Procedures


Safety Procedures

Methyl Jasmonate
1. First Aid Measures:
Inhalation and Ingestion Exposure: If swallowed, wash out mouth with water, provided person is conscious. Do not induce vomiting. Consult a doctor.
If inhaled, remove from exposure to fresh air.
Eye Contact: Flush with water for 15 min. Seek medical help if necessary.
Skin Contact: Wash area well with water. Seek medical help if necessary.
2. Fire Fighting Measures:
Extinguishing Media: CO2, Foam, Dry Chemical
3. Accidental Release Measures:
Material Released or Spilled: Standard absorbents can be used.
Waste Disposal: Incineration or sanitary landfill in accordance with local, state, and federal regulations.
4. Engineering Controls: A system of local and/or general exhaust is recommended to keep employee exposures low. Local exhaust ventilation is generally preferred because it can control the emissions of the contaminant at its source.
5. Protection: Gloves, apron, safety goggles.
6. Disposal: Methyl Jasmonate will be used up in experiment.
Coomassie Reagent (G-250 Dye)
1. First Aid Measures:
Inhalation and Ingestion Exposure: If swallowed, wash out mouth with water, provided person is conscious. Do not induce vomiting. Consult a doctor.
Inhalation: Remove to fresh air. Give oxygen and get medical attention for any breathing difficulty.
Eye Contact: Flush eyes thoroughly with gently running water, holding eyelids open while flushing, for five to ten minutes, or until no trace of chemical remains. Get medical attention if irritation develops.
Skin Contact: Remove contaminated clothing. Brush or wipe off dry material. Flush skin with plenty of running water until no evidence of chemical remains. Get medical attention if irritation develops.
2. Fire Fighting Measures:
Extinguishing Media: Extinguisher appropriate to the surrounding material which is burning.
Accidental Release Measures: Ventilate area of spill. Shut off all sources of ignition. Flush site with water.
3. Protection: Gloves, safety goggles, apron.
4. Disposal: Chemical will be saved for later experimentation.

Agrobacterium tumefaciens
Autoclave
1. Always wear goggles and aprons when using the autoclave.
2. The bottom of the auto clave 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. This allows the unit to operate at 16-21 psi range.


Cleaning up
1. After each day 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. Autoclave new equipment as needed.
4. Petri dishes and other disposable equipment will be placed in a plastic bag and autoclaved for 30 minutes at 20 psi, 120 degrees Celsius and disposed of.
5. All related glassware and related equipment would be autoclaved for 30 minutes at 20 psi, 120 degrees Celsius.
Disposal
1. Items of biological hazard must be disposed of after autoclaving.
2. Put on protective equipment (goggles, gloves, aprons).
3. Gather biological items to be autoclaved prior to disposal.
4. Put items into autoclavable bag. Put bag in autoclave.
5. Autoclave for thirty minutes at 20psi, 120 degrees C.
6. When able, remove from autoclave and transport to dumpster.

BSL 1 Aseptic Techniques
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, 120 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.
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.

Spectrophotometer
1. Always wear goggles and an apron.

Electrical Safety
Objects Used: Spectrophotometer, Environmental Chamber, Vaporizer, and Blender
1. Limit the use of high power devices around water.
2. Never use frayed or cut wires when plugging in a device.
3. Never overload the outlet and use a surge protector.
4. All electrical devices 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.
Sharp Object Safety
Objects Used: Blender and Syringe
1. Use caution when using a sharp object.
2. Restrict the use of "sharps" to a minimum.
3. Do not use bent or broken needles, knifes, or razor blades.
4. Cover or cap needle after every use.
5. Discard used syringes, needles, or scalpels into an approved sharps container.
6. Bring all needles in a sharps container to a local fire station for disposal.

Set Up
Growing Lycopersicon esculentum
1. Individual place about 140 tomato seeds in individual pots of moist soil.
2. Place pots in environmental chamber at room temperature and 12 hours of light.
3. Water each pot with one cup of water every other day.

Activating Agrobacterium tumefaciens
1. In a test tube, add 2ml of saline solution to Slant of Agrobacterium tumefaciens.
2. Incubate at room temperature for 10 minutes to allow multiplication of bacteria.

