Heat Shock Treatment of Chlamydomonas reinhardtii and Chlorella Cells

Materials and Reagents

1. Species:
   Three Chlorella species were used: C. vulgaris, isolated from soil samples of Livingston Island, the South Shetland Archipelago, Antarctic; C. vulgaris strain 8/1, isolated in 1968 from thermal springs in the region of Rupite, Bulgaria, and cultivated in our laboratory since 1975 and Chlorella kesslery a mesophile, from the Trebon collection.
   Cultivation: Chlorella species were cultivated on Tris Acetate Phosphate (TAP) medium under continuous light of 60 μmol/m²/s and a temperature of 23 °C ± 0.1 °C in a Phytotron GC 400 growth chamber. The species were cultivated at this temperature because it is well known, that eurythermal algae, could be grown at a wide range of temperatures.
   
2. TAP medium (see Recipes)
   
3. Sager–Granick medium (see Recipes)
   

Equipment

1. Phytotron GC 400 growth chamber (NUVE Ankara/Turkey 2009)
   
2. Microscope
   
3. Bürker chamber
   
4. Circulating water bath
   

Procedure

1. Cultivation
   1. Cultivate for 4-5 days algae strains or species on TAP medium (Harris, 1989) under continuous light of 60 μmol/m^2/s and a temperature of 23 °C ± 0.1 °C in a Phytotron GC 400 growth chamber to the end of exponential and early stationary phase of growth.
      
   2. Check under the microscope whether cell culture is not contaminated.
      
   3. Count cell number under the microscope using Bürker chamber.
      
   4. Centrifuge certain volume (depending on the cell density, counted in step A-3) of cell suspension at 1,200 x g by RT, resuspend in Sager-Granick medium or other appropriate medium, so to get 10 ml of cell suspension with a density of 1 x 10⁶ cells/ml for every sample.
      
      
2. Temperature treatment
   1. Keep 10 ml cell culture with a density 1 x 10⁶ cells/ml in an incubator under continuous shaking, at different temperatures:
      t = 39 °C for 30 min
      t = 42 °С for 5 min
      t = 45 °С for 5 min
      Notes:
      
      1. The same heating procedure could be done in a circulating water bath at the same three temperatures: t = 39 °С for 30 min, t = 42 °С for 5 min, and t = 45 °С for 5 min.
         
      2. Use 50 ml flasks to comply with the requirement that volume of the cell culture must be not more than 1/3 of the volume of the flask.
         
   2. Place on ice to stop the heating process.
      
   3. For HSP70B analysis keep cells for 2 and 4 h after the step II-2 at t = 23 °C ± 0.1 °C to allow cells to recover.
      
   4. Centrifuge 10 ml cell suspension at 1,200 x g for 5 min.
      

Recipes

1. TAP medium
   Stock solution for 1 L of TAP media
   

   1 M Tris base	20 ml
   Phosphate Buffer II (see 1-a)	1.0 ml
   Solution A (see 1-b)	10.0 ml
   Hutner's trace elements (see 1-c)	1.0 ml
   Glacial acetic acid (pH to 7.0)	1.0 ml

   1. Phosphate buffer II (Stock solution)
      Component (For 100 ml)
      

      K2HPO4	10.8 g
      KH2PO4	5.6 g

   2. Solution A
      Component (For 500 ml)
      

      NH4Cl	20 g
      MgSO4·7H2O	5.0 g
      CaCl2·2H2O	2.5 g

   3. Hutner's trace elements
      
      1. Dissolve 50 g of acid free EDTA in 250 ml of deionized. Heat to dissolve.
         
      2. Dissolve the following one by one in order. Heating to approximately 100 °C in 500 ml deionized H₂O.
         

         Component	Quantity
         H3BO3	11.4 g
         ZnSO4·7H2O	22.0 g
         MnCl2·4H2O	5.06 g
         FeSO4·7H2O	4.99 g
         CoCl2·6H2O	1.61 g
         CuSO4·5H2O	1.57 g
         Mo7O24(NH4)6·4H2O	1.1 g

      3. Mix the two solutions together. The resulting solution should be blue-green.
         
      4. Heat to 100 °C. Cool slightly, but don't let the temperature drop below 80 °C – 90 °C.
         
      5. Adjust pH to 6.5–6.8 with 20% KOH (approximately 83 ml).
         
2. Sager and Granick medium (adjust pH 6.8-7.0)
   

   Component	In 1 L stock	For 1 L media
   Trace elements*	--	1 ml
   NaCitrate·2H2O	100 g	5 ml
   FeCl3·6H2O	10 g	1 ml
   CaCl2·6H2O	58 g	1 ml
   MgSO4·7H2O	100 g	3 ml
   NH4NO3	100 g	3 ml
   KH2PO4	100 g	1 ml
   K2HPO4	100 g	1 ml

   *Trace Elements
   

   Component	In 1 L stock
   H3BO3	1.0 g
   ZnSO4·7H2O	1.0 g
   MnSO4·H2O	0.303 g
   CoCl2·6H2O	0.2 g
   Na2MoO4·2H2O	0.2 g
   CuSO4·5H2O	0.063 g

References

1. Chankova, S. G., Yurina, N. P., Dimova, E. G., Ermohina, O. V., Oleskina, Y. P., Dimitrova, M. T. and Bryant, P. E. (2009). Pretreatment with heat does not affect double-strand breaks DNA rejoining in Chlamydomonas reinhardtii. J Ther Biol 34(7): 332-336.
   
2. Chankova, S., Mitrovska, Z., Miteva, D., Oleskina, Y. P. and Yurina, N. P. (2013). Heat shock protein HSP70B as a marker for genotype resistance to environmental stress in Chlorella species from contrasting habitats. Gene 516(1): 184-189.