Eighth Grade Science Fair Projects Have Gotten Complicated

IMPACT OF CHLOROPHYLL CONCENTRATION LEVELS ON THE PROLIFERATION OF VIBRIO FISCHERI

Griffin Wagner
Storm Grove Middle School
September 27, 2015

I. Topic:

Vibrio fischeri (V. fischeri) is a naturally occurring microorganism in marine environments whose abundance may be influenced by variations in the levels of chlorophyll in its environment. The sensitivity of V. fischeri to chlorophyll will be analyzed by measuring its ability to reproduce in environments where levels of chlorophyll are systematically elevated from naturally occurring low levels to levels above those that naturally occur in the
Indian River Lagoon.

II. Problem Statement:

Will the reproductive capacity of V. fischeri be influenced by increasing levels of chlorophyll and exhibiting an increase in productivity, a decrease in productivity, or no change.

III. Hypothesis:

If I systematically increase levels of chlorophyll - from low levels that naturally occur in their environment, to a level above naturally occurring levels - then the abundance of V. fischeri will increase in numbers of colonies because providing an abundance of nutrients and resources will provide for more vigorous population growth.

IV. Background Statement:

Vibrio vulnificus and other pathogenic Vibrio are multiplying rapidly in the Indian River Lagoon. Vibrio fischeri, a nonpathogenic bacteria, is a close relative to these pathogenic bacteria. Nutrients and pollutants in the lagoon, such as nitrogen and phosphorous, contribute to Vibrio’s growth. This project will determine if chlorophyll is another nutrient that impacts their growth. Due to similarities in their genome structure to pathogenic species of Vibrio, the results of this experiment may be generalized to those pathogenic species of Vibrio. This is important because excess Vibrio proliferation may pose a health risk to people and other living organisms in the Indian River Lagoon community.

V. Variables:

A. Dependent Variable: Number of colonies of V. fischeri

B. Independent Variable: Differing levels of chlorophyll as compared to naturally occurring range
C. Controlled Variable: 0 ug/L of chlorophyll in agar

D. Constants: Temperature (room temperature)
Light (daily light cycle)
Agar/broth culture medium
Sterile water
Equipment and material

VI. Materials:

A. Pure Culture of V. fischeri
B. Pipettes
C. Mechanical pipetting pump
D. Graduated cylinder
E. Flasks
F. Sterile, Non-absorbent cotton plugs
G. TestTubes
H. Culture Plates
I. Growth Medium (agar, broth)
J. Inoculation loop
K. Tape
L. Sharpie marker
M. Grid; Colony counter
N. Sterile water
O. Antibacterial Agent
P. Spray bottle
Q. Bunsen burner/electronicsterilizer
R. Chlorophyll
S. Digital gram scale
T. Eye protection
U. Latex gloves
V. Tissues
W. Smock/Apron

VII. Safety Procedures:

A. Safety and handling procedures in the laboratory are designed to prevent accidents, and contamination with microbes. Aseptic technique will be used at all times to prevent self-contamination with microorganisms, ensure others in the laboratory do not become contaminated, prevent escape of organisms from the laboratory area, and to ensure that microbial cultures do not become contaminated with unwanted organisms.

B. The Centers for Disease Control (CDC), recommends biosafety levels for microorganisms. The International Science and Engineering Fair, and the Florida State Science and Engineering Fair rules, also stipulate genus and species of microorganisms that are acceptable and safe organisms for middle school science projects. Vibrio fischeri, is a nonpathogenic organism, classified as BSL (biosafety level) - 1 (agents not known to cause disease; no safety equipment required), by the CDC, and has been approved for use by the International Science and Engineering Fair, and the Florida State Science and Engineering Fair rules, for middle school science projects.

