Difference between revisions of "Team:NCTU Formosa/Protocol"
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Revision as of 16:32, 29 October 2017
Wet Lab Protocol
Experiment:
- Preparation for Potato dextrose agar (PDA) plates
Purpose:
To prepare solid medium to civilize the fungi
Purpose:
To prepare solid medium to civilize the fungi
Drugs and equipment:
- PDA powder
- ddH2O
- Petri dishes
- Serum bottle
- Microwave oven
- Autoclave
- Alcohol burner
Process:
- The ratio of PDA powder and ddH2O is 39g : 1000ml. To prevent the liquid spill out from Serum bottles, we usually have only 300ml of the liquid in a 500ml Serum bottle.
- Loosen the bottle cap for a bit, and put it into the autoclave.
- Screw the bottle cap tight to cool down. If the liquid becomes solid, heat it up with a microwave oven.
- Bring the bottle into the Hood, disinfect bottleneck with alcohol burner. Pour out the liquid to plates, each plate can hold 15~20ml of the PDA liquid. Shake the plate smoothly and make the liquid surface flat.
- Keep the plates in normal temperature or in 4 °C refrigerator
Experiment:
- Concentration test for spore suspension
Purpose:
To test concentration of spore suspension liquid and calculate germination rate.
Purpose:
To prepare solid medium to civilize the fungi
Drugs and equipment:
- 75% Alcohol
- ddH2O
- Alcohol burner
- Hemocytometer
- Fungi plates
- Glass Cell Spreaders
- Pipet
- Gauze
- Centrifuge
- Beaker
Process:
- Choose the fungi plate you want (age, growing situation…etc)
- Put the plate and equipment inside the Hood. If the spore is easy to fly in the air, please switch off the exhaust fan.
- Add ddH2O to the plate until water covers the surface of the plate. (You may use the pipet.) This step you can also use gauze to filter impurity.
- Disinfect the glass cell spreaders with alcohol burner, after cooling down, scrape the plate softly so the spore would be in the water.
- Remove the water in the plate to a beaker (You may use gauze to filter impurity). Now you got a spore suspension liquid with unknown concentration.
- Clean the Hemocytometer with 75%alcohol and wipe it with lens paper, so as not to make a scratch on it. Put the coverslip on the Hemocytometer, and inject 10ml spore suspension liquid from the tiny chamber beside. The spore suspension should cover all the square of the Hemocytometer.
- Put the Hemocytometer under a microscope, and observe the spore.
- Count the amount of the spore in the square, and calculate the concentration. Add water if it’s concentration is too high; centrifuge the liquid if the concentration is too low. Finally, you got a spore suspension liquid with a known concentration.
Experiment:
- Spore germination percentage
Purpose:
To test the concentration of spore suspension liquid and calculate germination rate.
Purpose:
To test the concentration of spore suspension liquid and calculate germination rate.
Drugs and equipment:
- 75% Alcohol
- 2% glucose solution
- ddH2O
- HEPES buffer
- Peptides
- Alcohol burner
- Hemocytometer
- Fungi plates
- Glass Cell Spreaders
- 10ul pipet
- Gauze
- Centrifuge
- Beaker*2
- Petri dishes
- Lens cleaning paper
- Microscope
- Slide glass with 2 Cavities
- Counter
Process:
- Choose fungi plates that have produced spores.
- (It is recommended to experiment in a laminar flow (hood). Add ddH2O to the plate until water covers the surface of the plate.
- Disinfect the glass cell spreaders with an alcohol burner, after cooling down, scrape the plate softly so the spore would be in the water.
- Remove the water in the plate to a beaker (You may filter out impurity with gauze). Now you got a spore suspension liquid with unknown concentration.
- Clean the Hemocytometer with 75%alcohol and wipe it clean with lens cleaning paper, so as not to make any scratch on it. Put the coverslip on the Hemocytometer, and inject 10ml spore suspension liquid from the tiny chamber beside. The spore suspension should cover all the square of the Hemocytometer.
- Put the Hemocytometer under a microscope, and observe the spore.
- Count the amount of the spore in the square, and calculate the concentration. Add water if it’s concentration is too high; centrifuge the liquid if the concentration is too low. Finally, you got a spore suspension liquid with concentration you know. We would like to prepare spore suspension liquid with the concentration of 1.5*10^5 spores/ml for our experiments.
- Mixed 5ul spore suspension liquid with 5ul 2% glucose solution and 5ul peptide together into a PCR tube (It is recommended for pipetting 50 times to ensure that there is a mix of uniform).
- Draw 15μl of the solution from PCR tube to the double concave slide, and put the slide into a Petri dish. Add some ddH2O around the slide, in order to create an environment of 100% relative humidity so that the liquid would note evaporate easily.
- After incubating under 20℃ for 6 hours, we observe and classify the spores into four grades according to the length of the germination tube, calculate the percentage of each germination grade of each sample.
