Biology 1081L Sect. 20, George R.

Rieveschl Hall 318 College Drive, Cincinnati, Ohio 45221

Proposal Submitted to: National Science Foundation, Box 000, Washington D.C. 00000
Principal Investigators: Stephanie Vandergriff, Teresa Rust, and Aaron Martinez
Analysis of the Presence or Absence of Life in Samples from the Deep Oceanic Trench
Introduction
The purpose of this proposal is to exhibit the most cost and time efficient, accurate and
reliable methodology to determine which samples that were obtained from the deep ocean
trenches contain evidence of life I order to pinpoint key areas for future study. Evidence of
prospering, high-productive communities in thermo genic trenches seem to have an energy
source of Methane-rich fluid of thermo genic origin (Subet & Olu, 1998). Using this information
and the samples collected more information about prospering species can be discovered. This
study will aid in the classification of the samples, which will then hasten the process of exploring
the deep seas trenches. The analysis and study of the samples may lead to further information of
organism and their methods of obtaining energy in such deep environment. The species
discovered through this experiment as S.K. Juniper suggests, as in his own similar
experimentation, will also give a plethora of information about the spreading rate and properties
of the environment, especially vent habitats. The information that is obtained from the samples
not only has biological significance, but also geological and environmental significance (S.K.
Juniper, 1997). C.L. Van Dover states the more specifically, information such as about how vent
water and sea water mix could also be obtained by studying the organisms and minerals found in
the samples based on their location. The organisms such as those that Van Dover described in
her study of the Mid-Atlantic Ridge near the Azores plateau and the communities that thrive
there (C.L. Van Dover, 1995). It is very important for the researchers who analyze the samples to

understand this or else valuable information will be passed over because the researchers will be
too focused on the biological aspect.
Methods

The operational definition being used for this procedure are that the sample will be tested
for being alive through the Tetrazolium test in order to find a metabolism, and if
metabolism is present, the test is positive and the sample is alive.

The operational definition being used for this procedure are that the sample will be tested
for being dead through the Tetrazolium test in order to find a lack of metabolism, the
test is negative and the sample is dead or inanimate.

The operational definition being used for this procedure are that the sample will be tested
for being organic through a burning test and if the mass significantly changes then at
least part of the substance is organic.

The operational definition being used for this procedure are that the sample will be tested
for being inorganic through a burning test and if the mass does not change significantly
then the sample is inorganic.

All 5,000 samples will be determined alive or not alive by performing the
Tetrazolium Test in order to find a metabolism, and if the result is positive then the
sample is alive and if the result is negative the sample is not alive.

The samples with a negative result from the Tetrazolium Test will be burned to
classify them as organic or inorganic. Positive results for burning will result in a
change in mass of the sample and will confirm that there is organic material within
the sample. Negative results for burning will result in no change in the mass of the
sample and will confirm that there is inorganic material within the sample.

The positive organic samples determined by burning will then be tested for DNA by
performing the Feulgen Test. Positive results declare that the samples were once
alive and are now dead, and negative results declare the samples as inanimate.

The Tetrazolium Test tests for metabolism, Burning tests for inorganic or organic
material, the Feulgen Test tests for DNA in dead samples, and we will use our
microscope to see if we find functions under it

The negative control for the Tetrazolium Test will be beans that were germinating
but then boiled to kill them. The positive control will be the beans that are
germinating in order to test positive for a metabolism. The positive control for the
Feulgen Test will be a bean root in order to test positive for DNA and the negative
control will be distilled water because there is nothing living in this processed
water. The positive control for our burning test will be a leaf of lettuce because it is
organic and will turn in mass, and our negative control will be a rock because it is
inorganic and will not change in mass. The controls in our experiment are necessary
in order to compare our replicates results to determine them dead, alive, inorganic,
or organic.