C. Laboratory Safety Procedure:?

1. Only materials pertinent to the project will be brought into the laboratory
2. Eating and drinking is not permitted in the laboratory
3. A lab coat or smock will be worn in the laboratory
4. Latex gloves will be worn while working with microorganisms
5. Eye protection will be worn in the laboratory
6. Close-toed shoes will be worn in the laboratory
7. Before beginning laboratory activities, and at the completion of laboratory activities, the work area will be wiped down with appropriate disinfectant
8. All disposable materials will be deposited in an appropriate biohazard receptacle
9. Extreme caution is to be used when working with an open flame or sterilizing equipment. Always make sure gas is turned off before exiting the laboratory
10. Any open wounds must be covered with a Band-Aid
11. Pipetting by mouth is prohibited

D. Disposal of Biological Wastes:

1. Disposable materials, (disposable petri dishes, disposable test tubes, disposable flasks, gloves, paper towels, tissues, etc.) will be disposed of in a biohazard container lined with a clear autoclave bag
2. Sharp objects, (slides, coverslips, disposable pipettes, broken glass, etc.) will be disposed of in a sharpkeeper

E. Surface Contamination

1. Cover spill with paper towels and saturate paper towels with disinfectant
2. Spray/pour a ring of disinfectant around the perimeter of the spill
3. Allow the disinfectant to act for at least 20minutes
4. Remove any solid materials from spill with forceps or scoop, and discard the waste in an appropriate manner, (see above)

F. Personal Contamination

1. Immediately clean exposed area with soap and water, eye wash (eyes), or saline, (mouth)
2. Apply first aid, and treat as an emergency

G. Aseptic Technique

1. Prior to beginning a laboratory activity, the work area is treated with disinfectant
2. Inoculating loops are sterilized by flaming it with a Bunsen burner until red-hot, before transferring any culture. Loop must be cooled (air cooling, dipping in agar), before picking up any culture
3. The mouth of any flask or test tube is flamed before introducing a cooled inoculating loop
4. After the culture is inoculated, the mouth of the flask or test tube is reflamed and recapped
5. At no time, after sterilization, is an instrument or cap laid on a surface. They are always held in one’s hand
6. After inoculation is complete, the inoculation loop is reflamed in the Bunsen burner until red-hot, and then air cooled
7. When opening, the petri plate cover is raised and held diagonally over the plate to protect it from airborne contamination.
8. The inoculating loop is gently dragged over the surface of the agar, without gouging the surface
9. The petri plate cover is then replaced, the plate inverted, and sealed with cellophane tape
10. The scientist’s name, the date, and type of organism is written on the base of the plate
11. The work area is disinfected again, when the activities for the day are completed

VIII. Safety Concerns

A. Working with microorganisms can lead to possible contamination of self and others. The following procedures are designed to remedy these concerns:
1. The organism selected for this experiment, V. fischeri, is designated as a BSL-1 organism by the CDC (an agent not known to cause disease)
2. Laboratory Safety Procedures (see above), designed prevent contamination of self and others, have been reviewed and practiced
3. Aseptic Technique (see above), designed prevent contamination of self and others, have been reviewed and practiced
4. Physical barriers to contamination (lab coat/smock; latex gloves; eye protection, close-toed shoes) will be used during all lab activities

B. Bunsen burner/electronic sterilizers can cause fires and burns.
1. Only after instruction will these items will be used, and with great care and only under direct supervision.
2. Fire extinguishers are available in the laboratory
3. At the conclusion of a laboratory activity using those tools, gas valves will be rechecked to insure they are closed, and electronic sterilizers will be unplugged

C. Glass materials present a risk of breakage and injury.
1. These materials will be used after direct instruction, and with great care.
2. Eye protection will be worn during all laboratory activities

IX. Procedure:

A. Agar Plates

1. Measure appropriate amount of powdered agar medium
2. Measure appropriate quantities of chlorophyll
a. Plates #1 - 5: Control plates - 0 ug/L chlorophyll added to agar medium
b. Plates #6 - 10: Experimental Plates A – 10 ug/L of chlorophyll added to agar medium
c. Plates #11 - 15: Experimental Plates B - 30 ug/L of chlorophyll added to agar medium
d. Plates #16 - 20: Experimental Plates C - 50 ug/L of chlorophyll added to agar medium
e. Plates #21 - 25: Experimental Plates D - 70 ug/L of chlorophyll added to agar medium
f. Plates #26 - 30: Experimental Plates E - 90 ug/L of chlorophyll added to agar medium
3. Measure appropriate quantity of sterile water
4. Thoroughly mix measured agar powder and sterile water in a sterile flask
5. Pour 25 ml. of agar solution into sterile culture plate
6. Cover plate, and allow agar to harden
7. Turn plate upside down and mark with student name and date made
8. Store plate upside down until needed