這裡有圖
這裡有圖
Experiment:
- Separation of infected plant tissue
Purpose:
To separate infected plant tissue and cultivate pathogen fungi
Purpose:
To separate infected plant tissue and cultivate pathogen fungi
Drugs and equipment:
- 75% Alcohol
- 0.5%~1% Sodium hypochlorite (NaClO)
- ddH2O
- PDA (Potato dextrose agar) plate
- Parafilm
- Alcohol burner
- Plants
- Knifes
- Tweezers
Process:
- Soak the plant tissue to sterilization: All parts of plant should soak in alcohol for 1 minute. For leaves, skim over NaClO for a few times; for roots, soak in NaClO for 1 minute; for mature stem parts, soak in NaClO for 20~30 seconds; for tender stems, skim over NaClO for a few times. All of these parts should be washed with ddH 2 O.
- Remove all the drugs and tools inside the Hood, disinfect knifes and tweezers with alcohol burner. You may take a PDA plate for cool down.
- Cut the infected plant tissue for an area of 5mm 2 , remove them all to new PDA plates. The side with hypha should put downward, and be towed on the plate. The amount of tissue which are put on one single plate depends on the range of cutting area.
- Seal up the plate with Parafilm, and label on the name of fungi, date and so on.
- Put the plate in the best environment to observe if the fungi or other species of microorganism grow.
Experiment:
- Concentration test for spore suspension
Purpose:
To test concentration of spore suspension liquid and calculate germination rate.
Purpose:
To test concentration of spore suspension liquid and calculate germination rate.
Drugs and equipment:
- 75% Alcohol
- ddH2O
- Alcohol burner
- Hemocytometer
- Fungi plates
- Glass Cell Spreaders
- Pipet
- Gauze
- Centrifuge
- Beaker
Process:
- Choose the fungi plate you want (age, growing situation…etc)
- Put the plate and equipment inside the Hood. If the spore is easy to fly in the air, please switch off the exhaust fan.
- Add ddH 2 O to the plate until water covers the surface of the plate. (You may use the pipet.) This step you can also use gauze to filter impurity.
- Disinfect the glass cell spreaders with alcohol burner, after cooling down, scrape the plate softly so the spore would be in the water.
- Remove the water in the plate to a beaker (You may use gauze to filter impurity). Now you got a spore suspension liquid with unknown concentration.
- Clean the Hemocytometer with 75%alcohol and wipe it with lens paper, so as not to make a scratch on it. Put the coverslip on the Hemocytometer, and inject 10ml spore suspension liquid from the tiny chamber beside. The spore suspension should cover all the square of the Hemocytometer.
- Put the Hemocytometer under a microscope, and observe the spore.
- Count the amount of the spore in the square, and calculate the concentration. Add water if it’s concentration is too high; centrifuge the liquid if the concentration is too low. Finally, you got a spore suspension liquid with a known concentration
Wet Lab Protocol
Experiment:
- Preparation for Potato dextrose agar (PDA) plates
Purpose:
To prepare solid medium to civilize the fungi
Purpose:
To prepare solid medium to civilize the fungi
Drugs and equipment:
- PDA powder
- ddH2O
- Petri dishes
- Serum bottle
- Microwave oven
- Autoclave
- Alcohol burner
Process:
- The ratio of PDA powder and ddH2O is 39g : 1000ml. To prevent the liquid spill out from Serum bottles, we usually have only 300ml of the liquid in a 500ml Serum bottle.
- Loosen the bottle cap for a bit, and put it into the autoclave.
- Screw the bottle cap tight to cool down. If the liquid becomes solid, heat it up with a microwave oven.
- Bring the bottle into the Hood, disinfect bottleneck with alcohol burner. Pour out the liquid to plates, each plate can hold 15~20ml of the PDA liquid. Shake the plate smoothly and make the liquid surface flat.
- Keep the plates in normal temperature or in 4 °C refrigerator
Experiment:
- Concentration test for spore suspension
Purpose:
To test concentration of spore suspension liquid and calculate germination rate.
Purpose:
To prepare solid medium to civilize the fungi
Drugs and equipment:
- 75% Alcohol
- ddH2O
- Alcohol burner
- Hemocytometer
- Fungi plates
- Glass Cell Spreaders
- Pipet
- Gauze
- Centrifuge
- Beaker
Process:
- Choose the fungi plate you want (age, growing situation…etc)
- Put the plate and equipment inside the Hood. If the spore is easy to fly in the air, please switch off the exhaust fan.
- Add ddH2O to the plate until water covers the surface of the plate. (You may use the pipet.) This step you can also use gauze to filter impurity.
- Disinfect the glass cell spreaders with alcohol burner, after cooling down, scrape the plate softly so the spore would be in the water.
- Remove the water in the plate to a beaker (You may use gauze to filter impurity). Now you got a spore suspension liquid with unknown concentration.
- Clean the Hemocytometer with 75%alcohol and wipe it with lens paper, so as not to make a scratch on it. Put the coverslip on the Hemocytometer, and inject 10ml spore suspension liquid from the tiny chamber beside. The spore suspension should cover all the square of the Hemocytometer.
- Put the Hemocytometer under a microscope, and observe the spore.
- Count the amount of the spore in the square, and calculate the concentration. Add water if it’s concentration is too high; centrifuge the liquid if the concentration is too low. Finally, you got a spore suspension liquid with a known concentration.
