were taken were all based on
visual assessment and were done under a careful
method of evaluation that the researchers thought was
best for this particular study.
Results
The 30% milk solution inhibited 26/40 milk
disc, and the 10% milk inhibited 35/40 milk disc. A ttest was conducted between the two solutions and a
value of p= 0.1475, therefore there was no significant
difference between the milk concentrations. The
control group showed no growth inhibition on the
mildew. The mildew appeared to be thicker and
denser in the control group. Around the agar plate
were areas that are not affected by the milk, mildew
around that area were smaller and less dense. There
was a visible change in the amount of powdery
mildew present in both the plants and the agar plates
that were treated with diluted milk concentrations.
The plant samples exhibited a reduced area of
mildew infection 48 hours after the milk treatment.
There was no significant difference between the
plants that were sprayed with the whole and nonfat
milk solutions. Overall the milk showed fungi
inhibition, but between the two milk solutions there
was not a significant difference in fungi inhibition.
46
Saddleback Journal of Biology
Spring 2007
25
Non-fat milk
Whole milk
20
Control
Number of Non-infected disk
15
10
5
0
30% milk solution
10% milk solution
Figure 1. The 10% non-fat milk solution proved to be most efficient by inhibiting growth of powdery mildew on all
the filter disk that were placed into the potato dextrose agar. The 30% whole milk solution proved to have the least
amount of fungicidal effect. The control group did not have any disc that inhibited the growth of the mildew. A t-test
was conducted and a value p= 0.1475 meaning that there is no significant difference between the solutions.
Discussion
The exposure of methionine, riboflavin and sulfur
rich amino acids to light resulted in the production of
the free radicals that was linked to the reduced density
of both the plant samples and the agar samples. The
glycoprotein lactoferrin alters the permeability of the
membranes in conidia. The conidia then ruptures due
to a disruption in osmotic balance inside the body
(Crisp 2006). The pronounced inhibition exhibited in
this experiment was produced by the compounds that
are present in milk. Compound like lactoferrin,
riboflavin, methionine and caseine are the main
components contributing in the tremendous inhibiting
properties of milk. Lactoferrin has an ability to resist
degradation (Crisp 2006), meaning that the substance
has a prolonged functional activity and efficiency.
The biological actions of lactoferrin under various
experimental conditions are wide-ranging and
include: inhibition of the survival or growth of many
different pathogenic organisms, activation or
stimulation of a variety of immune system cells and
regulation of normal cell growth (Hutton et. al 2002).
Lactoferrin’s characteristic as an innate defense
protein may also serve as the first line of defense in
protection against pathogens. The role of lactoferrin
in the immune system is highly crucial. Treatment of
milk on an infected plant might increase the plant’s
immunity to powdery mildew. Riboflavin, also
known as Vitamin B2, exists in trace amounts in milk.
In 100 grams of milk 0.18 grams (12%) is present.
Riboflavin ‘s known effect on pathogen was its ability
to inactivate pathogens (Dong 2000). Riboflavin is
substantially reactive when exposed to sunlight owing
to its decomposition to release oxygen radicals which
are toxic to fungi (Bettiol 1999). This explains why
milk products and byproducts inhibit the growth of
powdery mildew. Methionine is an essential sulfurcontaining amino acid (Troup et al 2006). The sulfur
in methionine might also be accountable for the
fungal inhibition. There are no definite studies
related to the specific amount of sulfur required to
produce the desired fungal inhibition. Whether or
not the sulfur present in methionine will have an
effect on powdery mildew depends on the amount of
sulfur in the amino acid. Further studies are needed
to determine which of the components of milk
inhibits fungal growth.
Investigation of other
substances present is required to assess their unknown
ability. Milk revealed a tremendous potential in
powdery mildew inhibition. Considering that it does
not possess any possible environment or contaminant
nor does it induce resistance in plants. The substance
might just increase its value and importance in both
agriculture and in every household for the following
years.
47
Saddleback Journal of Biology
Spring 2007
Literature Cited
Abbasi P A, Lazarovits G. 2006. Effect of Acidic
Electrolyzed Water on Viability of Bacterial and
Fungal Plant Pathogens and on Bacterial Spot Disease
of Tomato. Canadian Journal of Microbiology 52(10):
915-924.
Bettiol W. 1999. Effectiveness of Cow’s Milk
Against Zucchini Squash Powdery Mildew (sphaero
fuliginea) in Greenhouse Conditions. Elsevier Crop
Protection 18: 489-492.
Boas J, Crisp P, Hutton D, Mckinnon I, Scott E,
Troup G. 2002. EPR studies of milk products tested
for Grapevine Powdery Mildew. University of
California Davis : p19.
Crisp P, Scott E, Troup G, Wicks T. 2006. Mode of
Action of Milk and Whey in the Control of Grapevine
Powdery Mildew. Australasian Plant Pathology 35:
487-493.
Dong H, Beer S.V. 2002. Riboflavin Induces Disease
Resistance in plants by Activating a Novel Signal
Transduction Pathway. Department of Plant
Pathology, Cornell University, NY 14853.
Hall B, Hitch C, Wicks T. 2002. Controlling Powdery
Mildew- What to Spray and When. Southern
Australian Research and Development Institute 59(8):
132-139
Mims C, Hanlin R, Richardson E. 2006.
Ultrastructure of the Fungus Ophiodothella Vaccinii
in Infected Leaves of Vaccinium Arboretum.
Canadian Journal of Botany 84(8): 1186-1196.
Olsen M, Young D. 1998. Plant Disease
Management- Powdery Mildew. Cooperative
Extension 9(98).
The Effect of Various Brown Algae on Growth of Rhizopus stolonifer
Samir Al-Ayoubi and Abdullah Ibish
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Rhizopus stolonifer is a common black bread mold that is harmful to humans.
The objective was to use brown algae (phaophytes) as an antifungal agent to prevent
growth. Various researches indicate that phaophytic plants utilize defense
mechanisms to prevent fungal growth. Microbes in the biofilms produce inhibiting
compounds to retard or restrain organism growth. Two trials were completed. In
trial one, four varying species of phaophytes were placed on potato-dextrose agar
and inoculated with R. stolonifer. Samples were observed after 24 hours. Trial two
consisted of blending the two species that illustrated the most inhibition of fungal
growth and were mixed within the agar. The R. stolonifer solution was placed on the
mixed agar and again observed after 24 hours. Both trials were conducted at room
temperature. As a result, both trials established no inhibition factor with R.
stolonifer and brown algae.
INTRODUCTION
A range of mycotoxigenic fungi is known to
colonize bread. Furthermore, understanding that
mans oldest fungicide, produced by some certain
plant species from diverse families (William and
Cooper, 2004); will aid weeding out possible
retarding elements on R. stolonifer growth. The
experimental objective was to conduct an
experiment and examine the effects of these
algae on fungal growth when placed on potatodextrose agar. Four species of phaophytes were
collected from Laguna Beach, CA; Macrocystis
pyrifera (gaint kelp), Egregea menziesii( feather
boa), Eisenia arboria (sea palm), and Laminaria.
48
Saddleback Journal of Biology
Spring 2007
Algae have evolved a number of physical and
chemical defense mechanisms to prevent fouling
(fungal growth in marine environments), such as
the production of mucus, shedding of fouled
tissue, and the production of inhibitory
compounds (deNys, et. al., 1994). One such
example is the green algae Ulva australis, which
appears to rely on surface-associated microbes
for defenses against fouling organisms
(Kjelleberg and Steinberg, 2002). This biofilm
may be the conclusion in preventing molds and
fungus. The expected results may also bring rise
in the attempt to research these antifungal
compounds and use them for preventing fungal
growth that infect humans, such as, Trichophyton
rubrum (athlete’s foot) and Candida (yeast
infection) (Cateau, et. al., 2007). Comparing
growth inhibition with different types of brown
algae will help pinpoint the best candidate for the
best results. Such findings could possibly lead to
an organic method of preservation in bread rather
than using different compounds, such as,
Butylated hydroxytoluene (BHT). Brown algae
is also an abundant source enabling a cheaper
method to use on food products.
MATERIALS AND METHODS
Two trials were conducted. Trial one
consisted of a variety of brown algae speices that
were cut in circular disks and soaked in R.
stolonifer solution for 24 hours. Four sets of
seven plates were used for the inoculation
process. Each set included one species of algae
and had four disks (1 cm in diameter) in each
plate. The agar was saturated with 0.5 mL of R.
stolonifer solution. Isopropyl alcohol was used to
sterilize a glass spreader, which was used to
distribute the solution evenly over the agar. Trial
two was completed using the two species of
algae that resulted in the most inhibition of
fungal growth (M. pyrifera and E. arboria).
Approximatley 30 grams of each species was
blended along with the potato-dextrose agar
solution using an Osterizer™ blender. One extra
plate was used for both trials as the control plate,
consisting of only the agar and the R. stolonifer
solution. All experimental plates were placed in
one holding container at room temperature (21º
C) and exposure to ambient lighting conditions
(standard room lighting).
RESULTS
The hypothesis proved incorrect as none of
the four brown algae species illustrated any signs
of inhibition of R. stolonifer growth in both
trials. The dishes were inspected 24 hours after
the mold solution was placed on the agar before
spore development. Hyphae grew as abundant as
the control plate.
Figure One. Algae disks placed on the potatodextrose agar showing no inhibition in growth of
R. stolonifer.
M. pyrifera and E. arboria showed a miniscule
effect on the growth in trial one having a slight
halo of no growth around the algae disks when
seen through a dissection scope. Consequently,
M. pyrifera and E. arboria were used in trail two
using the second method. Blending the algae and
inoculating it in the agar yet again had no effect
n fungal growth.
DISCUSSION
M. pyrifera, E. menziesii, E. arboria, nor
Laminaria inhibited the growth of R. stolonifer.
It was hypothesized that brown algae contain
antifungal compounds that will inhibit growth of
R. stolonifer. After attempting two different
approaches, it was clear that hyphae and spore
germination were not inhibited nor retarded
when exposed to brown algae. There may be
several reasons why inhibition did not occur.
