I. Safety Procedures and Equipment:
Aseptic Techniques for Yeast and BSL1 Microorganisms:
1. Upon entering the lab, wash hands and arms up to the elbow with antibacterial soap.
2. Before and during experimentation wear rubber gloves, apron, and goggles at all times.
3. All glassware and material should be autoclaved for 30 minutes at 20 psi, 121 degrees Celsius.
4. Any open flask, container, or beaker should be covered with aluminum foil when placed in the autoclave.
5. Use 10 % bleach solution and wipe down the lab area and tabletop.
6. Transfer of any culture will be done with a mechanical pipette.
7. Place a biohazard sign in plain view for all to see. The room should be locked at all times.
8. Loops and Needles used to transfer the culture will be sterilized by flame heating until bright red prior to use. When not in use needles, will always be capped or covered. When not in use, place loops face up in a beaker. Never place loops on the counter.
9. The top of the culture-tube will be flame heated before inserting a needle or loop for culture transfer.
1. Upon entering the lab, wash hands and arms up to the elbow with antibacterial soap.
2. Before and during experimentation wear rubber gloves, apron, and goggles at all times.
3. All glassware and material should be autoclaved for 30 minutes at 20 psi, 121 degrees Celsius.
4. Any open flask, container, or beaker should be covered with aluminum foil when placed in the autoclave.
5. Use 10 % bleach solution and wipe down the lab area and tabletop.
6. Transfer of any culture will be done with a mechanical pipette.
7. Place a biohazard sign in plain view for all to see. The room should be locked at all times.
8. Loops and Needles used to transfer the culture will be sterilized by flame heating until bright red prior to use. When not in use needles, will always be capped or covered. When not in use, place loops face up in a beaker. Never place loops on the counter.
9. The top of the culture-tube will be flame heated before inserting a needle or loop for culture transfer.
Chemical Safety:
1. All chemicals (glucose, omega-3 fish oil) will be used in accordance with the MSDS.
2. The use of all chemicals will be done while wearing gloves, aprons, and goggles.
3. All chemicals that release fumes will be placed under the fume hood to prevent the release of fumes into the classroom.
4. A fire blanket, fume-hood, fire extinguisher, eyewash station, and shower are available for use.
5. Upon leaving the lab, hands will be washed and all chemicals will be stored in a locked ventilated chemical storeroom.
1. All chemicals (glucose, omega-3 fish oil) will be used in accordance with the MSDS.
2. The use of all chemicals will be done while wearing gloves, aprons, and goggles.
3. All chemicals that release fumes will be placed under the fume hood to prevent the release of fumes into the classroom.
4. A fire blanket, fume-hood, fire extinguisher, eyewash station, and shower are available for use.
5. Upon leaving the lab, hands will be washed and all chemicals will be stored in a locked ventilated chemical storeroom.
Safety Equipment:
1. Properly maintained biological safety cabinets are available. Palm Bay High School has the Air flow 6000 which is a Class II Biosafety cabinet. This cabinet includes a UV light with timer and alarm and closeable Plexiglas front entrance with a vertical flow Hepa filter.
2. PBHS also has a readily available autoclave which will reach and maintain a pressure of 20 psi and temperature of 121 degrees Celsius for 30 minutes.
3. Face protection (goggles, mask, face shield or other splatter guard) is used for anticipated splashes or sprays.
3. Protective coats, gowns, smocks, are worn while in the laboratory. This protective clothing is removed and left in the laboratory. All protective clothing is either disposed of in the laboratory or laundered by the institution; it should never be taken home by personnel.
4. Gloves are worn when hands may contact potentially infectious materials, contaminated surfaces or equipment.
1. Properly maintained biological safety cabinets are available. Palm Bay High School has the Air flow 6000 which is a Class II Biosafety cabinet. This cabinet includes a UV light with timer and alarm and closeable Plexiglas front entrance with a vertical flow Hepa filter.
2. PBHS also has a readily available autoclave which will reach and maintain a pressure of 20 psi and temperature of 121 degrees Celsius for 30 minutes.
3. Face protection (goggles, mask, face shield or other splatter guard) is used for anticipated splashes or sprays.
3. Protective coats, gowns, smocks, are worn while in the laboratory. This protective clothing is removed and left in the laboratory. All protective clothing is either disposed of in the laboratory or laundered by the institution; it should never be taken home by personnel.
