Difference between revisions of "Team:Pumas Mexico/Experiments"
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| + | <h5>Method and procedures </h5> <br>Obtaining of unialgal strain of Chlorella vulgaris<br> | ||
| + | The strain will be asked to “Algas continentales: Ecologia y taxonomia” (LACET) (Tercer piso, Edificio A, Facultad de Ciencias Universidad Nacional Autónoma de México, Ciudad Universitaria, Coyoacán, D.F. 0451 0, México). | ||
| + | <h5><br>Growing of C. vulgaris</h5><br> | ||
| + | Prepares 1 L of Bold Basal (BB) and will be inoculated with the strain, in asepsis conditions. Additionally, prepares 1 L of Bold basal with nitrates deficiency to cause nutritional stress. Later they are stored in “Laboratorio de Ambientes controlados” (Edificio B de Biología 3er piso, Facultad de Ciencias Universidad Nacional Autónoma de México Ciudad Universitaria, Coyoacán, D.F. 0451 0, México) at a temperature of 25°C +/- 1°C with light and dark cycles for 12 hours. Scaling 1L at a time according the water requirement of the alga.<br> | ||
| + | <h5><br>Calibration curve of C. vulgaris</h5><br> | ||
| + | They counted 20 µL of inoculation cell’s stock of C. vulgaris daily for two weeks with the help of a Neubauer cam and an optic microscope. The results will be analyzed in statistics form. | ||
| + | <h5><br>Growth under stress curve of C. vulgaris<h/5><br> | ||
| + | Simultaneously it will conduct an inoculation of 10ml of C. vulgaris stock in a liter of Bold Basal with nitrate deficiency. They will be counted daily for two weeks and the results will be compared in statistics form.<br> | ||
| + | <h5><br>Obtaining lipids from C. vulgaris in normal and stress nutritional conditions<h5><br> | ||
| + | Obtaining lipidsBegan with the dehydration of the biomass putting at 104° C for 4 hours, followed by a maceration which will remain in vortex at 500 rpm for 30 min in a solution of HCL 0.5 M. For the separation of the solid biomass.<br> | ||
| + | For the lipid obtaining from the cells they will be centrifuged at 8500 rpm for 5 min and washed with distillated water. The samples will be dried using a lyophilizer, the samples will be pulverized with a mortar and using a mixture of chloroform:methanol (2:1, v/v). They will be used 50ml of solvents for each dried sample. After mixing the sample with a mixer for 5 hours and ultrasonication for 30 minutes, the samples will be centrifuged at 3000 rpm for 10 minutes. The solid phase will be separated using filter paper. The solvent phase will be evaporated in a vacuum evaporator rotatory at 60° C. The process must be reply three times until the lipids are obtained.<br> | ||
| + | <h5><br>Quantification method of obtained lipids in C. vulgaris<h5><br> | ||
| + | The quantification of the lipid extract will be made with the next equations: | ||
| + | WBioPre is the weight of the biomass before the treatment (g), %RecupBIO is the percent of the recuperation in the process before the treatment of the biomass and WReal is the real weight added at the sample (g). | ||
| + | WLip is the weight of the lipid extract and %Extlip is the percentage of the lipids obtained.<br> | ||
| + | The description qualitative and quantitative also is produced with help of a chromatograph of gas mass. | ||
| + | |||
| + | |||
| + | |||
<h5>Modified and developed by Carolina Cruz, Alejandro Alarcón y Franklin Cruz</h5> <br> | <h5>Modified and developed by Carolina Cruz, Alejandro Alarcón y Franklin Cruz</h5> <br> | ||