One reason is that the microbes that are present
on the surface of phaophytes are species-specific
and do not produce the antifungal compounds for
prevention of R. stolonifer. Another reason is
that the microbes contained in the biofilms of
algae are only able to inhibit growth of types of
fungus that exist in marine environments (Bruhn,
et. al., 2007). Lastly, the environment that the
sample is grown in may effect growth, such as,
temperature or pH (Babich and Stotzky, 1977).
Disregarding the results of this study, the
anticipation for using antifungal compounds
from algae to protect against human infections,
still remains.
49
Saddleback Journal of Biology
Spring 2007
ACKNOWLEDGMENTS
The authors would like to thank Anthony
Huntley, PhD. from Saddleback College for
helping with preparing the potato-dextrose agar.
LITERATURE CITED
Arun Kumar K., and Rengasamy R.. 2000.
Evaluation of Antibacterial Potential of
Seaweeds Occurring along the Coast of Tamil
Nadu, India against the Plant Pathogenic
Bacterium Xanthomonas oryzae pv. oryzae
(Ishiyama) Dye. Vol 43 (5): 409-415.
Babich H. and Stotzky G. 1977. Effect of
Cadmium on Fungi and on Interactions Between
Fungi and Bacteria in Soil: Influence of Clay
Minerals and pH. Applied Environmental
Microbiology. 1977 May; 33(5): 1059–1066.
Bruhn JB, Gram L, and Belas R. 2007.
Production of antibacterial compounds and
biofilm formation by Roseobacter species are
influenced by culture conditions. Highwire Press
American Society for Microbiology. Vol. 73 (2):
442.
Cateau E, Levasseur P, Borgonovi M, and Imbert
C. 2007. The effect of aminocandin (HMR 3270)
on the in-vitro adherence of Candida albicans to
polystyrene surfaces coated with extracellular
matrix proteins or fibronectin. Synergy
Blackwell Journals . Vol. 13 (3): 311.
Franks A, Egan S, Holmstrom C, James S,
Lappin-Scott H, and Kjelleberg S. 2006.
Inhibition
of
fungal
colonization
by
Pseudoalteromonas
tunicata
provides
a
competitive
advantage
during
surface
colonization. Applied and Environmental
Microbiology. Vol. 72 (9): 6079.
Heier T., Jain S. K., Kogel K.H., and PonsKuhnemann J.. 2005. Influence of N-fertilization
and Fungicide Strategies on Fusarium Head
Blight Severity and Mycotoxin Content in
Winter Wheat. Journal of Phytopathology. Vol.
153 (9):551-557.
Kjelleberg S., and P. D. Steinberg. 2002.
Defenses against bacterial colonisation
of marine plants. Phyllosphere microbiology. p.
152–172
Martin M. Kulik. 1995. The potential for using
cyanobacteria (blue-green algae) and algae in the
biological control of plant pathogenic bacteria
and fungi. European Journal of Plant Pathology.
Williams J.S. and R.M. Cooper. 2004. The
oldest fungicide and newest phytoalexin - a
reappraisal of the fungi toxicity of elemental
sulfur. Journal of Plant Pathology. Vol. 53 (3):
264
deNys, R., Steinberg P.D., P. Willemsen,
Dworjanyn S. A., and C. L. Gabelish, and R. J.
King. 1994. Broad spectrum effects of secondary
metabolites from the red alga Delisea pulchra in
antifouling assays. Biofouling. Vol. 8: 259–271.
50
Saddleback Journal of Biology
Spring 2007
Contractile Vacuole Rates in Paramecia caudatum exposed to varying
Concentrations of Sucrose Solution
Daniel Rounds
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
The contraction rates of contractile vacuoles in Paramecia caudatum were
measured when placed into solutions containing varying concentrations of sucrose
in water. In order to regulate the follow of water into the center, the contractile
vacuoles expel water out of the cell at a constant rate. Also, when paramecia are
placed into solutions that are hypotonic to the cell, the paramecia absorbs water into
the cell via osmosis. When the control, 1%, and 3% solutions were added to the
Paramecia caudatum, the number of contractions per minute differed significantly
(7.8 x 10 -5 ANOVA). Twenty-two contractions/min was the average rate of
contraction for the control, 32 contractions/min for 3% solution, and 52.25
contractions/min for the 1% solution. It is only the 1% solution that differed
significantly from the two other groups because paramecia were in a hypoosmotic
solution causing an increase in water gain thus requiring the need to expel excess
water in fear of exploding.
Introduction
The life of a paramecium is one of movement,
absorption, and division. However, even though that
life may seem simple, there are very complex systems
at work to ensure the survival of life. Cells that live
in solutions can not absorb water and nutrients at will
with disregard of the quantity. If they do so, then the
cells will grow and swell until they reach a point
where they are not able to stay alive (Campbell et al.,
2006). The same is true for the lives of paramecia
cells. These cells are normally found in ponds in
shallow, still moving water (Headstrom, 1964). They
survive by swallowing nutrients along with water into
the cell through the membrane by osmosis.
Water will diffuse across the membrane of the cell
in limited quantities that can be predicted. There
exists a relationship between a cell and its solution
with regard to the movement of water. The tonicity,
cell’s ability to gain or lose water in a solution, is
affected by the concentration of a solute.
In this experiment, this solute will be adjusted to
create a hypotonic environment.
A cell in a
hypotonic solution has a greater concentration of
solute than the solution (Stock et al., 2002). The cell,
in an attempt to regulate itself to the solution, will
absorb water at a greater rate. The added mass of the
water will increase the cell’s volume until it pops
(lyses) and dies.
Cells are constantly regulating the amount of water
in the cell by pumping some of it out using their
vacuoles. Paramecia have two special types of
vacuoles to help expel water out of the membrane.
These are called contractile vacuoles. They are
shaped like little pinwheels with spokes twisting out
of the center. These spokes gather up water and move
it out of the cell constantly. It is this regulation that
keeps the cell from expanding to a point where it
would become too dilute to support life (Campbell, et
a.,l 2006).
Materials and Methods
Three solutions of different concentrations of
sucrose and water were placed on microscope glass
slides. The first solution was a 1% solution where 1
gram of sucrose was added to 100 mL of water. The
second solution was a 3% sucrose-water mixture.
The final solution was the control which a solution
where the Paramecia caudatum were bread in.
Organisms were obtains from Ward’s Biological
Supply Co.
Once all three of the solution had been prepared,
three drops of paramecia were added to each of the
samples. In order to observe the actual contractions
of the cell expelling water, Methyl Cellulose was
added to each slide to inhibit the movement of the
paramecia. After the slides have been readied, the
controlled solution was viewed underneath the
51
Saddleback Journal of Biology
Spring 2007
microscope. After finding a paramecium, the number
of contractions observed per minute was recorded.
Once a sample of the group was observed and
recorded, the next solution was placed under the
microscope and observed in the same way. After all
of the solutions were observed, the average number of
contractions per minute was calculated.
Results
The average contraction rate was recorded from
samples taken from the control (22 beats/min), 1%
(52 beats/min), and 3% (32 beats/min) sucrose
solutions. There was significant difference recorded
from the experiment (p=7.8 x 10-5 ANOVA). There
was a significant difference (Bonferroni Correction)
among two of the three groups in this experiment:
Control vs. 1%, and 1% vs. 3%.
Contractile Vacuole Contractions (beats/min)
60
50
40
30
contractions per minute. Both the control and the 3%
solution were well beneath the minimum level of
contractions per minute when compared to the
behavior in the 1%. This is normal because the lower
the concentration of sucrose in the solution is, the
more water the paramecium will have to absorb to
equal its concentration to the environment. However,
the amount of water that would be required to level
out the concentration inside the protist would have
caused it to lyse. In order to save itself, the
contractile vacuoles will continuously pump the water
out of the cell.
The more hypotonic (less solute concentration) a
solution is, the greater rate of osmosis will occur; thus
the contractile vacuoles will force water out at a
greater rate.
The more isotonic (more equal
concentration of solute) a solution is, the less water
will be required by the cell and vacuole activity will
decrease as well (Allen et al., 2005). If one were to
hypothesize that a certain solution contained a given
percentage of solute, one could determine that
percentage by creating varying solutions of that given
solute and adding Paramecia caudatum to them. By
comparing the contractions per minute of all solutions
to the unknown, one could find the solute percentage
of the unknown.
Literature Cited
20
Allen, Richard D., et al. 2005. Cell volume control in
Paramecium: factors that activate the control
mechanisms. J. of Exp Bio 154
10
0
Control
1% Sucrose
1
3% Sucrose
Environment Groups
Figure 1. The average mean results of the
Paramecia caudatum contractile vacuole rate
when placed in solutions with Sucrose levels of
1%, 3%, and the control. There was a
difference between the 1% vs. 3% and the 1%
and the control
Discussion
In this experiment, the 1% paramecia tested
showed significant differences in numbers of
Campbell, Neil A., Jane B. Reece, Martha R. Taylor,
and Eric J. Simon. Biology Concepts and
Connections. 5th ed. San Francisco, CA: Benjamin
Cummings, 2006.
Headstrom, Richard H. 1964 Adventures with
Freshwater Animals. New York: Dover Publications.
Stock, Christian, et al. 2002. Relationship between
the membrane potential of the contractile vacuole
complex and its osmoregulatory activity in
Paramecium multimicronucleatum. Journal of
Experimental Biology 10.
52
Saddleback Journal of Biology
Spring 2007
The Effect of Temperature on the Metabolic Rate of Goldfish (Carassius auratus)
Kelsey Quaranto
Department of Political Science
Saddleback College
Mission Viejo, CA 92692
Metabolic rate is how fast an organism consumes energy, uses it and then expels it. In
this experiment variations in temperature were used as an environmental factor in order to
test the effects it had on goldfish (Carssius aurutus) metabolism. Ten goldfish were placed
individually into beakers containing water temperatures at 8° Celsius and 20° Celsius. For
a length of five minutes, the dissolved oxygen content of the water was measured and noted
at its initial and final readings. It was hypothesized that at the warmer temperature of 20°
C the metabolic rate of the goldfish would be greater than it had been in the cooler water.