4. Gloves are worn when hands may contact potentially infectious materials, contaminated surfaces or equipment.
Electrical Power Safety
1. Be cautious with the use of the magnetic stirrer and the solutions, be careful not to spill solution on outlets around the device.
2. Never use frayed or cut wires when plugging in magnetic stirrer.
3. Never overload the outlet and use a surge protector.
4. All electrical devices, including the magnetic stirrer, should have an emergency cut off switch or “kill switch”.
5. Shut off and unplug all devices when not in use or when you leave the room unattended.
1. Be cautious with the use of the magnetic stirrer and the solutions, be careful not to spill solution on outlets around the device.
2. Never use frayed or cut wires when plugging in magnetic stirrer.
3. Never overload the outlet and use a surge protector.
4. All electrical devices, including the magnetic stirrer, should have an emergency cut off switch or “kill switch”.
5. Shut off and unplug all devices when not in use or when you leave the room unattended.
Laboratory Facilities (Secondary Barriers):
1. Area has lockable doors for facilities that house restricted agents
2. Laboratory is away from public areas.
3. Each laboratory contains a sink for hand washing.
4. No Carpets and rugs are located in the laboratory.
5. Bench tops are impervious to water and are resistant to moderate heat and the organic solvents, acids, alkalis, and chemicals used to decontaminate the work surfaces and equipment.
6. An eyewash station and shower is readily available.
1. Area has lockable doors for facilities that house restricted agents
2. Laboratory is away from public areas.
3. Each laboratory contains a sink for hand washing.
4. No Carpets and rugs are located in the laboratory.
5. Bench tops are impervious to water and are resistant to moderate heat and the organic solvents, acids, alkalis, and chemicals used to decontaminate the work surfaces and equipment.
6. An eyewash station and shower is readily available.
Autoclave:
1. Always wear goggles and aprons when using the autoclave.
2. The bottom of the autoclave should have about 2 inches of cleaned distilled water before operation.
3. Check the pressure release value prior to use (it should be clear)
4. Use potholders when opening or handling hot surfaces.
5. Opening the autoclave can be done only after the sterilizer after it has cooled (gauge should read zero.) and the all steam has been allowed to escape.
6. Place the control knob in the straight up position and the pressure valve should be down to lock in gasses. This allows the unit to operate at 16-21 psi range.
1. Always wear goggles and aprons when using the autoclave.
2. The bottom of the autoclave should have about 2 inches of cleaned distilled water before operation.
3. Check the pressure release value prior to use (it should be clear)
4. Use potholders when opening or handling hot surfaces.
5. Opening the autoclave can be done only after the sterilizer after it has cooled (gauge should read zero.) and the all steam has been allowed to escape.
6. Place the control knob in the straight up position and the pressure valve should be down to lock in gasses. This allows the unit to operate at 16-21 psi range.
II. Preparation Procedures:
Making Agar:
1. Nutrient Agar is to be prepared with a 4% glucose addition and by the procedures listed as “Preparing Nutrient Agar”. Any glassware used in the production of 4% glucose nutrient agar will be autoclaved.
1. Nutrient Agar is to be prepared with a 4% glucose addition and by the procedures listed as “Preparing Nutrient Agar”. Any glassware used in the production of 4% glucose nutrient agar will be autoclaved.
2. Store sterile agar in refrigerator.
3. Excess sterile agar will be wrapped in newspaper and disposed of by placing in a landfill.
Preparation of Nutrient Agar:
1. Measure out 23 grams of nutrient agar dehydrated media and 42.6 grams of glucose powder.
2. Measure out 1.0 L of distilled water in a graduated cylinder. Place in a 2 L Erlenmeyer flask.
3. Put flask with water onto hot plate and bring to a boil.
4. Once boiling, dissolve 23 grams of agar and 42.6 grams of glucose into the water. Boil for one minute to dissolve.
5. Pour liquid agar into reagent bottles.
6. Autoclave for 30 minutes at 20psi, 121 degrees C.
7. Store in refrigerator when cooled down after autoclaving.
General Procedure for Plating Microbes:
1. On the underside of each petri plate label with each plate with a wax pencil.
2. Heat the nutrient agar in a microwave or water bath to 50º C.
3. Pour a thin layer of agar on the bottom of each plate and cover immediately.
4. Let agar plate solidify. Liquid agar will solidify at about 42 º C.
5. After the medium agar has solidified, the plate is ready to be streaked.
6. Once the plates have been streaked (with solution identified in Experimental Procedures) invert and close the petri dish, tape the edges and incubate them at 25º C for 24-48 hours unless otherwise noted.