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To make salts:<br>Add 15.0g Ammonium chloride, 4.0g Magnesium sulphate heptahydrate, and 2.0g Calcium chloride to 1000mL distilled water.<br>To make phosphate solution.<br>Add 28.8g Dipotassium phosphate, and 14.4 g Monopotassium phosphate to 1000mL distilled water.<br>To make trace elements.<br>Add 8.82 Zinc sulfate heptahydrate, 1.44g Magnesium chloride tetrahydrate, 1.57g Copper sulfate pentahydrate to 1000mL distilled water. Autoclave is recquired to dissolve.<br>Important points:<br>If prepare a Growth medium (1000mL), add 2.42g Tris(hydroxymethyl)aminomethane, 25mL TAP, .375mL phosphate solution, 2mL trace elements and 1mL Acetic acid to 1000mL distilled wate. <br>Add 1mL Sulfuric acid to ajust pH.<br>To make a Nutritional stress growth medium add half nitrogen sources.<br>To make a solid growth medium add 15g Bacteriological to 1000mL growth medium. | To make salts:<br>Add 15.0g Ammonium chloride, 4.0g Magnesium sulphate heptahydrate, and 2.0g Calcium chloride to 1000mL distilled water.<br>To make phosphate solution.<br>Add 28.8g Dipotassium phosphate, and 14.4 g Monopotassium phosphate to 1000mL distilled water.<br>To make trace elements.<br>Add 8.82 Zinc sulfate heptahydrate, 1.44g Magnesium chloride tetrahydrate, 1.57g Copper sulfate pentahydrate to 1000mL distilled water. Autoclave is recquired to dissolve.<br>Important points:<br>If prepare a Growth medium (1000mL), add 2.42g Tris(hydroxymethyl)aminomethane, 25mL TAP, .375mL phosphate solution, 2mL trace elements and 1mL Acetic acid to 1000mL distilled wate. <br>Add 1mL Sulfuric acid to ajust pH.<br>To make a Nutritional stress growth medium add half nitrogen sources.<br>To make a solid growth medium add 15g Bacteriological to 1000mL growth medium. | ||
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Revision as of 01:12, 20 October 2016
Method and procedures
Obtaining of unialgal strain of Chlorella vulgaris
The strain will be asked to “Algas continentales: Ecologia y taxonomia” (LACET) (Tercer piso, Edificio A, Facultad de Ciencias Universidad Nacional Autónoma de México, Ciudad Universitaria, Coyoacán, D.F. 0451 0, México).
Growing of C. vulgaris
Prepares 1 L of Bold Basal (BB) and will be inoculated with the strain, in asepsis conditions. Additionally, prepares 1 L of Bold basal with nitrates deficiency to cause nutritional stress. Later they are stored in “Laboratorio de Ambientes controlados” (Edificio B de Biología 3er piso, Facultad de Ciencias Universidad Nacional Autónoma de México Ciudad Universitaria, Coyoacán, D.F. 0451 0, México) at a temperature of 25°C +/- 1°C with light and dark cycles for 12 hours. Scaling 1L at a time according the water requirement of the alga.
Calibration curve of C. vulgaris
They counted 20 µL of inoculation cell’s stock of C. vulgaris daily for two weeks with the help of a Neubauer cam and an optic microscope. The results will be analyzed in statistics form.
Growth under stress curve of C. vulgaris
Simultaneously it will conduct an inoculation of 10ml of C. vulgaris stock in a liter of Bold Basal with nitrate deficiency. They will be counted daily for two weeks and the results will be compared in statistics form.
Obtaining lipids from C. vulgaris in normal and stress nutritional conditions
Obtaining lipidsBegan with the dehydration of the biomass putting at 104° C for 4 hours, followed by a maceration which will remain in vortex at 500 rpm for 30 min in a solution of HCL 0.5 M. For the separation of the solid biomass.
For the lipid obtaining from the cells they will be centrifuged at 8500 rpm for 5 min and washed with distillated water. The samples will be dried using a lyophilizer, the samples will be pulverized with a mortar and using a mixture of chloroform:methanol (2:1, v/v). They will be used 50ml of solvents for each dried sample. After mixing the sample with a mixer for 5 hours and ultrasonication for 30 minutes, the samples will be centrifuged at 3000 rpm for 10 minutes. The solid phase will be separated using filter paper. The solvent phase will be evaporated in a vacuum evaporator rotatory at 60° C. The process must be reply three times until the lipids are obtained.