B. Culture

1. Label a sterile flask with researcher’s name, type of organism, and date cultured
2. Measure appropriate quantity of powdered Vibrio fischeri culture
3. Measure appropriate quantity of sterile water
4. Thoroughly mix culture and sterile water in a sterile flask
5. Seal mouth of flask with sterile, non-absorbent cotton plug
6. Incubate culture at room temperature for approximately 24 hours

C. Serial Dilutions

1. Fill a sterile test tube (test tube #1) with 9.9 ml of sterile water
2. Pipette 0.1 ml. of Vibrio fischeri culture from the prepared liquid culture
3. Wipe any excess solution from the tip of the pipette with sterile tissue, and dispose of tissue in biohazard receptacle
4. Transfer 0.1 ml. of pipetted culture into test tube #1. (1/100 dilution of the original prepared liquid culture)
5. Mix thoroughly
6. Fill a second sterile test tube (test tube #2) with 9.9 ml. of sterile water
7. Pipette 0.1 ml. of Vibrio fischeri culture from test tube #1
8. Wipe any excess solution from the tip of the pipette with sterile tissue, and dispose of tissue in biohazard receptacle
9. Transfer 0.1 ml. of solution pipetted from test tube #1 into test tube #2 (1/10,000 dilution of the original prepared liquid culture)
10. Fill a third sterile test tube (test tube #3) with 2.5 ml. of sterile water
11. Pipette 0.1 ml. of Vibrio fischeri culture from test tube #2
12. Wipe any excess solution from the tip of the pipette with sterile tissue, and dispose of tissue in biohazard receptacle
13. Transfer 0.1 ml. of solution pipetted from test tube #2 into test tube # (1/250,000 dilution of the original prepared liquid culture)

D. Inoculate Prepared Culture Plates with Vibrio fischeri

1. Light Bunsen burner
2. With gloved hand, pick up and flame an inoculating loop until the entire loop glows red (After this point, NEVER LAY THE INOCULATING LOOP DOWN)
3. Pick up and hold the flask with the diluted broth culture in one gloved hand and remove cotton plug with the hand holding the inoculating loop (DO NOT PUT PLUG ON THE TABLE)
4. Flame the mouth of the flask for 2 seconds
5. Reflame the inoculating loop until the entire loop glows red
6. Lift the lid of the culture plate just enough to insert the inoculating loop and jab the loop into the edge of the agar, away from where there will be any inoculation
7. Dip the inoculating loop into the broth culture withdrawing a sample of the culture
8. Reflame the mouth of the culture flask for 2 seconds and reinsert the cotton plug
9. Keeping the loop parallel with the agar surface, and starting at the 12 o’clock position, zigzag the loop back and forth across the surface of the agar until coving about 1/3 of the surface (area1)
10. Reflame the inoculating loop, and, again, stab the edge of the agar to cool the loop
11. Rotate the plate 90 degrees counter clockwise (the 12 o’clock position is now at the 9 o’clock position)
12. Zigzag the sterile loop through area 1, two or three times, picking up bacteria already on the plate, and cover about half of the remaining area creating area 2 (DO NOT TOUCH AREA 1 AFTER TWO OR THREE DRAGS THROUGH AREA 1)
13. Reflame the inoculating loop, and stab it into the edge of the agar to cool
14. Again rotate the plate counter clockwise 90 degrees, putting area 1 at the 6 o’clock position, and area 2 at the 9 o’clock position
15. Zigzag inoculating loop through area 2, two to three times spreading the bacterial solution across the remaining area of the plate - area 3 (DO NOT TOUCH AREA 2
AFTER THE FIRST 2 OR 3 ZIGZAGS, AND BE CAREFUL NOT TO ENTER AREA 1, AS
WELL)
16. Close the lid of the plate
17. Sterilize the inoculating loop
18. Tape the plate lid to the bottom of the plate
19. Invert the plate
20. Seal plate with tape
21. Write your name, date, type of bacteria, and treatment on the bottom of the plate
22. Incubate prepared plates for approximately 24 hours in appropriate conditions (room temperature; daily light cycle)

E. Counting Colony Forming Units (cfus)

1. Lay out the plates, continuing to maintain the inverted position, retaining the seal, in order of chlorophyll concentrations.
2. Place each plate on top of a grid, using the lines to organize the counting, and prevent overlooking a colony or counting the same colony more than once
3. Count every colony, regardless of size, and place a small dot with the Sharpie on each colony, as counted
4. Record counts on a table.