3. Excess sterile agar will be wrapped in newspaper and disposed of by placing in a landfill.
Preparation of Nutrient Agar:
1. Measure out 23 grams of nutrient agar dehydrated media and 42.6 grams of glucose powder.
2. Measure out 1.0 L of distilled water in a graduated cylinder. Place in a 2 L Erlenmeyer flask.
3. Put flask with water onto hot plate and bring to a boil.
4. Once boiling, dissolve 23 grams of agar and 42.6 grams of glucose into the water. Boil for one minute to dissolve.
5. Pour liquid agar into reagent bottles.
6. Autoclave for 30 minutes at 20psi, 121 degrees C.
7. Store in refrigerator when cooled down after autoclaving.
General Procedure for Plating Microbes:
1. On the underside of each petri plate label with each plate with a wax pencil.
2. Heat the nutrient agar in a microwave or water bath to 50º C.
3. Pour a thin layer of agar on the bottom of each plate and cover immediately.
4. Let agar plate solidify. Liquid agar will solidify at about 42 º C.
5. After the medium agar has solidified, the plate is ready to be streaked.
6. Once the plates have been streaked (with solution identified in Experimental Procedures) invert and close the petri dish, tape the edges and incubate them at 25º C for 24-48 hours unless otherwise noted.
General Procedures for Streaking Plates:
1. Sterilize work area and don safety equipment.
2. Take out plates from refrigerator and allow to warm up to room temperature.
3. Create solution of E. coli k-12 and Saccharomyces cerevisiae to be plated (create the solution in accordance with the Experimental Procedures explained below).
4. Dip sterile swab into solution.
6. Streak wet swab onto plate being sure to cover the entire area. Plate can be streaked multiple times in a sweeping motion to ensure plate overage.
7. When done streaking plates, securely tape and put into incubator upside down. Depending upon the microbes, growth can appear as soon as 24 hours later.
8. Dispose of materials. Autoclave inoculated plates after they have been analyzed. Plates are to never be reopened.
1. Sterilize work area and don safety equipment.
2. Take out plates from refrigerator and allow to warm up to room temperature.
3. Create solution of E. coli k-12 and Saccharomyces cerevisiae to be plated (create the solution in accordance with the Experimental Procedures explained below).
4. Dip sterile swab into solution.
6. Streak wet swab onto plate being sure to cover the entire area. Plate can be streaked multiple times in a sweeping motion to ensure plate overage.
7. When done streaking plates, securely tape and put into incubator upside down. Depending upon the microbes, growth can appear as soon as 24 hours later.
8. Dispose of materials. Autoclave inoculated plates after they have been analyzed. Plates are to never be reopened.
Preparing Yeast by Serial Dilution:
1. Weigh full package of yeast, then suspend contents in 100 ml of water. Mix thoroughly (magnetic stirrers) for 5-10 minutes.
2. Create data table in which plate number, specimen, its dilution factor, the aliquot plated, and a space for CFUs are all indicated and room for calculation of the total number of CFUs in the original sample.
3. Prepare dilution tubes: repipet 9.9 mL sterile dH2O into each of three sterile 16 x 150 mm capped test tubes. Label the tubes 2, 4, 6 (for 10-2, 10-4, and 10-6 dilutions respectively).
4. Perform a 106 serial dilution of the suspension as follow, using a fresh tip on the mechanical pipette with each stage:
a. Deliver 0.1 mL of original yeast suspension into first tube, (#2) and vortex to mix.
b. Deliver 0.1 mL from #2 to #4 tube, vortex to mix.
c. Deliver 0.1 mL from #4 to #6 tube, vortex to mix.
5. Label 4% glucose nutrient agar plates, on their BOTTOMS, with four bits of information: date, initials, mL aliquot plated (0.1 mL or 0.2 mL) and the dilution factor (ex. Df= 10-6).