Though the results show that there was no significant change, it is noted that other factors
may play into the effects on an organism’s metabolic rate. Thus, temperature may not be
an independent variable.
Introduction
Materials and Methods
Goldfish are poikilothermic organisms, meaning
their body temperatures change in accordance to their
environmental temperatures. When observing
metabolic rate, statistical analysis generally notes the
Q10 temperature coefficient, which states that for an
increase of 10° C in temperature the metabolic rate is
doubled (Gee et al., 1994). Ecologists study the
correlation between metabolic rate and temperature in
reference to aquatic activity such as migration as well
as ecosystem changes from such effects as climate
change. Varying temperature affects the migration
patterns of lobsters in during different seasons.
Schreiber et al. (2000) showed that an increase of 79° C in the lobster’s metabolic rate approximately
doubled and thus influenced the seasonal migration
patterns. As stated above researchers have also taken
note of the effects of climate change on aquatic
populations. Beamish et al., (2006) showed that
populations of the same species that are separated
latitudinally often have distinctive metabolic rates.
This demonstrates forced adaptations to temperature
variations. and how climate change and its effect on
sea temperatures will further force these aquatic
organisms to adapt (Beamish et al., 2006). Brown
(2001) observed that metabolic rate, which largely
effects the rate of biological development will be an
effective tool in predicting how quickly creatures will
develop, reproduce and die. This experiment observes
the relationship between metabolic rate and
temperature and its effect on goldfish.
Ten goldfish were placed in ten separate beakers.
Each beaker contained 300 mL of treated tap water at
the room temperature of 18° C. A thermometer was
placed in each beaker in order to monitor the water’s
temperature. The temperature of the water was
brought down to 8° Celsius through the use of an ice
bath. At this temperature, the beaker was removed
from the ice bath and placed into a container so that
the fish weren’t disturbed by outside activity. Using a
Pasco Xplorer GLX the dissolved oxygen sensors
measured the oxygen content of the 8° C water. Data
were taken at the initial reading and the final reading
– after five minutes. After the readings were retrieved
the water was warmed naturally to room temperature,
which had risen to 20° C. At room temperature, the
goldfish were removed from their beakers and put
into fresh, treated tap water so that they were in water
with “unused” oxygen. Again data were retrieved on
the amount of dissolved oxygen in the water for a
length of five minutes with an initial and a final
reading. The data were analyzed using a paired t-test
by measuring the average change in O2 levels. The
metabolic rate (mg/L/min) of each fish was calculated
by dividing the change in the dissolved oxygen level
by five minutes (the length of each run). Data were
analyzed with a paired t-test, p< 0.05 was considered
statistically different.
53
Saddleback Journal of Biology
Spring 2007
Discussion
Change in O2 concentration (mg/L)
1.4
1.2
1
0.8
0.6
0.4
0.2
0
8C
20C
1
Temperature (C)
Figure 1. There was no difference in the amount of
oxygen consumption for fish at 8°C and 20°C after
five minute (p=0.32, paired one tailed t-test).
Metabolic Rate (dissolved O2 mg/L/min)
0.3
0.25
0.2
The insignificant difference in the observed results
does not support the stated hypothesis; however it
must be noted that it does not nullify the hypothesis
either. Goldfish are generally found to have high
adaptation abilities in order to increase their chance of
survival. Within the experiment as the water
temperature was reduced to 8° C the goldfish were
present in the water, thus allowing for a more gradual
change and the opportunity to adapt. While the Q10
effect is generally used in such studies, James F.
Gillooly et al., comments that as much as a 15% error
can occur using the equation within the “biologically
relevant” temperature range (Brown et al., 2001).
This raises the question of temperature being more of
a dependent variable as opposed to an independent
variable. Suggested by both Gillooly and Brown, the
effect of metabolic rate on temperature is best
observed in correlation with the size of the organism.
Furthermore, with the beakers location in containers,
the goldfish were blocked from outside activity at all
times and were very still throughout nearly the entire
experiment. The measured metabolic rate was for the
most part, their resting metabolic rate.
Literature Cited
0.15
Beamish R., Carmichael T., Dempson B., King J.,
Power M., Prowse T., Reist J., Sawatzky C., Wrona
F. 2006. General effects of climate change on arctic
fishes and fish populations. AMBIO: A Journal of the
Human Environment. 35(7): 370-380.
0.1
0.05
0
8C
1
20C
Temperature (C)
Figure 2. Metabolic rate was calculated from the
change in dissolved O2 concentration. There was no
difference in metabolic rate between 8°C and 20°C
((p=0.32, paired one tailed t-test).
Results
There was no statistical difference (p= 0.32, paired
one-tailed t-test) in the change in the mean dissolved
oxygen concentration (Figure 1) between 8° C
(dissolved O2 0.98 mg/L) and 20° C (0.82 mg/L).
The mean metabolic rate for the water at 8° C was
0.196 mg/L/min and for the water at 20° C it was
0.164 mg/L/min. There was no significant statistical
difference (p=0.32, paired one-tailed t-test) between
the two groups (Figure 2).
Brown J., Charnov E., Gillooly J., Savage V., West
G. 2001. Effects of size and temperature on metabolic
rate. Science. 293(5538): 2248-2251.
Brown K. 2001 All fired up: a universal metabolic
rate. Science. 293(5538): 2191.
Cooke Schreiber SM., Jury S., Watson WH. 2000.
Seasonal differences in behavioral thermoregulation
in the lobster, Homarus americanus. American
Zoologist. 40(6): 925-1273.
Gee P., Stephenson D., Wright DE. 1994. Temporal
discrimination learning of operant feeding in goldfish
(Carassius auratus). Journal of the Experimental
Analysis of Behavior. 62(1): 1-13.
54
Saddleback Journal of Biology
Spring 2007
The Effect of Exercise on Carbon Dioxide Concentration Levels in Exhaled in Humans
Enas Abu Hammad
Department of Philosophy
Saddleback College
Mission Viejo, CA 92692
Carbon dioxide is produced during cellular respiration in humans. The paper at hand
studies the effect of exercise on carbon dioxide concentration levels in exhaled air utilizing
the relationship between exercise and cellular respiration rate. Exhaled air samples were
collected from five track runners before and after exercising and were measured for carbon
dioxide concentrations. The readings displayed an elevation in CO2 concentrations after
exercise. Hence, the results were consistent with the hypothesis of this paper.
Introduction
Carbon dioxide (CO2) is produced during cellular
respiration, the process through which energy is
synthesized in human body. Also, it is an aerobic
process; therefore necessitates the presence of oxygen
(O2) for its occurrence. Moreover, energy yielding in
cellular respiration involves; the breakdown of simple
sugars (glucose), consumption of oxygen, and
production of carbon dioxide and water as by
products. Below is the chemical equation of cellular
respiration:
C6H12O6 + 6O2
6CO2 + 6H2O + Energy (ATP)
Glucose Oxygen Carbon Water
Dioxide
Briefly, cellular respiration consists of three stages;
glycolysis, Kreb’s cycle, and electron transport chain.
In the first stage, two 3-carbon pyruvic acid
molecules result from breaking down 6-carbon
glucose molecule. During Kreb’s cycle, three O2
molecules are used to oxides each 3-carbon pyruvic
acid molecule. As a result, carbon dioxide is
formulated. The third stages involves electron
transport chain , which is a specified and specialized
set of electron carriers, that removes excess electrons
rising from reactions in glycolysis and Kreb’s cycle.
Gaseous exchange is a process during which the
human body expels CO2 and intakes O2. It takes place
in tiny sacs called alveoli that come at the end of the
bronchial branching and are the final destination of
inhaled air before it undergoes gaseous exchange.
Gaseous exchange occurs in accordance with the
concept of simple diffusion that requires a maintained
concentration gradient. There should be a sustention
of higher concentration level of O2 in the alveoli than
in the blood capillaries surrounding them and a lower
concentration level of CO2 in the blood capillaries
than in the alveoli to allow CO2 to diffuse from the
blood stream to the lungs and to be finally exhaled via
breathing. As for O2, the process occurs vice versa.
This study examines changes in carbon dioxide
concentration in exhaled air, before and after
moderate levels of exercise. Based on the fact that
exercise causes a rise in cellular respiration that
engages the production of carbon dioxide, it is the
study’s objective to prove an elevation in carbon
dioxide concentration after exercise.
Materials and Methods
Right before doing track, five female track runners
were given five Ziploc bags labeled B1, B2, B3, B4,
and B5. Each one of them slightly opened the bag
labeled with her number and exhaled in it. They
securely zipped their bags while they were still
exhaling to prevent any leakage or air mixing. The
same process was repeated after the same runners did
three laps around the track field. However, this time
with Ziploc bags labeled A1, A2, A3, A4 and A5.
Afterwards, the samples were taken to the lab and
measured for CO2 concentrations using Xplorer GLX
(PS-2002), which is a graphing datalogger device that
works with the pasport CO2 gas sensor to provide
readings of carbon dioxide concentrations in a certain
atmosphere. When measuring, each bag was opened
slight enough to place the rod-like sensor inside it and
was closed tightly afterwards. The readings were
given the needed time (96s-204s) to stabilize before
they were collected.
Results
Clearly, carbon dioxide concentration readings
after exercise witnessed an increase for each runner.
Average of carbon dioxide concentrations before and
after exercise were 32174.4 (ppm) and 41817.4( ppm)
respectively. It could be concluded that the results
supported the hypothesis investigated and propose
that exercise promotes an increase in carbon dioxide
concentration levels (Figure 1).
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Saddleback Journal of Biology
Spring 2007
Discussion
Cellular respiration provides the body with the
needed chemical energy that is transformed later into
many forms energy including kinetic during exercise.
Human body responds to higher levels of exercise by
increasing the rate of cellular respiration to yield
sufficient chemical energy. Due to this increase in
cellular respiration, carbon dioxide concentration in
cells rises. As a result, more carbon dioxide will
diffuse from cells to the blood stream for it to be
expelled through gaseous exchange. The directly
proportional relationship between exercise and
metabolic rate also suggests an increase in
concentrations of carbon dioxide in cells initially and
in exhaled air ultimately.
