Difference between revisions of "Team:Pumas Mexico/Experiments"

Method and procedures

Growing of C. vulgaris

Calibration curve of C. vulgaris

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.