6. Measure out 0.1 mL (and then 0.2 mL) aliquot of the original solution (100) using a mechanical pipette.
7. Apply aliquot to the surface of appropriate plates, being careful not to gouge the agar surface).
8. Spread aliquot evenly.
9. Repeat steps 6-8 to plate both amounts of aliquot of yeast and for differing dilutions of yeast suspension.
10. Tape all plates and incubate inverted at 37⁰C for 48 hours.
11. Count the number of colonies and record. Determine the appropriate level of dilution for experimentation and perform further dilution if necessary. Discard plates after being counted, plates will NOT be reopened.
Preparing Bacteria by Serial Dilution:
1. Obtain suspension of desired E. coli k-12.
2. Create data table in which plate number, specimen, its dilution factor, the aliquot plated, and a space for CFUs are all indicated and room for calculation of the total number of CFUs in the original sample.
3. Prepare dilution tubes: repipet 9.9 mL sterile dH2O into each of three sterile 16 x 150 mm capped test tubes. Label the tubes 2, 4, 6 (for 10-2, 10-4, and 10-6 dilutions respectively).
4. Perform a 106 serial dilution of the suspension as follow, using a fresh tip on the mechanical pipette with each stage:
a. Deliver 0.1 mL of original bacteria suspension into first tube, (#2) and vortex to mix.
b. Deliver 0.1 mL from #2 to #4 tube, vortex to mix.
c. Deliver 0,1 mL from #4 to #6 tube, vortex to mix.
5. Label 4%glucose nutrient agar plates, on their BOTTOMS, with four bits of information: date, initials, mL aliquot plated (0.1 mL or 0.2 mL) and the dilution factor (ex. Df= 10-6).
6. Measure out 0.1 mL (and then 0.2 mL) aliquot of the original solution (100) using a mechanical pipette.
7. Apply aliquot to the surface of appropriate plates, being careful not to gouge the agar surface).
8. Spread aliquot evenly.
9. Repeat steps 6-8 to plate both amounts of aliquot of bacteria and for differing dilutions of bacterial suspension.
10. Tape all plates and incubate inverted at 37⁰C for 48 hours.
11. Count the number of colonies and record. Determine the appropriate level of dilution for experimentation and perform further dilution if necessary. Discard plates after being counted, plates will NOT be reopened.
Cleaning up:
1. After each session of experimentation, clean the lab counter with 10% bleach.
2. Wash hands with 10% bleach.
3. Clean with 10% bleach all glassware and related items. New equipment should be autoclaved as needed.
4. Petri dishes and other disposable equipment will be placed in a plastic bag and autoclaved for 30 minutes at 20 psi, 121 degrees Celsius and disposed of.
5. All related glassware and related equipment would be autoclaved for 30 minutes at 20 psi, 121 degrees Celsius.
Disposal:
1. Items of biological hazard must first be sterilized before disposal and thus the autoclave method of sterilization will be used.
2. Put on protective equipment (goggles, gloves, aprons).
3. Gather biological items to be autoclaved prior to disposal.
4. Put items into autoclave-able bag; do not put any sharp objects into bag. The bag then should be tightly sealed by doubling over its end and sealing it shut with a twist tie. Put bag in autoclave.
5. Autoclave for thirty minutes at 20psi, 121 degrees C.
6. When able, remove from autoclave and transport to dumpster.
III. Experimental Procedures:
1. Prepare 400 4%Glucose nutrient agar plates. Divide these 400 into 4 groups of 100. Each of these groups will represent the different dietary treatments (4%Glucose/4%Omega-3 solution, 4%Glucose/0%Omega-3 solution, 0%Glucose/4%Omega-3 solution, and 0%Glucose/0%Omega-3 solution).
2. Label each plate throughout experimentation with the treatment type, date microbes were plated, initials, concentration of yeast plated and presence or absence of E. coli plated.
3. Divide each of these 100 plates into five groups of 20 plates each and label as follows: Control 1 (no yeast treatment), Treatment 1 (low concentration of yeast), Treatment 2 (high concentration of yeast), Control 2 (no bacteria plated, yeast at low concentration) and Control 3(no bacteria plated, yeast at high concentration).*
4. Control 1 (replace the micropipette tip after each step):
a. Aliquot 5 mL of bacterial suspension into 50 mL beaker.
b. Aliquot 5 mL of appropriate Glucose/Omega-3 solution depending on which diet treatment.
c. Aliquot 10 mL of distilled water to the solution.
d. Add stirrer and place on magnetic plate, turn on low (just enough to keep solution moving) and let sit for one hour.
e. Aliquot 1 mL of the solution to the surface of plate appropriately labeled for concentration of yeast*.
f. Streak plate (see Streaking Plates).
g. Repeat steps e and f once for each of 20 plates.