Infecting Lycopersicon esculentum with Agrobacterium tumefaciens
1. Individually make a 3 cm cut in the middle of the plant stem with a razor blade.
2. Dip a cotton swap into Agrobacterium tumefaciens solution.
3. Wipe dipped cotton swab down 3 cm slit on plant.
4. Allow time for Crown Gall to be produced.

Concentrating Methyl Jasmonate
1. Divide 1 gram Methyl Jasmonate into .6 of a ml and .3 of a ml.
2. Dilute each with 1 liter of ethanol to create a 1.34x10^-3 M per liter solution (Concentration A) and a 2.68x10^-3 M per liter concentration (Concentration B).

Preparing Leaf Liquid for Protein Concentration Quantitation
1. Gather 3 inches of stem from each of the 16 plants in each group.
2. Homogenize in blender with 10 ml of DI water.
3. Pour homogenized leaf liquid into Whatman filter paper.
4. Let liquid filter. Remaining liquid will be used for protein concentration assay.
5. Repeat for each group every first day of each treatment week for a three week period.

Preparing Coomassie Blue G-250 Brilliant Blue Solution
1. Dissolve 100 mg of Coomassie Brilliant Blue G-250 in 50 ml 95% ethanol
2. Add 100 ml 85% (w/v) phosphoric acid.
3. Dilute to 1 liter when the dye has completely dissolved, and filter through Whatman #1 paper just before use.

Standard Curve Protein Assay
1. Turn on the spectrophotometer and set the wavelength to 595 nm.
2. You are provided with a protein solution of BSA containing 100 ug/ml. Label seven cuvets and sequentially add your BSA solution, distilled water, and Coomassie Blue reagent according to the volumes indicated (ml) in Table 1. This will give a dilution series of the protein.
3. Mix the cuvets thoroughly immediately after adding the dye by placing parafilm over the top of each cuvet and inverting several times.
4. Use cuvet 1 as a protein blank to set the absorbance to .000. Measure the absorbance of the remaining cuvets (2-6) and record absorbance readings in Table 1.
5. Plot the results on graph paper, plotting the absorbance for each cuvet on the y-axis and the protein concentration on the x-axis.
Standard Curve was conducted at FIT under supervision of Dr. Carroll.

Table 1

Cuvet Number
Concentration
Of BSA (μg/ml)
Diluted Standard (³μl)
Coomassie Reagent Added (μl)
Protein Concentration
(μg/ml)
Absorbance at 595 nm
1
0
5
200
0

2
0.125
5
200
0.125

3
0.25
5
200
0.25

4
0.5
5
200
0.5

5
1.0
5
200
1.0

6
2.0
5
200
2.0




Design
Treating Lycopersicon esculentum Crown Gall with Methyl Jasmonate

Control Ethanol B
1. Place 16 infected plants in isolated chamber #5.
2. Withdraw three ml of Ethanol in syringe.
3. Carefully, inject full amount into Crown Gall directly in the center.
4. Repeat with each plant every Monday and Friday for three weeks.
5. Measure Crown Gall growth or regression every third day of each treatment week over a period of three weeks.

Control Ethanol A
1. Place 16 infected plants in isolated chamber #2.
2. Spray with three pumps of Ethanol every Monday and Friday for three weeks.
3. Measure Crown Gall growth or regression every third day of each treatment week over a period of three weeks.

Methyl Jasmonate Mist Concentration A
1. Place 16 infected plants in isolated chamber #3.
2. Spray with three pumps of 1.34x10^-3 Methyl Jasmonate Molar concentration A into the chamber every Monday and Friday for three weeks.
3. Measure Crown Gall growth or regression every third day of each treatment week over a period of three weeks.

Methyl Jasmonate Mist Concentration B
1. Place 16 infected plants in isolated chamber #4.
2. Spray three pumps of 2.68x10^-3 Methyl Jasmonate Molar concentration B into the chamber Monday and Friday for three weeks.
3. Measure Crown Gall growth or regression every third day of each treatment week over a period of three weeks.