Figure 1. Shown in the graph above are Carbon
Dioxide concentration readings for each runner
before and after exercise.
Literature Cited
Bingham, S. A., Coward, W. A., Cummings, J. H.,
Goldberg, G. R., Prentice, A. M. (1989). The effect of
exercise and improved physical fitness on basal
metabolic rate. British Journal of Nutrition, Volume
61, Number 2, pp. 155-173.
Carter, J. Stein (2004). Cellular Respiration and
Fermentation. Available at:
John Hopkins University. Alveoli. Available at:
http://oac.med.jhmi.edu/res_phys/Encyclopedia/Alve
oli/Alveoli.HTML
56
Saddleback Journal of Biology
Spring 2007
CAFFEINE’S AFFECT ON RESPIRATION
DURING EXERCISE. Samir R. Al-Ayoubi. Dept of
Biology, Saddleback College, 28000 Marguerite
Parkway, Mission Viejo, CA 92692, USA.
Caffeine is known to increase heart rate. It is
hypothesized that if heart rate increases, then
respiration rate will be directly affected and increase
as a result. The objective was to investigate whether
caffeine could increase one’s respiration rate to
compensate for the increase in heart rate; thus
providing enough oxygen for the body. An increase in
respirations results in a lack of adequate oxygen
intake. This forces the body to undergo anaerobic
respirations and results in lactic acid fermentation,
causing more muscle soreness and a prolonged
healing process. Ten male subjects were put to
exercise on a stationary bicycle for ten minutes. Each
subject was tested twice, once without caffeine
(control) and a second under the influence of 200 mg
of caffeine. Expired volume of carbon dioxide
(VCO2), heart rate, and number of respirations, were
recorded throughout the ten-minute duration of their
exercise. An increase in CO2 reflects an increase in
respiration rate. The means for CO2 exhaled, heart
rate, and numbers of respirations, prior to caffeine
intake were 144731.1 parts per million (ppm), 147.5
beats per minute (b ⋅ m-1), and 120 respirations.
THE EFFECT OF PREMOLAR EXTRACTION
ON MAXILLARY WIDTH Omid Niavarani and
Ross Johnson. Dept. of Dental Surgery, Saddleback
College, Mission Viejo, CA 92692
Removal of premolars is common practice
throughout the field of orthodontics. However, the
removal of these premolars can alter the maxillary
width in individuals who are receiving treatment.
Hence, the purpose of this study: to discover the
change in maxillary width as a result of premolar
extraction. The control group (n= 36) of this study
consisted of individuals without premolar extraction.
The experimental group (n= 22) contained individuals
with premolar removal. Both experimental groups
included individuals older than 13 years of age.
Analysis of the data showed that the mean maxillary
width of females without premolar extraction (43.25
+/- 0.83 mm) were greater than that of females with
premolars absent (38.70 +/- 0.9341 mm). Males
exhibited similar results with the mean maxillary
width of those with premolars present (45.75 +/1.127 mm) being greater than subjects without
premolars (38.5 +/- 0.773 mm). A one-tailed t-test of
the data showed that there was a significant difference
between that of females and males with premolars
present and absent (p= 4.75 x 10-4 and p= 4.13 x 10-5,
respectively).
GOLDFISH LONG TERM MEMORY (Carassius
auratus). Courtney Alexander and Colleen Worne.
Dept. of Biology, Saddleback College, Mission Viejo,
California 92692, USA
Goldfish are thought to have a memory of only
three seconds. This test determined whether goldfish
have a long term memory. Via food and red colored
rings around precut holes the goldfish were trained to
swim through and obstacle course. With repeated
trails the fish associate the red rings with food, thus
triggering their memory that red rings equal reward.
They also exhibited social learning behavior. The
ultimate result being the fish are able to swim through
the entire course. The experiment ran for one week
with two trails each day. There was a significant
difference between the first day’s trials and the last
day’s trials (p= 0.0033 Paired t-Test). The goldfish
averaged an improved time of 241 seconds, or four
minutes between each day. The overall trend showed
a decrease in time of swimming through the course.
THE EFFECT OF FOOD ON METABOLIC
RATE IN GOLDFISH (Carassius auratus). Hasib
Rahmani* and Avishan Kolahoduz Nasiri. Dept.
Biology, Saddleback College, 28000 Marguerite
Parkway, Mission Viejo, CA, 92692, USA
Metabolism is the biochemical modification of
chemical compounds in living organisms and cells.
The total metabolism are all biochemical processes of
an organism. When an animal senses that there is a
lack of food, their metabolic systems decrease activity
in order to conserve energy. When a lack of energy is
overcome and food is available and consumed by
animals, their metabolic activity increases. To study
if food as a form of chemical energy affects metabolic
rate, we measured opercular pumping rates in
goldfish (Carassius auratus). If the measure of water
is unvarying as it enters through the mouth and exists
through the operculum, the rate of opercular pumping
is relative to the oxygen demand. We used this fact to
indirectly measure metabolic rate as a function of
opercular pumping rate. To support the idea that
metabolic rate increases with food intake, ten goldfish
were fasted for four days. At the end of the fast,
opercular pumping was measured right before
feeding. The fish were then fed one flake of food of
equal mass per fish and allowed to consume the food
for 20 minutes.
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Saddleback Journal of Biology
Spring 2007
ARE OVERWEIGHT INDIVIDUALS MORE
LIKELY
TO
TEST
POSITIVE
FOR
GLYCOSURIA? Jennifer Wilden and Kevin
Laitipaya. Department of Biological Science,
Saddleback College, Mission Viejo, CA 92692, USA.
The rise in diabetes as one of the leading causes of
death has been closely related to obesity, and onethird of the people with diabetes are undiagnosed and
a-symptomatic. This study examined non-diabetic
participants to determine if there is a relationship
between glycosuria and weight. Between October 23
and November 16, 2006, 34 participants (aged 16-53)
were tested for glycosuria using Benedict’s copper
reduction tests. Each subject willingly provided their
age, height, and weight in writing, and their body
mass index (BMI) was calculated (kg/m2) and
categorized as normal (<25), overweight (>25 or
<30), or obese (>30). Results determined that 8.8%
(n=3) of the subjects tested positive for glycosuria
(0.1 – 0.5 g/dl) of which 66.7% (n=2) were classified
as overweight, and 33.3% (n=1) were normal weight.
There was no statistical significant difference for BMI
between subjects who tested positive vs. negative for
glycosuria. However, glycosuria testing is a costeffective measure of screening for diabetes that
should not be limited to overweight individuals.
COMPARATIVE VARIATION OF WATER
LOSS IN MALES AND FEMALES DURING
INTENSE PHYSICAL ACTIVITY.
Nicholas
Davison and Erinn Deters. Dept. of Biological
Sciences, Saddleback College, Mission Viejo, CA.
The human body is subject to various rates
of evaporative water loss as well as trans-epidermal
water loss, depending on the intensity of physical
activity. Since there are many other physiological
differences between males and females it was
predicted that a similar difference in evaporative
water loss as well as trans-epidermal water loss would
be apparent. Participants were placed on stationary
bikes and instructed to pedal for 30 minutes each
whilst maintaining a heart rate of 150. Heart rate was
measured using integrated monitors and total water
loss, including both evaporative and trans-epidermal
water loss, was measured using a scale to the closest
tenth of a pound. There was a visible amount of
water loss apparent in both the male and female
groups in comparison to the control, which had no
water loss. As much as 1.2 lbs, 0.54 kg, and as little
as 0.2 lbs, 0.09 kg, were lost in the active participants.
Although the males appeared to have heavier water
loss, statistical analysis revealed no significant
difference between the two groups.
PEDALING CADENCE EFFICIENCY AS
MEASURED BY DISTANCE TRAVELED
VERSUS CARDIAC OUTPUT. Jason Beltz and
David Stapleton, Department of Biological Sciences,
Saddleback College, Mission Viejo, CA 92692.
There have been numerous studies of pedaling
cadence efficiency conducted with professional
cyclists that claim levels of efficiency at 80, 90 or 100
pedal revolutions per minute, but these cyclists are
finely tuned machines capable of attaining speeds and
exhibiting endurance far in excess of most normal
recreational cyclists. The question we asked: Is there
any significant efficiency to be gained at 80, 90 or
100 pedal rpm to be gained by recreational cyclists?
Average pulse rate, distance traveled and mean
arterial pressure were recorded at each cadence.
Cardiac output was calculated and a ratio of cardiac
output to distance traveled provided a measure of
efficiency at each cadence. Mean efficiencies were
between 0.027 ± 0.003 km·L-1 and 0.031 ± 0.004
km·L-1at the measured cadences. As a group the
analysis of the data collected showed that there was
not a significant difference in the measured efficiency
at any of the cadences. We can conclude that while
there may exist a level of efficiency in professional
riders at specific cadences, this may not hold true for
recreational cyclists pedaling over shorter distances.
HOW EFFECTIVE ARE ANTI-BACTERIAL
CLEANERS
AGAINST
BACTERIAL
GROWTH? Mayra Mejia and Annie John. Dept. of
Biology, Saddleback College, Mission Viejo,
California 92692, United States.
Bacterial growth has been looked upon as a major
problem in society. In order to eradicate that problem,
several products have been manufactured to help
control or eliminate bacterial growth. Two antibacterial products, 10% Clorox® and 100% Lysol®
solutions, were used to test the hypothesis formulated,
which was Lysol® would have a higher area of
inhibition than Clorox®. To assess the hypothesis,
chads, holes punched out of filter paper, were soaked
in the respective cleaners and then placed onto
Staphylococcus aureus culture plates. The control for
the experiment was chads soaked in sterile water. The
mean area of inhibition of Lysol® as compared to
Clorox® and sterile water (P=0.00011) showed that
there was a statistical difference between the groups.