To perform the Tetrazolium Test 4mL of 1% Tetrazolium solution per test sample,
test tubes for each sample, and the 37C water bath are needed. To perform the
Feulgen Test a plastic micro centrifuge tube for each sample, goggles for each
experimenter, .5 mL of 3:1 ethanol/acetic acid per sample, transfer pipets, .5mL of
1M HCl, 60C water bath, and Schiffs reagent are needed. To burn the samples a
crucible, crucible cover, thermal ceramic pads, balance, tongs, Bunsen burner,
gloves, a pair of goggles for each experimenter, and matches are needed. To view the

samples under a microscope a microscope, slides, slide covers, water, and pipets are
needed.

The microscope will be used to see if the substance is a mixture. It will be put in
water, then the outer structure will be broken apart if necessary, and observe it
under the microscope.

Justification
The purposes of this study are to categorize all of the samples gathered from the newly
found trenches and use the results to gather more information about unknown species and deepsea communities. The rationale behind the order of methods stated above is to narrow down as
many species as alive before performing anything else so then further testing can determine the
specifics of organisms that were found not living. The overarching idea behind the purposed
methodology is to be as accurate as possible while using the least amount of resources. Research
in this field that has previously been documented about such as the shrimp which T. M. Shank
discover through research. The shrimp that live deep in the sea around hydrothermal
vents were discovered using COI gene or Cytochrome c Oxidase subunit I and based off of
the genetics of the shrimp and biogeography, it was determined that sea floor spreading
did not affect the shrimp community living around the vents. The COI gene that was
analyzed indicated that they did not originate in the Mesozoic era; they originated in the
Miocene era (T.M. Shank, 1998). Research, such as the research done by Shank, leads to the
belief that new organisms will be identified and more information about accepted species will be
elaborated on.
The proposal is the most reasonable and cost effective approach because organisms will
first be tested if they are alive as opposed to dead, inanimate and inorganic or organic. The tests

will be conducted in such a way to reduce the amount of testing to a minimum while retaining
accuracy. Each test will have three replicates of each sample in order to insure the correct
classification. If these tests are inconclusive further tests will be administered. If the NSF
chooses to present the grant to this proposal it is recommended that the findings will be
published in multiple peer reviewed scientific journals in order to give the greatest amount of
international scientists access to the data to aid their related studies. V. Tunnicliffe stated that in
recent decades, hundreds of new species previously undocumented and unknown to humans
were discovered and fourteen new families as of the date when his paper was published through
taking samples from the sea floor (V. Tunnicliffe, 1988). This is very important, a lot of
pressure will be on the researchers who receive this grant to analyze the samples
well. The information that not only the once living samples are studied but that the inorganic
and organic samples, which will shine light on the geographical and ecological aspects of the
area in which they were collected from. R.R. Hessler states from his own experimentation that he
found the recent evolution of many species, especially invertebrates, could be crucial in forming
up-to-date biogeographic patterns because they give information about the Cenozoic tectonic
history and oceanic circulation patterns (R.R. Hessler, 1991).
Budget
The primary goal of the methods used is to ensure the correct classification of each
sample, and the secondary goal is to use the methods in the best way possible to minimize
costs. The costs below are based on preliminary research performed by BIO 1081L sect.
20. Based on preliminary research, the estimate is that 8% of the 5,000 samples (400) will
test positive for metabolism after the Tetrazolium Test, and 92% (4,600) of the 5,000
samples will test negative for metabolism therefore classifying them as not alive. They are