Quantification method of obtained lipids in C. vulgaris
The quantification of the lipid extract will be made with the next equations:
WBioPre is the weight of the biomass before the treatment (g), %RecupBIO is the percent of the recuperation in the process before the treatment of the biomass and WReal is the real weight added at the sample (g).
WLip is the weight of the lipid extract and %Extlip is the percentage of the lipids obtained.
The description qualitative and quantitative also is produced with help of a chromatograph of gas mass.
Modified and developed by Carolina Cruz, Alejandro Alarcón y Franklin Cruz
Bold Basal Growth Medium
To make A solution:
Add 10g sodium nitrate, 0.03g Magnesium sulphate heptahydrate, 0.1g Sodium chloride, 0.03g Dibasic potassium phosphate, 0.07g Monobasic potassium phosphate and 0.01g Calcium chloride to 400mL distilled water >br>To make trace elementes
Add 8.82g Zinc sulfate heptahydrate, 1.44g Magnesium chloride tetrahydrate and 1.57g Copper sulfate pentahydrate to 1000mL distilled water. Autoclave is recquired to dissolve.
To make B solution
Add 50g Ethylenediaminetetraacetic acid, 31g Potassium Hydroxide and 4.98g Heptahydrate iron sulfate.
Important poinst:
If prepare growth medicum add 10mL A solution, 1ml trace elements and 1mL C solution to 936mL distilled water
Add 1mL Sulfuric acid to ajust pH
To make a Nutritional stress growth medium add half nitrogen sources.
To make a solid growth medium add 15g Bacteriological to 1000mL growth medium.
TAP growth medium
To make salts:
Add 15.0g Ammonium chloride, 4.0g Magnesium sulphate heptahydrate, and 2.0g Calcium chloride to 1000mL distilled water.
To make phosphate solution.
Add 28.8g Dipotassium phosphate, and 14.4 g Monopotassium phosphate to 1000mL distilled water.
To make trace elements.
Add 8.82 Zinc sulfate heptahydrate, 1.44g Magnesium chloride tetrahydrate, 1.57g Copper sulfate pentahydrate to 1000mL distilled water. Autoclave is recquired to dissolve.
Important points:
If prepare a Growth medium (1000mL), add 2.42g Tris(hydroxymethyl)aminomethane, 25mL TAP, .375mL phosphate solution, 2mL trace elements and 1mL Acetic acid to 1000mL distilled wate.
Add 1mL Sulfuric acid to ajust pH.
To make a Nutritional stress growth medium add half nitrogen sources.
To make a solid growth medium add 15g Bacteriological to 1000mL growth medium.
Simultaneously it will conduct an inoculation of 10ml of C. vulgaris stock in a liter of Bold Basal with nitrate deficiency. They will be counted daily for two weeks and the results will be compared in statistics form.
Obtaining lipids from C. vulgaris in normal and stress nutritional conditions
Obtaining lipidsBegan with the dehydration of the biomass putting at 104° C for 4 hours, followed by a maceration which will remain in vortex at 500 rpm for 30 min in a solution of HCL 0.5 M. For the separation of the solid biomass.
For the lipid obtaining from the cells they will be centrifuged at 8500 rpm for 5 min and washed with distillated water. The samples will be dried using a lyophilizer, the samples will be pulverized with a mortar and using a mixture of chloroform:methanol (2:1, v/v). They will be used 50ml of solvents for each dried sample. After mixing the sample with a mixer for 5 hours and ultrasonication for 30 minutes, the samples will be centrifuged at 3000 rpm for 10 minutes. The solid phase will be separated using filter paper. The solvent phase will be evaporated in a vacuum evaporator rotatory at 60° C. The process must be reply three times until the lipids are obtained.
Quantification method of obtained lipids in C. vulgaris
The quantification of the lipid extract will be made with the next equations:
WBioPre is the weight of the biomass before the treatment (g), %RecupBIO is the percent of the recuperation in the process before the treatment of the biomass and WReal is the real weight added at the sample (g).
WLip is the weight of the lipid extract and %Extlip is the percentage of the lipids obtained.
The description qualitative and quantitative also is produced with help of a chromatograph of gas mass.