5. Low Concentration Yeast (replace micropipette tip after each step):
a. Aliquot 5 mL of the low concentration* yeast suspension into test tube.
b. Aliquot 5 mL of bacterial suspension into 50 mL beaker.
c. Aliquot 5 mL of appropriate Glucose/Omega-3 solution depending on which diet treatment.
d. Aliquot 5 mL of distilled water to the solution.
e. Add stirrer and place on magnetic plate, turn on low (just enough to keep solution moving) and let sit for one hour.
f. Aliquot 1 mL of the solution to the surface of plate appropriately labeled for concentration of yeast*.
g. Streak plate (see Streaking Plates).
h. Repeat steps f and g once for each of 20 plates.
6. High Concentration Yeast (replace micropipette tip after each step):
a. Aliquot 5 mL of the high concentration* yeast suspension into 50 mL beaker.
b. Aliquot 5 mL of bacterial suspension into test tube.
c. Aliquot 5 mL of appropriate Glucose/Omega-3 solution depending on which diet treatment.
d. Aliquot 5 mL of distilled water to the solution.
e. Add stirrer and place on magnetic plate, turn on low (just enough to keep solution moving) and let sit for one hour.
f. Aliquot 1 mL of the solution to the surface of plate appropriately labeled for concentration of yeast*.
g. Streak plate (see Streaking Plates).
h. Repeat steps f and g once for each of 20 plates.
7. Control 2 (replace micropipette tip after each step):
a. Aliquot 5 mL of the low concentration* yeast suspension into 50 mL beaker.
b. Aliquot 5 mL of appropriate Glucose/Omega-3 solution depending on which diet treatment.
c. Aliquot 10 mL of distilled water to the solution. Stir/mix and let sit for 30 minutes.
d. Add stirrer and place on magnetic plate, turn on low (just enough to keep solution moving) and let sit for one hour.
e. Aliquot 1 mL of the solution to the surface of plate appropriately labeled for concentration of yeast*.
f. Streak plate (see Streaking Plates).
g. Repeat steps e and f once for each of 20 plates.
8. Control 3 (replace micropipette tip after each step):
a. Aliquot 5 mL of the high concentration* yeast suspension into 50 mL beaker.
b. Aliquot 5 mL of appropriate Glucose/Omega-3 solution depending on which diet treatment.
c. Aliquot 10 mL of distilled water to the solution. Stir/mix and let sit for 30 minutes.
d. Add stirrer and place on magnetic plate, turn on low (just enough to keep solution moving) and let sit for one hour.
e. Aliquot 1 mL of the solution to the surface of plate appropriately labeled for concentration of yeast*.
f. Streak plate (see Streaking Plates).
g. Repeat steps e and f once for each of 20 plates.
9. Securely tape all plates and incubate all plates inverted for 48 hours at 37⁰C.
10. For all plates, following the 48 hours incubation, count the number of yeast cells and the number of bacterial cells and record, be careful to denote at which concentration yeast is present and which concentrations of additives are present.
11. Record size of yeast colonies, number of E. coli colonies within one millimeter of the yeast cells and number of E. coli not within one millimeter of the yeast colonies (this will go to determine the correlation between the size of the yeast colony and the number of bacteria that agglutinate, adhere, to it).
*all dilution levels are to be determined from the serial dilution procedures, dilution levels will be chosen that represent the high and low levels but that still show visible and discernable yeast colonies; these will then be applied in testing.
11. Repeat entire procedure four times. Each time varying the glucose/omega-3 concentrations and remembering to denote such changes.
IV. Data Analysis Procedures:
Control 1 Data Analysis Procedures:
1. Data should be recorded properly in Log denoting date of incubation, diet additives and their concentrations present, microorganisms cultured (this group is cultured without yeast), and the total number of colonies of E. coli k-12.