either dead, inanimate, inorganic, or organic. The following tests will further classify these
samples. The Tetrazolium test alone costs $6 per sample; based on the data previously
collected the cost of the first test would cost $90,012 to test the 5,000 samples. The next
test that will be performed is the Feulgen test, to test whether DNA is present signifying
that the sample was once alive, but is now dead or if the sample is not alive and never was
alive. Based on previous data collected by BIO 1081L sect. 20, 64% (2,944) of the samples
will test negative in the Feulgen test for DNA and 36% (1,656) of the samples will test
positive for DNA in the Feulgen test. The Feulgen test alone costs $9 per sample; based on
the data previously collected the cost of the second test would cost $220,818 to test the
estimated 4,600 samples. The samples which tested negative for DNA will then be further
tested for organic mass in the sample. Based on previous testing, 36% (1,060) of the
samples burned will test negative for organic material, classifying them as inorganic and
64% (1,884) of the samples burned will test positive for organic material. The burning test
alone costs $16 per sample; based on the data previously collected the cost of the third test
would cost $79,520 for burning the estimated 2,944 samples. Overall, the testing will cost
$390,350, based on previous research. In each calculation, testing the controls was added
into the cost.
The labor will be completed by 100 professional scientists and graduate students;
ideally at least 50 scientists whom are experienced in lab work and experts in the field of
geology, environmental science, oceanology, biology (insert more if desired) and the other
50 can be a combination of graduate students studying these fields and other scientists
interested in the research. On average, these participants will be employed and paid by an
hourly basis of $41 per hour. Ideally around 50 undergraduate students would also

participate as a volunteer position. The paid employees would be assigned different tasks
and the undergraduate students would be assigned to aid the employees and analyze the
samples. Based on previous experimentation, two samples fully analyzed takes around two
hours. If each paid employee were given 50 samples to analyze using their specific test, the
classifying of samples should take less than 2,500 hours. The labor would then cost, at
most based on the previous data collected, $102,500. This does not include if any
employee works over-times or if the experiment is set back and for some reason or another
takes longer than expected for the accuracy of classifying the samples is most important.
Overall, based on the previous data collected, the cost of analyzing all 5,000 samples,
with three replicates of each sample and a sufficient amount of controls would be
$492,850. In the previous research, no extra supplies were needed, not one piece of
glassware broke, and not one sample needed to be re-tested, but in order to account for the
possibility of mishaps, $580,000 would be ideal in completing this experiment with the
most reliable results. The extra $7, 150 would be ideal factoring in that the paid
researchers overall would work 100 extra hours which totals $4,100 and if replacement
crucibles or crucible lids must be purchased an estimate of 20 crucibles and lids which
costs $360. Factoring in the inevitable human error, 100 additional Tetrazolium tests
would cost an estimate of $600, 50 additional burning tests would cost $800, and 125
additional Fleugen tests would cost $1,125. The Fleugen test is much more sensitive and if
the sample is stained it must be analyzed quickly, or else the sample could test a false
positive. The burning test is very straightforward and any significant change in mass
signifies organic matter, so there are fewer aspects that could possibly go wrong, therefore

fewer additional tests were factored in. The estimated extra costs above totals $6,985 so
the remaining $165 would be set aside for other unexpected costs.
References
C. L. Van Dover, in Hydrothermal Vents and Processes, L. M. Parson, C. L. Walker, D. R.
Dixon, Eds. (Geological Society Special Publication 87, Geological Society, London,
1995), pp. 257294
M. Sibuet, K. Olu, Deep-Sea Res. Part II 45, 517 (1998).
R. R. Hessler, P. F. Londale, Deep-Sea Res. 38, 185 (1991)
S. K. Juniper, V. Tunnicliffe, Philos. Trans. R. Soc. London Ser. A 355, 459 (1997)
T. M. Shank, R. A. Lutz, R. C. Vrijenhoek, Mol. Mar. Biol. Biotechnol. 7, 88 (1998)
V. Tunnicliffe, Proc. R. Soc. London Ser. B 233, 347 (1988)

Tables and Figures

Figure 1: Results

Figure 2: Budget Table

Expense

Cost of the Expense (Dollars)

Microscopic Examination

80,000

Tetrazolium Test

90,012

Feulgen Test

220,818

Burning Test

79,520

Labor

102,500

Total

572,850

Figure 3: Breakdown of the Ideal Budget

Expense

Cost of the Expense (Dollars)