F. Analysis

1. Colony forming units (cfus) will be counted at each chlorophyll concentration level,
(0; the naturally occurring lowest concentration level in the Indian River
Lagoon (IRL); at the 25th percentile between the lowest naturally occurring concentration level and the highest naturally occurring concentration level in the IRL; at the 50th percentile between the lowest naturally occurring concentration level and the highest naturally occurring concentration level in the IRL; at the 75th percentile between the lowest naturally occurring concentration level and the highest naturally occurring concentration level in the IRL; at the highest naturally occurring concentration level in the IRL; and 25% above the highest naturally occurring concentration level in the IRL

2. A comparison will be made among the number of cfus at each concentration level including:
a. Raw count of cfus of each plate
b. The mean number of cfus at each chlorophyll concentration level
c. The range number of cfus at each chlorophyll concentration level
d. The standard deviation of the number of cfus at each chlorophyll concentration level
e. A correlation coefficient between cfus and chlorophyll concentration levels

G. Conclusions

The conclusions will attempt to determine if the chlorophyll concentration in Vibrio fischeri’s environment has a direct impact upon its ability to proliferate, and if so, in what direction - positively or negatively - or no observable/measurable impact. These results may demonstrate the importance of controlling the levels of pollutants entering the Indian River Lagoon system - particularly nitrogen and phosphorous - which have a direct relationship upon the growth of algae in the Lagoon’s waters. These algae are an important source of chlorophyll in the Lagoon’s waters.

X. Bibliography:
Works Cited
Print

Benson, Harold J., and Alfred E. Brown. Benson's Microbiological Applications: Laboratory Manual in General Microbiology. 11th ed. Boston: McGraw-Hill, 2012. Print.

Cappuccino, James, and Natalie Sherman. Microbiology: A Laboratory Manual. 10th ed. San Francisco: Pearson-Benjamin Cummings, 2013. Print.

Treadway, Tyler. "VIBRIO KILLS: Is Salt the Solution?" Indian River Press Journal 6 Sept. 2015, Sunday ed., sec. A: 1A, 14A. Print.

Websites

"Aliivibrio Fischeri ( ATTC 7744)." ATCC Product Sheet. American Type Culture Collection, 2 Aug. 2013. Web. 11 Sept. 2015. <https://www.atcc.org/~/ps/7744.ashx>.

"Biosafety in Microbiological and Biomedical Laboratories." Ed. L. Casey Chosewood, M.D. and Deborah Wilson, DrPH. U.S. Dept. of Health and Human Services, 2009. Web. 11 Sept. 2015.

"Complete Genome Sequence of Vibrio Fischeri: A Symbiotic Bacterium with Pathogenic
Congers." Proceedings of the National Academy of Sciences of the United States. PNAS, 31 Dec. 2004. Web. 11 Sept. 2015.

"Continuous Sensor-based Water Quality Data." St. John's Water Management District, 2015. Web. 14 Sept. 2015. <https://webapub.sjrwmd.com/agws10/hdswq/>.
Ewald, Heather, James Brashears, Christine Huynh, and Eric Freeman. "Micro-organisms For Education." Collaborative Pre-university Science Projects. Department of Biology, The College of William & Mary, 1999. Web. 11 Sept. 2015.

Garland, Ed. "The Indian River Lagoon: An Estuary of National Significance." Water Bodies, Watersheds, and Storm Water. St. John's Water Management District, 4 Oct. 2013. Web. 14 Sept. 2015.

McCarthy, Peter J., and Gabby Barbarite. "Pathogenic Vibrio Bacteria in the Indian River Lagoon and Their Potential Threat to Human Health." FAU-Vibrio Research and Practical Information. Harbor Branch Oceanographic Institute. Web. 23 Sept. 2015.

"Potentially Hazardous Biological Agents." Student Science. Society for Science and the Public, 2015. Web. 11 Sept. 2015.

Takemura, Alison, Diana Chien, and Martin Polz. "Associations and Dynamics of Vibrionaceae in the Environment, from the Genus to the Population Level." Frontiers in Microbiology. NCBI, 11 Feb. 2014. Web. 10 Sept. 2015.

Interviews

McCarthy, Peter J. "Microbiology and Vibrio fischeri." Personal interview. 23 Sept. 2015.

Vogt, Charles, Indian River County Environmental Health Officer. "Bacteria Dynamics in the Indian River Lagoon." Personal interview. 21 Sept. 2015