The difference in the areas of inhibition shows that
Lysol® had a higher area of inhibition than Clorox®.
58
Saddleback Journal of Biology
Spring 2007
ENERGY
PRODUCTION
THROUGH
MICROBIAL
OXIDATION-REDUCTION
REACTION. Erik Olmos. Isobel Santos. Vyron
Tayag. Department of Biology, 28000 Marguerite
Parkway Mission Viejo, California 92692.
A new prototype called the Microbial Fuel Cell
(MFC) is a seemingly promising process that involves
the use of bacteria not only for a cost-effective energy
production but also for a highly effective wastewater
treatment. Its advantages made the MFC paradigm
practical and convenient. The process entails a
minimal amount of energy input as opposed to a
relatively high amount of energy production, a
byproduct of water compared to carbon dioxide which
makes it environmentally efficient and is used to treat
wastewater plants. The goal of this experiment is to
be able to produce a structure that would be relatively
efficient by comparing the energy production from a
soil-based MFC in opposition to a wastewater MFC.
Power generation can be attained through the wellknown oxidation-reduction process in organisms.
The bacteria that are known to proliferate and are
most abundant in environments such as soil and
wastewater are the Shewanella putrefaciens,
Geobacter
metallireducens
and
Rhodoferax
ferrireducenss(Liu, Ramnarayanan, Logan 2004).
These bacteria are placed in an anaerobic
environment (referred to as the anode
ANALYSIS OF THE ACCURACY OF SUGAR
CONCENTRATION IN SPORTS DRINKS
THROUGH FERMENTATION Blair Leaf, Heidi
Schaff and Nasrene Hosseini. Department of
Biological Science. Saddleback College. Mission
Viejo, CA 92692
The accuracy of the sugar content displayed
in the nutrition facts labels of sports drinks has been
subject to questioning. Previous studies show that the
intake of carbohydrates has varying affects on
metabolism and athletic performance. Unnecessary
sugar consumption is often a consideration when
selecting a sports beverage, thus determining sugar
content from nutrition labels is significant. To
determine the accuracy of nutrition labels, the sugar
concentrations in the two popular sports beverages,
Powerade® and Gatorade®, were measured. Through
the process of fermentation it was concluded that
there was not a significant difference in the mean
amount of carbon dioxide production, indicating no
difference in the content of sugar in the two beverages
(p=0.49). No difference in the mean amount of carbon
dioxide production indicates that the two substrates
had relatively similar amounts of sugar per serving.
This supports the hypothesis that nutrition labels of
the sports beverages accurately reveal the amount of
sugars that they contain.
COMING CLEAN: HAND SANITIZERS ARE
MORE
EFFECTIVE
AGAINST
STAPHYLOCCOCUS
AUREUS
THAN
ANTIBACTERIAL SOAP. Amantha Bagdon. Dept.
of Biological Sciences, Saddleback College, Mission
Viejo, CA 92692
The purpose of this experiment was to determine
whether alcohol-based hand sanitizer is more
sufficient in inhibiting the growth of Staphylococcus
aureus than antibacterial soap. The study streaked
nutrient agar plates with S. aureus and incubated the
samples for 48 hours to produce bacteria growth.
After the incubation period, small sterilized discs of
filter paper soaked in hand sanitizer, soap, and
sterilized water were distributed evenly over the
bacteria and allowed to incubate and allowed to
incubate for another 72 hours. After the final
incubation stage, the area of inhibited growth was
recorded. The results demonstrate that while both
hand sanitizer and antibacterial soap are more
productive against S. aureus than sterilized water,
hand sanitizer is the most effective means of
destroying bacteria. Antibacterial soap reduced
bacteria growth by an average area of 0.468 cm²
while the hand sanitizer reduced growth by an
average area of 0.571 cm². Sterilized water on the
other hand, only reduced the growth by an average
MUSIC AFFECTS HUMAN HEART RATE.
Dominique Alex and Andrew Starsiak. Department of
Biology. Saddleback College. Mission Viejo, CA
92692
The tempo of music affects the human heart rate
by stimulating receptor cells in the inner ear, exciting
neurons in the temporal lobe of cortex, sending out
signals to systems such as the autonomic nervous
system. They may excite the sympathetic and
parasympathetic nervous systems and make the heart
beat faster or slower, and increase or decrease the
pulse. Due to this, it was predicted that the higher the
beats per minute (bpm) in the song, the greater the
increase in heart rate. Resting heart rate without
music, heart rate aroused by a high bpm musical piece
and heart rate aroused by a low bpm musical piece
were used to assess this. Of a sample of 20 subjects,
there was a mean resting heart rate of 67 bpm, a mean
of 87 bpm for the faster tempo music, and a mean of
74 bpm for the slower tempo. There was a mean
increase of 20 bpm for the faster tempo music and 6
bpm for the slower tempo music when comparing it
to the mean heart rate. An ANOVA single factor
analysis was run in Microsoft Excel® for the data and
found a statistical difference with (p = 4.6x10-7).
Bonferroni correction isolated the data discrepancies
in fast vs. slow tempos, and fast tempo
59
Saddleback Journal of Biology
Spring 2007
THE EFFECTS OF A HIGHER DOSE OF
NITROGEN VERSUS A HIGHER DOSE OF
PHOSPHATE IN THE MARIGOLD FLOWER,
TAGETES PATULA. April Jones and Stephanie
Olamendi. Department of Biological Science,
Saddleback College 28000 Marguerite Parkway,
Mission Viejo, California 92692.
Plants rely heavily on Nitrogen (N) for
photosynthesis, which is directly related to plant
health. Available phosphate (P2O5) is vital for plant
metabolism, energy production, and other important
plant processes. This study focused on the effects of
N and P2O5 in the plant, Tagetes patula, and how they
effect plant growth. Weekly measurements of stem
height and leaf lengths were taken to determine
growth. The hypothesis being tested was that plants
receiving more N would show a favorable difference
in stem height and leaf lengths than plants receiving
more P2O5. Thirty pots were used in this study
containing four seeds each. Ten pots received more
N, ten received more P2O5, and ten were the control
receiving only DI water.
By week four of
measurements, stem height and leaf lengths showed
no significant difference between plants receiving
more N and the control; however, showed a
significant difference between plants that received
more P2O5 and the control, and also between the N
group and the P2O5 group. (ANOVA = P <0.05, with
THE RATE OF OXYGEN CONSUMPTION OF
GOLDFISH BASED ON SIZE. Matin Khoshnevis
and Mortaza Aghazadah.
Dept. of Biology,
Saddleback College, 28000 Marguerite Parkway,
Mission Viejo, CA 92692.
This experiment was done to look at the effects of
size on the rate of oxygen consumption of goldfish.
This experiment was done using an oxygen electrode,
Xplorer GLX, made by the PASCO Company which
measures the oxygen consumption in a 100 mL
beaker of water. The procedure involved leaving the
goldfishes in the beaker for a period of 3 minutes. All
the goldfishes started initially with the same amount
of dissolved oxygen in the beaker (20mg/ L). The
dissolved oxygen was measured before and after the
fish was in the beaker. The rate of oxygen
consumption was greater in large goldfishes (19.78
mg/ L) comparing to small goldfishes (19.20 mg/
L).There is no significant difference between the
average rate of oxygen consumption of large
goldfishes from the initial dissolved oxygen (p=0.92).
In contrasts, there is a significant difference between
the rate of oxygen consumption of small goldfishes
from the initial oxygen consumption (p=0.02). This
experiment stressed the importance of size on the rate
of oxygen consumption, while leaving a broad impact
on other undiscovered factors that are now open for
further research.
EFFECTS OF VARIOUS ENERGY DRINKS ON
ATHLETIC
PERFORMANCE
OF
ADOLESCENTS
DURING
STRENUOUS
SWIMMING ACTIVITIES Gimena Altamirano
and David M. R. Quijano. Department of Chemical
Sciences and Department of Biological Sciences.
Saddleback College Mission Viejo, CA 92692
It is believed that energy drinks will increase the
quality in performance of athletes and others under
physical stress. This study was performed to
determine the effects of energy drinks on athletes,
specifically adolescent female swimmers. 10 subjects
were chosen for the test and each was given eight
ounces of a specific energy drink or supplement to
consume before completing a 100 meter freestyle
heat. The subjects would then have their heart rate
and lap time measured and recorded. On another day,
to insure no overlap in the effects, the subjects were
given a different drink and were instructed to
complete the heat again. The effects of each drink on
performance were determined after all data were
collected. The results showed that energy drinks do
not have effects on the performance of athletes.
However, the heart rate of each athlete was
significantly increased and adverse reactions during
digestion of the drinks were noticed by the subjects.
EFFECTS
OF
CRYSTALLOID
FLUID
THERAPY ON THE HEMATOCRIT OF
HOSPITALIZED CANINE PATIENTS. Heather
Rufino and Stephanie Reed. Dept. of Biology, Dept.
of Landscape Design, Saddleback College, 28000
Marguerite Pkwy, Mission Viejo, California 92692
When administering fluid therapy, special attention
must be given to the microcirculation and factors
controlling fluid movement across capillaries. The
central circulation is regulated mostly by a need to
maintain normal effective plasma volume, which
plays a role in determining whether the capillary
space and serious body cavities gather or release
surplus solute and water. Because of Starling forces
on the fluid dynamics into and out of the capillary
space, it was predicted that fluid therapy would cause
an initial drop in hematocrit, followed by a
transitional response that would attempt to correct the
drop. Sampling intervals before fluid therapy, twice
during fluid therapy, and after 12 hours of fluid
therapy in hospitalized canines were analyzed to
assess this. The ratio of fluid to body mass affected
the change in hematocrit, such that maintenance level
fluid therapy (1.25mL•Lb-1•hr-1) caused a mean drop
of .42 g•dL-1, and 2.5 x maintenance level caused a
mean drop of 5.17 g•dL-1. Between the 8-hr and 12-hr
sampling intervals the 2.5 x maintenance group.