2. Find the arithmetic mean of the colony counts of the E. coli k-12 for each group of 20 plates with differing concentrations of additives and record.
Low Yeast Concentrations Data Analysis Procedures:
1. Data should be recorded properly in Log denoting date of incubation, concentrations of glucose and omega-3, concentration of yeast cultured, bacteria cultured and the following data points.
2. Find the arithmetic mean of the colony counts of E. coli k-12 in CFUs for each group of 20 plates with differing concentrations of glucose and omega-3 and record.
3. Find the arithmetic mean of the colony counts of S. cerevisiae in CFUs for each group of 20 plates with differing concentrations of glucose and omega-3 and record.
4. Find the arithmetic mean of the size of yeast colonies present on plates in mm2 for each group of 20 plates with differing concentrations of glucose and omega-3 and record.
5. Find the arithmetic mean of the number of E. coli k-12colonies in CFUs adhered to (within 1 mm of) yeast colonies for each group of 20 plates with differing concentrations of glucose and omega-3 and record.
6. Determine ratio of E. coli k-12 colonies adhered to yeast colonies as being the number of E. coli k-12 colonies in CFUs per mm2 of yeast colony area for each group of 20 plates with differing concentrations of glucose and omega-3 and record.
7. Find the arithmetic mean of the number of E. coli k-12 colonies not adhered to (within 1 mm of) yeast colonies for each group of 20 plates with differing concentrations of glucose and omega-3 and record.
8. Determine ratio of E. coli k-12 colonies not adhered to yeast colonies as being the number of not adhered colonies per mm2 of yeast colony area for each group of 20 plates with differing concentrations of glucose and omega-3 and record.*
* this is necessary because the ratio of the number of E. coli k-12 adhered to yeast colonies could be misleading if the ratio of those not adhered also increases but is not taken into consideration
High Yeast Concentrations Data Analysis Procedures:
1. Data should be recorded properly in Log denoting date of incubation, concentrations of glucose and omega-3, concentration of yeast cultured, bacteria cultured and the following data points.
2. Find the arithmetic mean of the colony counts of E. coli k-12 in CFUs for each group of 20 plates with differing concentrations of glucose and omega-3 and record.
3. Find the arithmetic mean of the colony counts of S. cerevisiae in CFUs for each group of 20 plates with differing concentrations of glucose and omega-3 and record.
4. Find the arithmetic mean of the size of yeast colonies present on plates in mm2 for each group of 20 plates with differing concentrations of glucose and omega-3 and record.
5. Find the arithmetic mean of the number of E. coli k-12 colonies in CFUs adhered to (within 1 mm of) yeast colonies for each group of 20 plates with differing concentrations of glucose and omega-3 and record.
6. Determine ratio of E. coli k-12 colonies adhered to yeast colonies as being the number of E. coli k-12 colonies in CFUs per mm2 of yeast colony area for each group of 20 plates with differing concentrations of glucose and omega-3 and record.
7. Find the arithmetic mean of the number of E. coli k-12 colonies not adhered to (within 1 mm of) yeast colonies for each group of 20 plates with differing concentrations of glucose and omega-3 and record.
8. Determine ratio of E. coli k-12 colonies not adhered to yeast colonies as being the number of not adhered colonies per mm2 of yeast colony area for each group of 20 plates with differing concentrations of glucose and omega-3 and record.*
Control 2 Data Analysis Procedures:
1. Data should be recorded properly in Log denoting date of incubation, concentrations of glucose and omega-3, concentration (low) of yeast cultured, bacteria cultured (no bacteria are grown on these plates), and the following data points.
2. Find the arithmetic mean of the colony counts of S. cerevisiae for each group of 20 plates with differing concentrations of glucose and omega-3 and record.
3. Find the arithmetic mean of the size of yeast colonies present on plates in mm2 for each group of 20 plates with differing concentrations of glucose and omega-3 and record.
Control 3 Data Analysis Procedures:
1. Data should be recorded properly in Log denoting date of incubation, concentrations of glucose and omega-3, concentration (low) of yeast cultured, bacteria cultured (no bacteria are grown on these plates), and the following data points.
2. Find the arithmetic mean of the colony counts of S. cerevisiae for each group of 20 plates with differing concentrations of glucose and omega-3 and record.