Methyl Jasmonate Injection Concentration A
1. Place 16 infected plants in isolated chamber #5.
2. Withdraw three ml of 1.34x10^-3 Methyl Jasmonate Molar concentration A.
3. Carefully, inject full amount into Crown Gall directly in the center.
4. Repeat with each plant every Monday and Friday for three weeks.
5. Measure Crown Gall growth or regression every third day of each treatment week over a period of three weeks.

Methyl Jasmonate Injection Concentration B
1. Place 16 infected plants in isolated chamber #6.
2. Withdraw three ml of 2.68x10^-3 Methyl Jasmonate Molar concentration B.
3. Carefully, inject full amount into Crown Gall directly in the center.
4. Repeat with each plant every Monday and Friday for three weeks.
5. Measure Crown Gall growth or regression every third day of each treatment week over a period of three weeks.

Protein Assay Concentration A in Mist
1. Take 5 ul of leaf extract and mix with 200 ul of Coomassie Blue in a cuvet.
2. Add 795 ul of RO water
3. Measure the absorbance of your unknown and record the absorbance.
4. Determine the concentration of leaf extract from the standard curve. Record this number.
5. Repeat everday for 1 week.

Protein Assay Concentration B in Mist
1. Take 5 ul of leaf extract and mix with 200 ul of Coomassie Blue in a cuvet.
2. Add 795 ul of RO water
3. Measure the absorbance of your unknown and record the absorbance.
4. Determine the concentration of leaf extract from the standard curve. Record this number.
5. Repeat everday for 1 week.

Protein Assay Concentration A in Injection
1. Take 5 ul of leaf extract and mix with 200 ul of Coomassie Blue in a cuvet.
2. Add 795 ul of RO water
3. Measure the absorbance of your unknown and record the absorbance.
4. Determine the concentration of leaf extract from the standard curve. Record this number.
5. Repeat everday for 1 week.


Protein Assay Concentration B in Mist
1. Take 5 ul of leaf extract and mix with 200 ul of Coomassie Blue in a cuvet.
2. Add 795 ul of RO water
3. Measure the absorbance of your unknown and record the absorbance.
4. Determine the concentration of leaf extract from the standard curve. Record this number.
5. Repeat everday for 1 week.


Protein Assay Ethanol in Mist
1. Take 5 ul of leaf extract and mix with 200 ul of Coomassie Blue in a cuvet.
2. Add 795 ul of RO water
3. Measure the absorbance of your unknown and record the absorbance.
4. Determine the concentration of leaf extract from the standard curve. Record this number.
5. Repeat everday for 1 week.


Protein Assay Ethanol in Injection
1. Take 5 ul of leaf extract and mix with 200 ul of Coomassie Blue in a cuvet.
2. Add 795 ul of RO water
3. Measure the absorbance of your unknown and record the absorbance.
4. Determine the concentration of leaf extract from the standard curve. Record this number.
5. Repeat everday for 1 week.

Data Collection

Measuring Crown Gall Growth or Regression
1. Using a Vernier Caliper, measure the height of the Crown Gall (vertical, top to bottom).
2. Using a Vernier Caliper, measure width of the Crown Gall (horizontal, side to side)
3. Using a Vernier Caliper, measure the thickness of the Crown Gall. Measure the thickness of stem, then the thickness of Crown Gall with stem. Subtract Stem thickness from Stem and Crown Gall thickness to calculate Crown Gall thickness.
4. Repeat with each plant in each group Wednesday before treatment, and following Wednesdays for three week period.

Standard Curve for Protein Assay
1. Turn on the spectrophotometer and set the wavelength to 595 nm.
2. You are provided with a protein solution of BSA containing 100 ug/ml. Label seven cuvets and sequentially add your BSA solution, distilled water, and Coomassie Blue reagent according to the volumes indicated (ml) in Table 1. This will give a dilution series of the protein.
3. Mix the cuvets thoroughly immediately after adding the dye by placing parafilm over the top of each cuvet and inverting several times.
4. Use cuvet 1 as a protein blank to set the absorbance to .000. Measure the absorbance of the remaining cuvets (2-6) and record absorbance readings in Table 1.
5. Plot the results on graph paper, plotting the absorbance for each cuvet on the y-axis and the protein concentration on the x-axis.