100 additional hours worked by paid

4,100

researchers
125 additional Feulgen Tests

1,125

100 additional Tetrazolium Tests

600

50 additional Burning Tests

800

20 Replacement Crucibles and Lids

360

Unexpected costs

165

Total

7,150
Figure 4: Ideal Budget

Expense

Cost of the Expense (Dollars)

Total Budget

572,850

Ideal Additional Amount

7,150

(In case extra test, hours, and supplies are needed)

Ideal Total

580,000

Figure 5: Protocol Flow

See physical attached paper
Appendix
The classifications of the samples are as follows: sample 2 contains inorganic material and is
inanimate. Sample 5 contains organic material and is now dead, but previously alive. Sample 10
is a mixture of dead, but once alive organic material and inanimate inorganic material. Sample
20 contains organic material and was previously alive, but is now dead.
In group fours collected data all samples tested negative after the Tetrazolium test for
metabolism. Samples 5, 10, and 20 tested positive for containing organic material after the burn
test. Sample 2 tested negative for containing organic material after the burn test. Samples 5, 10,
and 20 tested positive for DNA in the Feulgen test and sample 2 tested negative for DNA in the
Feulgen test.
The operational definitions for the preliminary study were different from the proposed
study. The operational definitions for the preliminary study were well thought out, but needed
adjustments such as the operational definition of dead. The definition was far too long and did
not include inanimate in the possible results of the Tetrazolium test. The operational definitions
for the preliminary studies and the proposed studies for alive, organic, and inorganic were
concise and well thought out and as a result few changes need to be made. One of the few
changes was re-working the definitions to be in third person, limited omniscient point of view.

During preliminary testing in BIO 1081L samples 16, 5, 19, 20, 10, 21, 15, and 23 were
classified as dead, which is two thirds of the total samples. Sample 7 was the only sample
classified as alive. One third of the samples, samples 13, 3, 15, and 2, were classified as
containing inorganic material. Five twelfths of the total samples, samples 10, 7, 19, 20, and 5,
were classified as containing organic matter.
In order to classify the samples, certain tests were selected by each lab group. The
methodology described above is the most conclusive compared to other groups because
inconclusive results were recorded using other tests. This testing sequence went from most
general to most specific, narrowing the possible classifications. Other tests used, such as the
Iodine test do give valuable information when used appropriately, but other tests are not the
optimal tests for the purpose of analyzing the samples and classifying them as alive, dead but
once alive, inanimate, organic, or inorganic. The iodine test was inconclusive for one of the
samples which does not aid in classifying the samples, if anything it inhibits the process of
classification because further tests are needed. Benedicts test is used to test if monosaccharides
and reducing disaccharide sugars are present. In the methods presented above, the Feulgen test
and Tetrazolium test are used instead to differentiate a sample between dead but once was alive
or alive, and inanimate. Some groups in Dr. Beyettes BIO 1081L section 20 used Benedicts
test to determine if the specimen is alive or was once living, but the Feulgen test for DNA is
more dependable being that all living organisms have to contain DNA. The Buiret test, used by
some groups in the BIO 1081L to test for the presence of polypeptide bonds, signifying
proteins. Again, in the methods presented above, the Feulgen test is used to determine if DNA is
present, a very reliable way of determining if a sample is dead but once was alive or alive, and
inanimate. Another test that could be used is the test for lipids, but again the Feulgen test is

better suited for classifying the organisms because all living organisms must contain
DNA. Overall, the methodology as presented above is the most relative and reliable for
classifying the 5,000 samples.
Based on preliminary testing by Dr. Beyettes BIO 1081L section 20, 92% of the 5,000
samples (4,600 samples) will test negative, classifying them as not alive: dead, inanimate,
organic, or inorganic. 8% of the 5,000 samples will test positive, classifying them as alive. It is
predicted that 64% of the 4,600 samples (2,944 samples) that tested negative for metabolism will
test negative for DNA. The remaining 36% will test positive for DNA classifying them as dead
but once alive. Of the 2,944 samples that will test negative for DNA, 64% will test positive for
organic matter and 36% tested negative for organic matter, identifying them as inorganic.