Modified and developed by Carolina Cruz, Alejandro Alarcón y Franklin Cruz
Bold Basal Growth Medium
To make A solution:
Add 10g sodium nitrate, 0.03g Magnesium sulphate heptahydrate, 0.1g Sodium chloride, 0.03g Dibasic potassium phosphate, 0.07g Monobasic potassium phosphate and 0.01g Calcium chloride to 400mL distilled water >br>To make trace elementes
Add 8.82g Zinc sulfate heptahydrate, 1.44g Magnesium chloride tetrahydrate and 1.57g Copper sulfate pentahydrate to 1000mL distilled water. Autoclave is recquired to dissolve.
To make B solution
Add 50g Ethylenediaminetetraacetic acid, 31g Potassium Hydroxide and 4.98g Heptahydrate iron sulfate.
Important poinst:
If prepare growth medicum add 10mL A solution, 1ml trace elements and 1mL C solution to 936mL distilled water
Add 1mL Sulfuric acid to ajust pH
To make a Nutritional stress growth medium add half nitrogen sources.
To make a solid growth medium add 15g Bacteriological to 1000mL growth medium.
TAP growth medium
To make salts:
Add 15.0g Ammonium chloride, 4.0g Magnesium sulphate heptahydrate, and 2.0g Calcium chloride to 1000mL distilled water.
To make phosphate solution.
Add 28.8g Dipotassium phosphate, and 14.4 g Monopotassium phosphate to 1000mL distilled water.
To make trace elements.
Add 8.82 Zinc sulfate heptahydrate, 1.44g Magnesium chloride tetrahydrate, 1.57g Copper sulfate pentahydrate to 1000mL distilled water. Autoclave is recquired to dissolve.
Important points:
If prepare a Growth medium (1000mL), add 2.42g Tris(hydroxymethyl)aminomethane, 25mL TAP, .375mL phosphate solution, 2mL trace elements and 1mL Acetic acid to 1000mL distilled wate.
Add 1mL Sulfuric acid to ajust pH.
To make a Nutritional stress growth medium add half nitrogen sources.
To make a solid growth medium add 15g Bacteriological to 1000mL growth medium.
Obtaining lipidsBegan with the dehydration of the biomass putting at 104° C for 4 hours, followed by a maceration which will remain in vortex at 500 rpm for 30 min in a solution of HCL 0.5 M. For the separation of the solid biomass.
For the lipid obtaining from the cells they will be centrifuged at 8500 rpm for 5 min and washed with distillated water. The samples will be dried using a lyophilizer, the samples will be pulverized with a mortar and using a mixture of chloroform:methanol (2:1, v/v). They will be used 50ml of solvents for each dried sample. After mixing the sample with a mixer for 5 hours and ultrasonication for 30 minutes, the samples will be centrifuged at 3000 rpm for 10 minutes. The solid phase will be separated using filter paper. The solvent phase will be evaporated in a vacuum evaporator rotatory at 60° C. The process must be reply three times until the lipids are obtained.
Quantification method of obtained lipids in C. vulgaris
The quantification of the lipid extract will be made with the next equations:
WBioPre is the weight of the biomass before the treatment (g), %RecupBIO is the percent of the recuperation in the process before the treatment of the biomass and WReal is the real weight added at the sample (g).
WLip is the weight of the lipid extract and %Extlip is the percentage of the lipids obtained.
The description qualitative and quantitative also is produced with help of a chromatograph of gas mass.
Modified and developed by Carolina Cruz, Alejandro Alarcón y Franklin Cruz
Bold Basal Growth Medium
To make A solution:
Add 10g sodium nitrate, 0.03g Magnesium sulphate heptahydrate, 0.1g Sodium chloride, 0.03g Dibasic potassium phosphate, 0.07g Monobasic potassium phosphate and 0.01g Calcium chloride to 400mL distilled water >br>To make trace elementes
Add 8.82g Zinc sulfate heptahydrate, 1.44g Magnesium chloride tetrahydrate and 1.57g Copper sulfate pentahydrate to 1000mL distilled water. Autoclave is recquired to dissolve.