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Saddleback Journal of Biology
Spring 2007
THE EFFECTS OF pH ON BACTERIAL
GROWTH INHIBITION OF Staphylococcus
aureus. Tom Caldwell and Mitra Foroutan. Dept. of
Biology, Saddleback College, Mission Viejo, CA
92692 USA
It is understood that bacterial contamination
in food products is a major problem today. Food
requires proper care to prevent such contamination,
but there could be new and better ways to keep food
products fresher. Bacteria have a certain pH range
within which lies the optimum condition for their
growth, an environmental pH out of their desired
range should inhibit the growth of bacteria
(Staphylococcus aureus); so we hypothesize that a pH
of 9 should be outside of the desired range for most
bacteria, and it should inhibit bacterial growth. To
test this hypothesis, a solution of nutrient agar was
prepared basic to a pH of 9 with 1% NaOH (sodium
hydroxide) for the experimental factor while another
solution of nutrient agar was prepared for the control
factor at a pH of 7. Using the two solutions, the
bacterial samples of the two groups were incubated at
37oC for 48 hours, after which time the samples were
taken out and the number of colonies in each Petri
dish was counted. The average of the bacterial
colonies for the neutral group was 532.95 ± 46.05
colonies per Petri dish (mean ± standard error, N =
20) and the average for the basic group was 487.65 ±
26.30 colonies per Petri dish (mean ± standard error,
N = 20); a one-tailed t-test was conducted on the data,
calculating a p-vale of
EFFECTS OF ACID RAIN ON PLANT
GROWTH AND THE ENVIRONMENT. Jackie
Olvera. Department of Biological Sciences.
Saddleback College, Mission Viejo, CA 92692
The emergence of industrial pollution has
caused acid rain that can harm the natural
environment by inhibiting plant growth. This study
examined the effects of acid rain in a simulated
environment on Brassica juncea. Germinated seeds
were watered with solutions of pH 5.6 and 3.5,
respectively to simulate normal and acid rain. The
average growth rates after four after four weeks of
acid watering were 0.074cm/day and 0.053 cm/day
for the respective control and experimental groups.
There was a significant difference between the growth
rates of the control and experimental plants (unpaired
t-test, p=0.047). These results supported the
hypothesis that acid rain would inhibit growth
potential for this species. Furthermore, a connection
might exist between the acidic tolerance of these
plants and their growth limit.
THE
EFFECT
OF
ENVIRONMENTAL
TEMPERATURE ON EXERCISE EFFICIENCY.
Kristin Harm and Jana Stverakova. Department of
Biological Science, Saddleback College, Mission
Viejo, CA 92692
Heart rate is a major determinant of cardiac
function. It is recommended that persons of moderate
functional capacity exercise at an intensity that will
elevate their heart rate to at least 65% percent of their
maximum heart rate, in order to yield maximum
results.
This experiment was designed to test if
exercising in a heated environment would prove more
beneficial, by enabling exercisers to reach their target
heart rates more quickly, than exercising in a cold
environment. The time needed to reach the calculated
target heart rate was recorded after the participants
jumped roped at a moderate pace in the warm
environment (that averaged 31.84˚C) and in the cool
environment (that averaged 10.28˚C). The results
demonstrated that there failed to be a significant
difference in the time needed to reach the calculated
target heart rate between the two exercise
environments.
CAFFEINE STIMULATION AND EFFECTS ON
COGNITIVE MEMORY RECALL OF MICE
(Mus musculus). Jonathan Castillo and Melissa
Molina. Dept. of Biology, Saddleback College,
Mission Viejo, California.
Caffeine stimulation has obvious effects on
metabolic rate, but with the increase in metabolic rate,
the need for an equivalent increase in cognitive
response develops. Memory is one of the main
functions that may be altered by caffeine stimulation.
Using caffeine to test whether there is a correlation
between caffeine stimulation and increased memory
retention, it was predicted that giving the common
mouse a proportion of caffeine equivalent to three
cups of coffee to a human would increase memory
retention and cognitive recognition. In doing so, 10
mice were placed into a maze and the time it took to
reach the finish was recorded. Three trials per mouse
were done: introduction to the maze, caffeinated trial,
and post-caffeine trial. The weights of each mouse
ranged from 27.1g-31.2g (N=10, mass 29.15±2.05,
mean ±S.E.M.) and caffeine was proportional to body
weight (0.1mg). Mus musculus was designated a
specific number in order to identify changes in time.
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Saddleback Journal of Biology
Spring 2007
THE EFFECT OF MICROWAVE (MW)
RADIATED FOOD ON THE OXYGEN
CONSUMPTION OF MUS MUSCULUS. Cristine
M. Kreitzer and Derrick Tran. Dept. of Biology,
Saddleback Community College, Mission Viejo, CA.
92691, United States
The effects of MW radiated food on mammals is
relatively unknown. It is debated that there are no
harmful effects because after the food is MW
radiated, the gamma rays come off of the food a few
minutes after the process is complete so when
ingested, there is no radiation. It is also debated that
the effects must be harmful because the food is
undergoing a radioactive process. In this experiment,
the effects of MW radiated food were studied in Mus
musculus. For a period of one month, a group of ten
male M. musculus were fed MW radiated food. Rate
of oxygen consumption was measured with a Sable
Systems FOXBOX CO2 and O2 Analysis System
using Ascarite as a CO2 absorber and Drierite as an
O2 absorber. The oxygen consumption rate was
measured at a resting state for five minutes and at a
post-active state for five minutes. The activity prior to
post-active state measurements consisted of the M.
musculus running on a treadmill for the duration of
two minutes at 0.2330 m/s. The results showed that
MW radiated food did provide a difference in the rate
of oxygen consumption in M. musculus (one-tailed,
paired t-test p = 6.68669 x 10-22).
THE EFFECT OF WATER TEMPERATURE
ON THE METABOLIC RATE OF GOLDFISH.
Billy Belgen, Jennifer Hipolite, and Jerry Kephart.
Dept. of Biology, Saddleback College, Mission Viejo,
CA, 92692
Changes in water temperature have a significant
affect on the metabolic rates of goldfish (Carassius
auratus). Dissolved [O2] was monitored for 10
goldfish in three varying water temperatures: 32º, 22º,
12º C to determine if a 10º C increase in water
temperature will induce an increase in metabolic rate.
Initial O2 level readings were recorded and each fish
was placed in each of the three water temperatures for
a 5 min period, after which a dissolved [O2] reading
was recorded in mg/l/min. The results showed that
the increase in metabolic rate was systematically
correlated with a 10º C rise in water temperature. As
the temperature increased by 10º C, the mean
dissolved [O2] decreased, showing that metabolic rate
had increased due to an increase in O2 intake. Our
dissolved [O2] values were as follows: 12º was 0.93
mg/l/min, 22º was 0.86 mg/l/min and 32º was 0.76
mg/l/min. Although an increase in metabolic rate was
observed with a 10° C increase in water temperature,
calculations of Q10 did not double (Q10 = 1.1 for the
12º to 22º C and for 22º to 32º range).
GENDER DIFFERENCES IN DIRECTED
ATTENTION AND HEMISPHERIC BRAIN
DOMINANCE IN HUMANS. Jennifer Van Malsen
and Kaitlyn Baker.
Department of Biology,
Saddleback College, Mission Viejo, CA 92692.
The ability to inhibit or stop one response in order
to complete another task is the cognitive mechanism
known as directed attention. Students were tested on
their ability to react to colors in the presence of
conflicting word stimuli, commonly called the Stroop
test. A measure of the effect of simultaneous word
and color stimuli was taken by timing one hundred
randomly sampled students from Saddleback College,
Mission Viejo, CA (50 females and 50 males). A
written test to determine hemispheric dominance was
also given to subjects after completing the Stroop test.
It was hypothesized that males would complete the
Stroop test with a faster time than females. The
average time for males was 20.7 sec (N=50). The
average time for females was 24.4 sec (N=50). The
study revealed that the mean times for males were
significantly different from the mean times for
females (p=2.4 x 10-3, one-tailed student t-test).
Further test determined that there were no significant
differences in times among left hemisphere, right
hemisphere or balanced brain dominant subjects.
THE EFFECT OF RED AND BLUE LIGHT ON
CHLOROPHYLL
CONCENTRATION
IN
JALAPENO PLANTS CAPSICUM ANNUUM.
Krystal N. Prince and Shaya Malekoshoarai.
Department of Biological Science, Saddleback
College, Mission Viejo, Ca 92677 United States.
Sunlight energy is utilized by plants to perform
photosynthesis. The chlorophyll molecules found in
plants mostly absorb visible light in the red and blue
part of the electromagnetic spectrum. If a plant is set
under certain wavelengths then their chlorophyll
concentrations will be affected. The objective of this
experiment was to study chlorophyll molecules in
plants and the affect of their concentration when
placed under different wavelengths of the
electromagnetic spectrum. Ten plants were set under
blue light, ten were set under red light and ten were
under white light. Twenty 6 mm plant disks were
punched out and placed into scintillation vials
containing 5 mL of 80% acetone. The vials were
placed into a 4oC environment for twelve days to
ensure complete chlorophyll extraction. The
concentrations of the vials were measured using a
Beckman DU 730 spectrophotometer. There were
significant differences among the red (78.84 ± 7.70
mg/L) and white (265.23 ± 11.10 mg/L) light groups,
and the red and blue (262.47 ± 12.71 mg/L) light
62
Saddleback Journal of Biology
Spring 2007
THE EFFECT OF CARBONATED BEVERAGES
ON A TOOTH’S ENAMEL. Kristopher Peterson &
John Lew. Department of Biological Sciences.
Saddleback College, Mission Viejo, CA 92692
Tooth Enamel is said to be one of the
toughest substances in the human body.32 teeth were
used in the experiment, 16 test tubes were filled with
coke. With a Ph of 3.4, coke is a very acidic soft
drink. The other 16 test tubes were filled with regular
bottled water as a control. Bottled water has a ph of
7.1. Each tooth was soaked in 25mL of its solution for
168 hour sessions plus or minus a few hours. We
repeated this for a total of 3 sessions totaling 504
hours plus or minus a few hours (because we did not
measure necessarily at the same time of the day).