3. Find the arithmetic mean of the size of yeast colonies present on plates in mm2 for each group of 20 plates with differing concentrations of glucose and omega-3 and record.
Comparative Analysis:
1. For colony counts of E. coli k-12 compose a bar graph of the treatment levels of yeast concentration versus the average colony counts of the bacteria, each type of glucose and omega-3 treatment should have its own separate bar graph and then a triple bar graph should be compiled showing all three conditions bacteria were cultured in (no yeast, low yeast, high yeast) versus the concentrations of the glucose and omega-3 treatments and how the colony counts reflect that.
2. For ratio of E. coli k-12 adhered (CFUs/mm2) compose a bar graph of the treatment levels of yeast concentration versus the ratios of the adhered bacteria, each type of glucose and omega-3 treatment should have its own separate bar graph and then a triple bar graph should be compiled showing all three conditions bacteria were cultured in (no yeast, low yeast, high yeast) versus the concentrations of the glucose and omega-3 treatments and how the ratios of the adhered bacteria reflect that.
3. For ratio of E. coli k-12 not adhered (CFUs/mm2) compose a bar graph of the treatment levels of yeast concentration versus the ratios of the not adhered bacteria, each type of glucose and omega-3 treatment should have its own separate bar graph and then a triple bar graph should be compiled showing all three conditions bacteria were cultured in (no yeast, low yeast, high yeast) versus the concentrations of the glucose and omega-3 treatments and how the ratios of the not adhered bacteria reflect that.
V. Statistical Analysis Procedures:
Standard Deviations:
Standard Deviations will be calculated for each arithmetic mean taken in the Data Analysis procedures.
1. Standard deviations will be calculated, using a Microsoft Office Excel Spreadsheet, for each arithmetic mean of data collected (should be a total of 20 standard deviations for each glucose and omega-3 treatment versus yeast concentration).
Standard deviation equals the square root of the quantity of the summation of the square of all of the calculated values (here represented as x) minus the arithmetic mean divided by the square root of the total number of data points.
2. The deviation will be calculated numerically and also represented in graph form.
Correlations:
A. Correlation for concentration of yeast versus colony counts of E. coli k-12 within each agar formula treatment (the means for no bacteria plated do not need to be considered here).
1. The independent variable will be identified as the increasing concentration of S. cerevisiae cultured with bacteria and this value will be known x.
2. The dependent variable will be identified as the average colony counts of the E. coli k-12 and known as y. This statistical data will be represented visually in a chart and further calculations will be done to determine the correlation coefficient of the data.
3. x2 will be calculated for each concentration of yeast.
4. y2 will be calculated for each average colony count as it relates to the concentration of yeast.
5. xy will also be calculated and used to find the correlation coefficient (r).
6. Using the formula for the correlation coefficient, (r) is calculated:
A. Correlation for concentration of yeast versus colony counts of E. coli k-12 within each agar formula treatment (the means for no bacteria plated do not need to be considered here).
1. The independent variable will be identified as the increasing concentration of S. cerevisiae cultured with bacteria and this value will be known x.
2. The dependent variable will be identified as the average colony counts of the E. coli k-12 and known as y. This statistical data will be represented visually in a chart and further calculations will be done to determine the correlation coefficient of the data.
3. x2 will be calculated for each concentration of yeast.
4. y2 will be calculated for each average colony count as it relates to the concentration of yeast.
5. xy will also be calculated and used to find the correlation coefficient (r).
6. Using the formula for the correlation coefficient, (r) is calculated:
where n is the number of pairs of data.
7. If the r value is close to zero, there is no correlation.
8. If the r value is close to positive 1, there is a positive correlation. As one goes up the other goes up.
9. If the r value close to negative 1, there is a negative correlation. As one goes up the other goes down.
B. Correlation for concentration of yeast versus number of adhered E. coli k-12 colonies:
1. The independent variable will be identified as the increasing concentration of S. cerevisiae cultured with E. coli within each agar formula and this value will be known x.
2. The dependent variable will be identified as the number of E. coli k-12 colonies adhered to (within 1 mm of) yeast colonies and known as y. This statistical data will be represented visually in a chart and further calculations will be done to determine the correlation coefficient of the data.
3. x2 will be calculated for each concentration of yeast.
4. y2 will be calculated for each adhered E. coli k-12 colony count as it relates to the concentration of yeast.