Experiment:
- Spore germination percentage
Purpose:
To test the concentration of spore suspension liquid and calculate germination rate.
Purpose:
To test the concentration of spore suspension liquid and calculate germination rate.
Drugs and equipment:
- 75% Alcohol
- 2% glucose solution
- ddH2O
- HEPES buffer
- Peptides
- Alcohol burner
- Hemocytometer
- Fungi plates
- Glass Cell Spreaders
- 10ul pipet
- Gauze
- Centrifuge
- Beaker*2
- Petri dishes
- Lens cleaning paper
- Microscope
- Slide glass with 2 Cavities
- Counter
Process:
- Choose fungi plates that have produced spores.
- (It is recommended to experiment in a laminar flow (hood). Add ddH2O to the plate until water covers the surface of the plate.
- Disinfect the glass cell spreaders with an alcohol burner, after cooling down, scrape the plate softly so the spore would be in the water.
- Remove the water in the plate to a beaker (You may filter out impurity with gauze). Now you got a spore suspension liquid with unknown concentration.
- Clean the Hemocytometer with 75%alcohol and wipe it clean with lens cleaning paper, so as not to make any scratch on it. Put the coverslip on the Hemocytometer, and inject 10ml spore suspension liquid from the tiny chamber beside. The spore suspension should cover all the square of the Hemocytometer.
- Put the Hemocytometer under a microscope, and observe the spore.
- Count the amount of the spore in the square, and calculate the concentration. Add water if it’s concentration is too high; centrifuge the liquid if the concentration is too low. Finally, you got a spore suspension liquid with concentration you know. We would like to prepare spore suspension liquid with the concentration of 1.5*10^5 spores/ml for our experiments.
- Mixed 5ul spore suspension liquid with 5ul 2% glucose solution and 5ul peptide together into a PCR tube (It is recommended for pipetting 50 times to ensure that there is a mix of uniform).
- Draw 15μl of the solution from PCR tube to the double concave slide, and put the slide into a Petri dish. Add some ddH2O around the slide, in order to create an environment of 100% relative humidity so that the liquid would note evaporate easily.
- After incubating under 20℃ for 6 hours, we observe and classify the spores into four grades according to the length of the germination tube, calculate the percentage of each germination grade of each sample.
這裡有圖
這裡有圖
Experiment:
- Separation of infected plant tissue
Purpose:
To separate infected plant tissue and cultivate pathogen fungi
Purpose:
To separate infected plant tissue and cultivate pathogen fungi
Drugs and equipment:
- 75% Alcohol
- 0.5%~1% Sodium hypochlorite (NaClO)
- ddH2O
- PDA (Potato dextrose agar) plate
- Parafilm
- Alcohol burner
- Plants
- Knifes
- Tweezers
Process:
- Soak the plant tissue to sterilization: All parts of plant should soak in alcohol for 1 minute. For leaves, skim over NaClO for a few times; for roots, soak in NaClO for 1 minute; for mature stem parts, soak in NaClO for 20~30 seconds; for tender stems, skim over NaClO for a few times. All of these parts should be washed with ddH 2 O.
- Remove all the drugs and tools inside the Hood, disinfect knifes and tweezers with alcohol burner. You may take a PDA plate for cool down.
- Cut the infected plant tissue for an area of 5mm 2 , remove them all to new PDA plates. The side with hypha should put downward, and be towed on the plate. The amount of tissue which are put on one single plate depends on the range of cutting area.
- Seal up the plate with Parafilm, and label on the name of fungi, date and so on.
- Put the plate in the best environment to observe if the fungi or other species of microorganism grow.
Experiment:
- Concentration test for spore suspension
Purpose:
To test concentration of spore suspension liquid and calculate germination rate.
Purpose:
To test concentration of spore suspension liquid and calculate germination rate.
Drugs and equipment:
- 75% Alcohol
- ddH2O
- Alcohol burner
- Hemocytometer
- Fungi plates
- Glass Cell Spreaders
- Pipet
- Gauze
- Centrifuge
- Beaker
Process:
- Choose the fungi plate you want (age, growing situation…etc)
- Put the plate and equipment inside the Hood. If the spore is easy to fly in the air, please switch off the exhaust fan.
- Add ddH 2 O to the plate until water covers the surface of the plate. (You may use the pipet.) This step you can also use gauze to filter impurity.
- Disinfect the glass cell spreaders with alcohol burner, after cooling down, scrape the plate softly so the spore would be in the water.
- Remove the water in the plate to a beaker (You may use gauze to filter impurity). Now you got a spore suspension liquid with unknown concentration.
- Clean the Hemocytometer with 75%alcohol and wipe it with lens paper, so as not to make a scratch on it. Put the coverslip on the Hemocytometer, and inject 10ml spore suspension liquid from the tiny chamber beside. The spore suspension should cover all the square of the Hemocytometer.
- Put the Hemocytometer under a microscope, and observe the spore.
- Count the amount of the spore in the square, and calculate the concentration. Add water if it’s concentration is too high; centrifuge the liquid if the concentration is too low. Finally, you got a spore suspension liquid with a known concentration