After each session the teeth were transferred to test
tubes filled with De-ionized water and soaked for a 24
hours. We had the sugar molecules left over in the
teeth diffuse out of the teeth and into the De-ionized
water. Succeeding this session the teeth were dried
and placed into a drying oven at 60 degrees. We then
weighed the teeth in grams and recorded the data into
our Lab notebook and laptop for comparison. Results
demonstrated that carbonated beverages do have a
significant amount of denamalization on a tooth’s
enamel whereas water did not. (p = 0.158, one tailed
value).
DNA QUANTITY OF SEEDED AND SEEDLESS
RED GRAPES AND THE POSITIVE EFFECTS
ON CELLULAR ACTIVITIES (Vitis spp.). April
Tegtmeier and Chris Glendinning. Department of
Biological Science, Saddleback College, Mission
Viejo, CA 92692.
Red grape seed extract has been utilized for its
bioflavonoid, known as proanthocyanidins, and its
other beneficial antioxidants in nutritional
supplements. These extracts have been used in
chemoprevention of cellular damage, and an inhibitor
of UVB radiation-induced photocarcinogenesis in
mice. In humans, it has been shown to cause apoptotic
death in prostate carcinoma DU145 cells.
Procyanidins are known to exercise antiinflammatory, anti-arthritic and anti-allergenic
properties, and prevent skin aging, so they can be
described as anti-carcinogenic. Seedless red grapes
are not used in the prevention of cancers. In this
experiment, these two types of grapes had their DNA
isolated and extracted and then the DNA quantity was
evaluated by spectral analysis. The results showed
that the red seeded grapes (Vitis spp.) have more
DNA content, per mg, than red, seedless grapes. The
quantity of DNA in the seeded grapes ranged from
2.7 mg/mL to 3 mg/mL. The seedless grapes had a
DNA content range of 1.5 mg/mL to 2.25 mg/mL.
THE EFFECTS OF CARBON DIOXIDE ON
BIOMASS
DURING
RADISH
SEED
GERMINATION (Raphanus sativus). Milad
Danesh. Dept. of Biological Sciences. Saddleback
College, 28000 Marguerite Parkway, Mission Viejo,
California 92692.
The presence of CO2 is required during plant
growth and development. Increasing the [CO2]
surrounding a plant would consequently increase its
biomass. Thus, it has been proven that there is a
correlation between carbon dioxide concentration and
biomass in plants. It has been predicted [CO2] would
have a strong correlation with the biomass of radish
seeds during seed germination. Two groups of seeds
were germinated in the experiment. The first group,
the control group, was exposed to the CO2
concentration of the room, which was approximately
505 parts per million (ppm). The second group, the
experimental group, was exposed to a higher [CO2] of
38,000 ppm. The differences between the initial and
final masses were taken. After a data analysis of the
differences in biomass of the two groups, it was
shown that there indeed was a strong correlation
between CO2 concentration and the biomass of radish
seeds during germination. The results of the
experiment have shown that there is a negative
correlation between CO2 concentration and the
biomass of the radish seeds.
EFFECTS OF SUPER THRIVE GROWTH
HORMONE ON CO2 CONSUMPTION AND
OVERALL GROWTH AND DEVELOPMENT
ON RED RADISHES Raphanus sativus. Stephanie
Anstadt and Greg Nelson. Department of Biological
Science, Saddleback College, Mission Viejo, Ca
92692
Super thrive growth hormone contributes to minor
amounts of growth and production on red radishes.
Growth and development of the plant was measured
by using a metric ruler on a frequent basis. As soil
became thirsty the plants were watered with ¼ gallon
water and miracle grow solution. The experimental
group received the same solution with added Super
thrive growth hormone. The average length of sprouts
after 27 days for the experimental group was 12.9 cm.
The average length of sprouts of the control group
was 12.6 cm. These results show that the effect of the
growth hormone was not significant in the stem
growth of radish plants. Further tests using the Pasco
data logger determined the difference in CO2
consumption between the two groups. The group with
the added growth hormone consumed more CO2
within a 35 minute time period than the group not
given the hormone. However, there was no significant
difference between the two. A paired T-test showed
that T> 0.02445, the mean CO2.
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Saddleback Journal of Biology
Spring 2007
THE EFFECTS OF SPORTS DRINKS ON
HEART
RATE
DURING
EXERCISE
COMPARED TO WATER IN HUMANS. Meilad
J. Hojati and Taran S. Sondhu. Department of
Biological Sciences, Saddleback College, Mission
Viejo, CA 92692.
Consuming sports drinks is one of the ways
athletes keep hydrated and maximize performance
while exercising. Many people also drink water while
exercising to keep hydrated and maximize
performance. In this experiment the effects of sports
drinks on heart rate during exercise were compared to
the effects water has on heart rate during exercise in
Homo sapiens by recording the pulse rate of the
subjects while they rode for 60 minutes at a constant
rate of 85-90 rpm on a stationary bicycle. Two
separate trails occurred, one for water and one for
Gatorade®, where the heart rate of the subjects was
recorded every minute for the 60 minute period of
exercise. Prior to the period of exercise ample time
was given for the absorption of the test liquid (one
serving of test liquid for every 100 lbs of body
weight), followed by a warm up period before
beginning the actual exercise. The result indicated
that there was statistically no difference between the
heart rates of the subjects while consuming
Gatorade® compared to consuming water during the
exercise period (p=0.307, two-tailed t-test).
EFFECT
OF
TEMPERATURE
ON
OPERCULAR PUMPING RATE OF SMALL
AND LARGE GOLDFISH. Avelon Felsinger and
Zachary Beam. Dept. of Biological Sciences,
Saddleback College, Mission Viejo, CA 92692.
Opercular pumping rate of goldfish can vary with
changes in the temperature (Ta) of their environment.
Changes in Ta were used to determine the difference
in opercular pumping rate between small and large
sized goldfish. All goldfish were acclimated at 10ºC
and 30ºC and their opercular beats per minute were
recorded. Since larger organisms consume more
oxygen, we hypothesized that opercular pumping rate
should be higher at higher Ta. The results showed an
increase in opercular pumping rate with increased Ta
(p=1.0x10-10), as well as a significant difference
between sizes with each temperature. When
temperature increased to 30C, overall opercular bpm
for larger goldfish (157.8±1.14 bpm) was
significantly higher (p=1.1x10-6) than small goldfish
(134.3±2.54 bpm). However, mass-specific opercular
pumping rate of larger goldfish (13.2±0.48 bpm) was
significantly lower than small goldfish (26.05±0.95
bpm) with a 42.7% difference at 30C (p=1x10-8).
These differences indicate that with each increase in
temperature, opercular pumping rate will increase, but
as body size increases, opercular pumping rate per
gram will decrease.
COMPARISON OF THE METABOLIC RATES
BEFORE AND AFTER CONSUMPTION OF
CAFFEINE IN MUS MUSCULUS. Matthew
Kovach and Chan Kim. Dept. of Biological Science,
Saddleback College, 28000 Marguerite Parkway,
Mission Viejo, C.A. 92692, United States
Metabolism is the sum of the chemical reactions
that occur in the cell that yield energy and nutrients
necessary for life. This experiment was designed to
test the claims that caffeine increases one’s
metabolism, along with its well-known effects of
increasing alertness, wakefulness and focus as a CNS
stimulant. When caffeine is overdosed, over
stimulation the CNS occur, which leads to muscle
twitching, restlessness, rapid heart beat, and other
symptoms that are related to increase in metabolic
rate (MR). The effect of caffeine on the MR was
measured by examining the changes on the MR of
mice before and after the consumption of caffeine via
their food for about two hours. The MR of mice were
measured in the time it took to consume 10 cc of
oxygen, O2/g/hr and standardized for pressure,
temperature and dry air (STPD). The result showed
that after caffeine consumption for only about two
hours, significant increase in metabolic rates was
found in all ten mice
COMPARING THE RESTING AND ACTIVE
HEART RATE AND CO2 PRODUCTION OF A
NON-ATHLETE TO AN ACTIVE ATHLETE.
Katie R. Rauschl and Jessica Lai. Dept. of Biology,
Saddleback College, Mission Viejo, CA 92692.
Exercise plays a major role in developing a
body that operates efficiently. It strengthens organs
such as the heart and the lungs, as well as increasing
their capacity. These larger organs are able to contain
larger volumes, and should contract less frequently. In
this experiment, the resting and active heart rates
along with exhaled CO2 of three Active Athletes
(exercising more than five days a week) were
compared to three Non-Athletes (exercising less than
once a week). The resting heart rates were measured
after five minutes of lying down, while active heart
rates were measured after running 800 meters in 4.5
minutes. Using a CO2 monitor, the same process was
applied to find the resting and active concentrations
of CO2. Results rejected the null hypothesis in three
out of four comparisons, showing significant
differences between the Active Athlete and the NonAthlete in resting and active heart rates, as well as
significant differences in active CO2 concentrations.
Exercise was not found to significantly affect
concentrations of resting CO2.
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Saddleback Journal of Biology
Spring 2007
pH
NEUTRALIZATION
THROUGH
SUGARLESS GUM. Cathy O’Connor* and Chris
Yang*. Dept. of Biology, 28000 Marguerite Parkway,
Mission Viejo, California 92692
Dentists have suggested brushing 3X a day and
specifically advised brushing after meals to avoid
dental caries, which are caused by acid producing
bacteria that drop the pH of saliva. This study of pH
neutralization through sugarless gum has served to
shed light on the issue of dental caries and oral
hygiene by objectively quantifying the pH of saliva
after different hygiene measures. pH was measured in
23 healthy adults between 18-30 years currently
without braces. Participants spat 2 mL of saliva into
plastic cups and measured with pH strips. All subjects
consumed one Snickers® within 3 minutes and pH of
their saliva was measured again. Ten participants
each chewed one piece (3 g) of Koolerz® sugarless
gum. The remaining 10 chewed no gum. Every five
minutes for the next 30 minutes the pH of all the
subjects’ saliva were measured. The mean pH result
after the consumption of the Snickers® was 7.47 ±
0.09. After 30 minutes the two-sample assuming
unequal variance with one tailed t test was p =
0.0006. The gum chewers’ mean pH was 7.0 ± 0.03
and the mean pH of those without gum was 6.67 ±
0.08. Sugarless chewing gum is an efficient and
effective way of balancing oral pH after meals.