5. xy will also be calculated and used to find the correlation coefficient (r).
6. Using the formula for the correlation coefficient, (r) is calculated (as done above).
1. The independent variable will be identified as the increasing concentration of S. cerevisiae cultured with E. coli within each agar formula and this value will be known x.
2. The dependent variable will be identified as the number of E. coli k-12 colonies adhered to (within 1 mm of) yeast colonies and known as y. This statistical data will be represented visually in a chart and further calculations will be done to determine the correlation coefficient of the data.
3. x2 will be calculated for each concentration of yeast.
4. y2 will be calculated for each adhered E. coli k-12 colony count as it relates to the concentration of yeast.
5. xy will also be calculated and used to find the correlation coefficient (r).
6. Using the formula for the correlation coefficient, (r) is calculated (as done above).
C. Correlation for concentration of yeast versus number of not adhered E. coli k-12 colonies:
1. The independent variable will be identified as the increasing concentration of S. cerevisiae cultured with E. coli within each agar formula and this value will be known x.
2. The dependent variable will be identified as the number of E. coli k-12 colonies not adhered to (within 1 mm of) yeast colonies and known as y. This statistical data will be represented visually in a chart and further calculations will be done to determine the correlation coefficient of the data. Again, this data is important to consider because the correlation of the number of apparently adhered E. coli k-12 colonies could be misleading if that is positive and the correlation for the not adhered E. coli k-12 is also positive but not considered in data analysis.
3. x2 will be calculated for each concentration of yeast.
4. y2 will be calculated for each not adhered E. coli k-12 colony count as it relates to the concentration of yeast.
5. xy will also be calculated and used to find the correlation coefficient (r).
6. Using the formula for the correlation coefficient, (r) is calculated (as done above).
1. The independent variable will be identified as the increasing concentration of S. cerevisiae cultured with E. coli within each agar formula and this value will be known x.
2. The dependent variable will be identified as the number of E. coli k-12 colonies not adhered to (within 1 mm of) yeast colonies and known as y. This statistical data will be represented visually in a chart and further calculations will be done to determine the correlation coefficient of the data. Again, this data is important to consider because the correlation of the number of apparently adhered E. coli k-12 colonies could be misleading if that is positive and the correlation for the not adhered E. coli k-12 is also positive but not considered in data analysis.
3. x2 will be calculated for each concentration of yeast.
4. y2 will be calculated for each not adhered E. coli k-12 colony count as it relates to the concentration of yeast.
5. xy will also be calculated and used to find the correlation coefficient (r).
6. Using the formula for the correlation coefficient, (r) is calculated (as done above).
Inferential statistics:
A z-value will be used to determine if differences in colony counts observed with varying concentrations of yeast are due to chance or are due to introduction of the independent variable.
A z-value will be used to determine if differences in colony counts observed with varying concentrations of yeast are due to chance or are due to introduction of the independent variable.
1. A Null Hypothesis will be stated for each inferential statistics test performed: a z-value will be calculated to compare colony counts of E. coli k-12 grown without yeast and with low concentration of yeast an again with high concentration of yeast for each glucose and omega-3 treatment (total of 10 z-values). Furthermore a z-value will be calculated to compare colony counts of E. coli k-12 grown with identical concentrations of yeast but under varying glucose and omega-3 treatments (total of 12 z-values). Furthermore a z-value will be calculated to compare colony counts of S. cerevisiae grown in identical concentrations (without bacteria) but under varying glucose and omega-3 treatments (total of 8 z-values). Each z-test relates a treatment group’s arithmetic mean to that of a comparable control group, it does not relate two treatment group’s arithmetic means to one another.
2. Standard deviations will be calculated (see Standard Deviations) for each arithmetic mean of data collected (should be a total of 25 standard deviations for each condition bacteria or yeast is grown in)
3. Z-value will be calculated using the control and treated groups averages and standard deviation of the control group:
Z equals the quantity of sample mean minus the population mean divided by the standard deviation of the control.
4. In a one tailed z-test any value above z=1.65 or below z=-1.65 is cause for rejection of the null hypothesis (and thus acceptance of the hyptothesis).
5. Rejecting the null hypothesis indicates that the difference between the treated group’s average and the average of the control are due to the introduction of the independent variable or treatment and thus are statistically significant.