To make B solution
Add 50g Ethylenediaminetetraacetic acid, 31g Potassium Hydroxide and 4.98g Heptahydrate iron sulfate.
Important poinst:
If prepare growth medicum add 10mL A solution, 1ml trace elements and 1mL C solution to 936mL distilled water
Add 1mL Sulfuric acid to ajust pH
To make a Nutritional stress growth medium add half nitrogen sources.
To make a solid growth medium add 15g Bacteriological to 1000mL growth medium.
TAP growth medium
To make salts:
Add 15.0g Ammonium chloride, 4.0g Magnesium sulphate heptahydrate, and 2.0g Calcium chloride to 1000mL distilled water.
To make phosphate solution.
Add 28.8g Dipotassium phosphate, and 14.4 g Monopotassium phosphate to 1000mL distilled water.
To make trace elements.
Add 8.82 Zinc sulfate heptahydrate, 1.44g Magnesium chloride tetrahydrate, 1.57g Copper sulfate pentahydrate to 1000mL distilled water. Autoclave is recquired to dissolve.
Important points:
If prepare a Growth medium (1000mL), add 2.42g Tris(hydroxymethyl)aminomethane, 25mL TAP, .375mL phosphate solution, 2mL trace elements and 1mL Acetic acid to 1000mL distilled wate.
Add 1mL Sulfuric acid to ajust pH.
To make a Nutritional stress growth medium add half nitrogen sources.
To make a solid growth medium add 15g Bacteriological to 1000mL growth medium.
The quantification of the lipid extract will be made with the next equations: WBioPre is the weight of the biomass before the treatment (g), %RecupBIO is the percent of the recuperation in the process before the treatment of the biomass and WReal is the real weight added at the sample (g). WLip is the weight of the lipid extract and %Extlip is the percentage of the lipids obtained.
The description qualitative and quantitative also is produced with help of a chromatograph of gas mass.
Modified and developed by Carolina Cruz, Alejandro Alarcón y Franklin Cruz
Bold Basal Growth Medium
To make A solution:
Add 10g sodium nitrate, 0.03g Magnesium sulphate heptahydrate, 0.1g Sodium chloride, 0.03g Dibasic potassium phosphate, 0.07g Monobasic potassium phosphate and 0.01g Calcium chloride to 400mL distilled water >br>To make trace elementes
Add 8.82g Zinc sulfate heptahydrate, 1.44g Magnesium chloride tetrahydrate and 1.57g Copper sulfate pentahydrate to 1000mL distilled water. Autoclave is recquired to dissolve.
To make B solution
Add 50g Ethylenediaminetetraacetic acid, 31g Potassium Hydroxide and 4.98g Heptahydrate iron sulfate.
Important poinst: If prepare growth medicum add 10mL A solution, 1ml trace elements and 1mL C solution to 936mL distilled water Add 1mL Sulfuric acid to ajust pH
To make a Nutritional stress growth medium add half nitrogen sources.
To make a solid growth medium add 15g Bacteriological to 1000mL growth medium.
TAP growth medium
To make salts:
Add 15.0g Ammonium chloride, 4.0g Magnesium sulphate heptahydrate, and 2.0g Calcium chloride to 1000mL distilled water.
To make phosphate solution.
Add 28.8g Dipotassium phosphate, and 14.4 g Monopotassium phosphate to 1000mL distilled water.
To make trace elements.
Add 8.82 Zinc sulfate heptahydrate, 1.44g Magnesium chloride tetrahydrate, 1.57g Copper sulfate pentahydrate to 1000mL distilled water. Autoclave is recquired to dissolve.
Important points:
If prepare a Growth medium (1000mL), add 2.42g Tris(hydroxymethyl)aminomethane, 25mL TAP, .375mL phosphate solution, 2mL trace elements and 1mL Acetic acid to 1000mL distilled wate.
Add 1mL Sulfuric acid to ajust pH.
To make a Nutritional stress growth medium add half nitrogen sources.
To make a solid growth medium add 15g Bacteriological to 1000mL growth medium.