Table 1

Cuvet Number
Concentration
Of BSA (μg/ml)
Diluted Standard (³μl)
Coomassie Reagent Added (μl)
Protein Concentration
(μg/ml)
Absorbance at 595 nm
1
0
5
200
0

2
0.125
5
200
0.125

3
0.25
5
200
0.25

4
0.5
5
200
0.5

5
1.0
5
200
1.0

6
2.0
5
200
2.0



Determining Concentration of Leaf Extract in Treatment Groups
1. Take 5 ul of leaf extract and mix with 200 ul of Coomassie Blue in a cuvet.
2. Add 795 ul of RO water
3. Measure the absorbance of your unknown and record the absorbance.
4. Determine the concentration of leaf extract from the standard curve. Record this number.
3. Determine the concentration of your unknown from the standard curve. Record this number in table 1.

Data Analysis

1. For each group there will be a bar graph for each of the two treatment weeks. One bar graph with each mean of each group’s regression percentage for week one. And a bar graph with each mean of each group’s regression percentage for week two. And one double bar graph with both week’s mean regression percentage. Then a bar graph with all group’s total individual mean regression percentage. It will show Treatment Group versus Percentage of Regression.
2. There will be a standard curve line graph to show what I used to determine the concentration of proteins for the plants. And there will be 2 line graphs to show the increase over the 2 week treatment period. One for week one, and one for week two. It will show Treatment Group versus Concentration of Proteins.

Statistical Analysis Procedures
Standard Deviations:
Stard 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 24 standard deviations for each treatment versus regression group and treatment versus protein concentration).
Standard deviation equals the square root of the quantity of the summation of the square of all 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 the treatment group versus gall regression (the control groups A and B do not need to be considered here).
1. The independent variable will be identified as the increasing concentration of Methyl Jasmonate increase and spray as appose to injection, this value will be known as x.
2. The dependent variable will be identified as gall regression 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. x^2 will be calculated for each Methyl Jasmonate concentration and spray appose to injection.
4. y^2 will be calculated for gall regression as it relates to concentration of Methyl Jasmonate and form of treatment.
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 is close to negative 1, there is a negative correlation. As on goes up the other goes down.
B. Correlation for the treatment group versus gall regression (the control groups A and B do not need to be considered here).
1. The independent variable will be identified as the increasing concentration of Methyl Jasmonat increase and spray as appose to injection, this value will be known as x.
2. The dependent variable will be identified as protein concentraion 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. x^2 will be calculated for each Methyl Jasmonate concentration and spray appose to injection.
4. y^2 will be calculated for protein concentration as it relates to concentration of Methyl Jasmonate and form of treatment.
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.
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 is close to negative 1, there is a negative correlation. As on goes up the other goes down.

Inferential Statistics:
A z-value will be used to determine if differences in gall regression observed with varying concentrations of Methyl Jasmonate (spray & injection) 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 gall regression without Methyl Jasmonate and with low concentrations of Methyl Jasmonate and again with high concentrations of Methyl Jasmonate for each group (total of 6 z-values). Furthermore, there will be a z-value calculated for to compare protein concentration without Methyl Jasmonate and with low concentrations of Methyl Jasmonate and again with high concentrations of Methyl Jasmonate for each group (total of 6 z-values). Each z-test relates a 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 12 standard deviations for each concentration of Methyl Jasmonate Lycopersicon esculentum were exposed to).
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 hypothesis).
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.



Disposal

Methyl Jasmonate will be used up in experiment.

Coomassie Blue G-250 will be saved for later experimentation.

Agrobacterium Tumefaciens:
1. Items of biological hazard must be disposed of after autoclaving.
2. Put on protective equipment (goggles, gloves, aprons).
3. Gather biological items to be autoclaved prior to disposal.
4. Put items into autoclavable bag. Put bag in autoclave.
5. Autoclave for thirty minutes at 20psi, 120 degrees C.
6. When able, remove from autoclave and transport to dumpster.
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