THE EFFECT OF BACTERIAL RESISTANCE
TO A DISANFECTANT. Maribeth Johnson and
Michelle Nazarifar.Dept. of Biology, Saddleback
College, 28000 Marguerite Parkway, Mission Viejo,
California 92692
Lysol® and Clorox Bleach® are common
household disinfectants that are used to eliminate
bacteria in homes. But do these products truly
eliminate bacteria? We tested Escherichia coli’s
resistance to 10 % Lysol® and 10% Clorox Bleach®.
We exposed E. coli broth to the aforementioned
solutions, using aseptic technique for three
consecutive runs. Zones of inhibition were measured
after each run. Lysol’s® was 28% more effective than
Clorox® (p=0.03). After run two, Lysol® was 39%
more effective than Clorox® (P=0.02). After three,
Lysol® was 54% more effective than Clorox® (p=1.5
x 10 –8). From this study, Lysol® was more effective
in controlling E. coli growth than Clorox ®. A 10%
bleach solution may not be as effective as once
thought in controlling bacterial growth. E. coli was
able to adapt and evolve to the Clorox Bleach®
solution more effectively, confirming resists after
three generation. Therefore Lysol® is a better
disinfectant to use in order to eliminate E. coli
bacteria.
SHORT TERM EFFECTS OF CAFFEINE ON
METABOLIC
RATE
OF
GOLDFISH
(CARASSIUS AURATUS). Lauren C. Ferris and
Brook A. Thomas. Ecology and Evolutionary
Biology, History. Department of Biological Sciences .
Saddleback College. 28000 Marguerite Parkway,
Mission Viejo, CA 92692.
Caffeine, a xanthine alkaloid compound and
central nervous system stimulant, is known to
increase metabolic rate in most organisms. This
experiment measured the oxygen consumption (mg/L)
of common goldfish (Carassisus auratus) over five
minutes to determine the average metabolic rate (mg/
L/ min) in a non-stressful environment. Oxygen
consumption was measured over five minutes after
fifteen minutes of exposure in a 100mg/700mL
caffeine/dechlorinated water solution. Results
indicated a significant difference in metabolic rate
between the caffeinated and non-caffeinated groups.
The caffeinated group exhibited higher rates of
oxygen consumption and therefore a higher overall
metabolic rate.
THE COMPARISON OF CHLOROPHYLL IN
SHADE AND SUN LEAVES OF THE
LEMONDADE
BERRY
(RHUS
INTEGRIGOLIA). Ryan C. Clark and Josue J.
Mandujano. Department of Biology, Saddleback
College, Mission Viejo, CA 92692
Chlorophyll is a green, photosynthetic
pigment that absorbs sunlight, and uses this energy to
produce carbon dioxide and water. It is, therefore, the
foundation for the life functions of all plants.
Chlorophyll content varies with different plants but
can vary in different leaf types of a certain plant. The
amount of chlorophyll in a leaf depends on the
quantity of sunlight the leaf in question receives.
Given that photosynthesis occurs with more
efficiency if it has more sunlight, it was predicted that
the sun leaves, which receive more direct sunlight
than the shade leaves would contain higher
chlorophyll content than shade leaves.
A
spectrophotometer and chlorophyll extraction were
used to determine whether sun or shade leaves would
contain more chlorophyll. 5 mL of 80% concentrated
acetone were mixed with leaf two 6 mm leaf chads in
scintillation vials. A 3 mL solution was inserted via
quartz cuvette into the spectrophotometer for analysis.
It was discovered that in a majority of the samples
taken, the shade leaves of the Rhus
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Saddleback Journal of Biology
Spring 2007
POOL TEMPERATURE AND ITS EFFECT ON
COMPETITIVESWIMMERS’ PERFORMANCE.
Asami Uzawa. Dept. of Kinesiology, Saddleback
Community College, Mission Viejo, California
92692, United States.
Increase in athletic performance raises body
temperature and heart rate, swimming in varying pool
temperatures will alter a swimmer’s performance and
heart rate. At 27o C, the average heart rate for males,
ages 15 & 16, was 182.8 and for females, ages 16 to
18, was 181.6. While at a pool temperature of 31o C,
male heart rates were an average of 193.6 and female
average heart rates were 192.2. The use of heart rate
monitors and swim times show that a swimmers
performance does not alter time wise but heart rate is
greatly influenced by the temperature of the pool
THE EFFECTS OF NICOTINE ON HEART
RATE AND BLOOD PRESSURE. AJ Bezer and
Arash Shahrokhshahi. Dept. of Biology. Saddleback
College. Mission Viejo, CA 92692
This study investigated the effects of nicotine on
blood pressure and cardiac rate on 20 male and
female human subjects. In order to collect sufficient
data that would yield noticeable results on these
subjects, we reasoned that it would most likely be
necessary to begin data collection after about four
hours of no prior nicotine consumption.
We
requested that each subject should remain free of any
nicotine exposure for four hours. Following this four
hour period, we introduced nicotine into the subjects
through the lungs with the use of a gaseous agent
containing nicotine; in this case, an inexpensive and
simple method was preformed issuing cigarettes,
Camel Brand. The systolic and diastolic pressure and
cardiac rate was determined using a blood pressure
monitoring kit, and a Polar CS200, a device
containing a strap-around apparatus to determine
cardiac rate. Data were collected while each subject
was in a stand up position, with very minimal body
movement. A set of data for blood pressure and
cardiac rate were collected before nicotine treatment.
An additional data were collected after about five
minutes of constant nicotine treatment. Following the
five minute period of constant nicotine treatment,
each subject was exposed to nicotine about every
three minutes for fifteen minutes.
THE EFFECTS OF ENERGY DRINKS ON
OXYGEN SATURATION. Payam Hedayat and
Sandra Guirguis. Dept of Biology. Saddleback
College. Mission Viejo, CA 92692.
A great deal of people depend on energy drinks to
get through their day; however, most of these people
don’t understand the consequences of relying on these
caffeinated drinks to keep themselves energized and
awake. It is vital to gain an understanding of how
energy drinks can affect one’s body. This experiment
contributed to the science field by providing evidence
of how caffeine might influence the amount of
oxygen saturation in the blood flow system before
and after the subject’s were given caffeinated drink.
We hypothesized that if caffeine is introduced in the
blood system, the level of O2 saturation will increase.
In this experiment, the rate of the oxygen saturation in
the blood system was recorded from sixteen adults
before and after exercise while simultaneously given
caffeine. The subjects’ O2 saturation level was
recorded before they performed the first segment of a
required exercise and after they performed the
required exercise. After that, they consumed a can of
Red Bull (8.3 oz) and waited for 20 minutes and then
repeated the required exercise, and then the level of
O2
THE EFFECTS OF GATORADE VERSUS
WATER ON HUMAN HEART RATE DURING
PROLONGED EXERCISE. Tony Shofer and
Trevor Bodmer. Saddleback College, Mission Viejo,
CA 92692. Department of Biological Sciences
Prolonged physical activity without proper fluid
intake occurs often in humans participating in
organized sports. This causes a multitude of physical
handicaps including increased heart rate, dehydration
and water loss while the body works to bring in
oxygen and maintain competitive pace. The purpose
of this study was to determine how water, the
traditional form of hydration, functioned in
comparison to the marketed sports drink, Gatorade on
human heart rate trends. It is believed that the intake
of Gatorade, full of various electrolytes and
carbohydrates, causes less physiological stress on an
exercising human than water, reflected by lower heart
rate trends during exercise. Subjects consisted of 11
recreationally active young adults between the ages of
19 and 22 running for a total of two miles. Calculated
p-value (p=.230) shows that there is not a significant
difference (p>.05) in the use of water or Gatorade.
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Saddleback Journal of Biology
Spring 2007
EFFECTS OF MODERATE DOSES OF CAFFEINE ON
SIMPLE COGNITIVE FUNCTION. Tiffanie L. Hand and
L. Jane Stewart. Department of Biological Sciences,
Saddleback College, Mission Viejo, CA 92692
This study investigates the effects of caffeine on
simple cognitive processes in an attempt to show its
benefits in an academic setting on college-age
students.
Twenty students completed a timed
cognitive task, and received either a placebo or 400
mg of caffeine both in the form of iced coffee. They
then completed another version of the same task
again, and their times were compared to show a
percentage of improvement.
On average, both
groups improved, but the experimental group had a
statistically significant higher rate of improvement
(p= 0.0137). Caffeine seems to have a positive effect
on the mental performance of college age students.
THE EFFECTS OF DRINKING HOT AND
COLD
TEA
ON
HUMAN
BODY
TEMPERATURE. Michelle Chawner. Dept of
Biology. Saddleback College. Mission Viejo, CA
92692
Human body temperature can be affected or
partially regulated by drinking hot and cold liquids.
Cold liquids cause the body to speed up metabolism
to heat the liquid to body temperature for proper
digestion, therefore increasing energy and heat
radiated from the body. Using this information, it was
predicted that drinking cold liquid would increase
overall core body temperature. In this experiment,
humans (Homo sapiens) were used as test subjects to
find out the overall differences in body temperature
when drinking hot and cold liquids. Temperatures
were measured after 45 minutes of ingestion and
digestion of either a hot or cold liquid. The results
showed that the hypothesis configured was null and
further testing would be needed. There were no
overall differences in the effects of drinking hot or
cold liquids on human (Homo sapiens) body
temperature. Temperature variation could be due to a
number of different variables, such as different rates
of metabolism, weight difference between test
subjects, and different rates of digestion. Differences
could also be due to the high specific heat of water,
which was used as a base for the tea. There were
major differences in both the hot and cold liquid
groups, as well as in the female and male temperature
differences.
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Saddleback Journal of Biology
Spring 2007