Induction, Isolation and Counting of Akinetes in Aphanizomenon ovalisporum

Materials and Reagents

1. Erlenmeyer flasks of appropriate volume
   
2. 50 ml tubes (SARSTEDT AG & Co, catalog number: 62.547.004 )
   
3. 250 ml centrifuge bottle (TermoFisher SCIENTIFIC, catalog number: 3141-0250 )
   
4. Aphanizomenon ovalisporum, strain ILC-164 [isolated from Lake Kinneret, Israel (Banker et al., 1997)]
   
5. BG11 growth medium (Stanier et al., 1971)
   
6. BG/-K akinete induction medium
   Note: BG11 medium in which the K₂HPO₄ component was substituted with Na₂HPO₄.
   
7. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
   
8. Ethylenediaminetetraacetic acid disodium salt dehydrate (Na-EDTA) (Sigma-Aldrich, catalog number: E5134 )
   
9. Magnesium chloride (MgCl₂) (Sigma-Aldrich, catalog number: M8266 )
   
10. Lysozyme from chicken egg white (Sigma-Aldrich, catalog number: L6876 )
    Note: It is also named as “Mucopeptide N-acetylmuramoylhydrolase” or “muramidase”.
    
11. Lugol’s solution (Sigma-Aldrich, catalog number: L6146 )
    
12. SYTOX (Invitrogen, catalog number: S7020 )
    Note: Currently, it is “SYTOX^® Green Nucleic Acid Stain-5 mM Solution in DMSO” (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: S7020).
    
13. Coomassie brilliant blue R-250 staining solution (BIO RAD catalog number: 1610436 ).
    
14. Molecular weight markers [All blue pre-stained protein standards (M. W. 10-250 kD)] (Bio-Rad Laboratories, catalog number: 1610373 )
    
15. Tris (Sigma-Aldrich, catalog number: 252859 )
    Note: It is also named as “Tris (hydroxymethyl) aminomethane” on Sigma-Aldrich website.
    
16. Tris buffer(see Recipes)
    
17. Tris-EDTA-Mg buffer (see Recipes)
    
18. Tris-Mg buffer (see Recipes)
    
19. TE buffer (see Recipes)
    
20. 0.5 M EDTA stock solution (see Recipes)
    
21. 1 M MgCl₂ stock solute (see Recipes)
    
22. 1 M NaCl (see Recipes)
    

Equipment

1. Spectrophotometer (such as Uvikon XS SECOMAM)
   
2. Sorvall centrifuge RC 6 Plus and appropriate rotor for the centrifuge tubes or bottles
   
3. Vortex
   
4. Sonicator [e.g., Sonifier 450 (Branson Ultrasonics)]
   
5. Orbital shaker (e.g., MaxQ^TM 2000 and 3000 benchtop orbital shaker, TermoFisher SCIENTIFIC, model number: SHKA2000 )
   
6. Incubator (regulated temperature and light)
   
7. Bright field/fluorescent inverted microscope (e.g., Zeiss Axioobserver Z1)
   Note: Use the following filter sets: for chlorophyll (EX-425-443 nm; BS-452 nm; EM-496 nm LP), for Phycobilins (EX-510-550 nm; BS-565 nm; EM-582 nm LP) and for SYTOX (EX-445-495 nm; BS-500 nm; EM-505-555 nm).
   
8. Utermohl sedimentation chamber (Aquatic Research Instruments, http://www.aquaticresearch.com/sedimentation_chamber.htm)
   
9. Thermostatic water bath
   

Procedure