Media and buffers

HL5
FM medium
SM medium
Bonner's salts
DB (Development Buffer)
KK2
Lower Pad Solution (LPS)
PDF (Development buffer)
Upper Pad Solution (LPS)

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• HL-5 (Axenic Medium) (1 liter)  Watts and Ashworth (1970)
  
  • 5 g proteose peptone
  • 5 g Thiotone E peptone
  • 10 g glucose
  • 5 g yeast extract
  • 0.35 g Na₂HPO₄·7H₂O
  • 0.35 g KH₂PO₄
  
  
  Bring to a volume of 1 liter.
  Adjust pH with HCl to pH 6.4 - 6.6.
  Autoclave to sterilize.
  
  Notes:
  pH should be 6.7.
  MES (1.3 g/l) can be substituted for phosphate. This facilitates phosphate labeling.
  HL-5 medium must be stored at 4°C, but can keep one bottle at room temperature for regular use.
  To insure consistent growth rates, subculture Dictyostelium cells using medium warmed at 22°C.
  Some batches of peptone seem to be lacking certain nutrients and results in unusually long doubling time. This problem has been successfully overcome by adding vitamins (from the FM medium recipe).
  Antibiotics (ampicillin, streptomycin) may be added to reduce bacterial contamination.

• FM medium

• SM broth (1 liter)
  Sussmann (1966) in Methods in Cell Physiology, Vol. 2. Edited by D. Prescott, Academic Press, NY pp. 397-410.

• 10 g glucose
• 10 g Oxoid proteose peptone
• 1 g Oxoid yeast extract
• 1 g MgSO₄·7H₂0
• 1.9 g KH₂PO₄
• 0.6 g K₂HPO₄


Adjust pH to 6.0 - 6.4 with KOH.

• SM plates
  
  • Add 20 g/l bacto agar to SM broth.
  
  
  Autoclave.
  Pour thick plates (35 ml per 100 mm Petri dish); 1 liter should give around 30 plates.
  For best results, let plates dry overnight at room temperature and then store at 4°C. If the plates are too dry the bacteria won't grow well.

• SM/2 (1 liter)
  
  • 1 g MgSO₄·7H₂0
  • 1 g Na₂HPO₄
  • 2.2 g KH₂PO₄
  • 5 dextrose
  • 5 bacteriological peptone
  • 0.5 g yeast extract
  • 15 g agar

• Bonner's salts (1 liter)  Bonner (1947)
• 0.6 g NaCl
• 0.75 g KCl
• 0.3 g CaCl₂


Autoclave for long-term storage.

• DB (Development Buffer)
  
  Note: This buffer is lower in ionic strength than PDF. The high ionic strength of PDF seems to retard development. Dictyostelium grows essentially in fresh water in dirt. Therefore this buffer may be more physiological.
  
  
  • 5 mM Na₂HPO₄
  • 5 mM KH₂PO₄
  • 1 mM CaCl₂
  • 2 mM MgCl₂
  
  
  Adjust pH to 6.5.

• 10x KK2 buffer (1 liter)
  
  • 22g KH₂PO₄ (monobasic)
  • 7.0g K₂HPO₄ (dibasic)
  
  
  Autoclave for long-term storage.
  Use at 1x concentration.



• KK2 plates
  
  • To 1x KK2, add 15 g/l of agar
  
  
  Pour about 20 ml/plate.
  Develop 2-4x10⁶ cells/cm², ie can develop up to 2x10⁸ cells/plate.

• Lower Pad Solution (LPS) plates (1 liter)  Sussman 1987
  
  • 1.14 g Na₂HPO₄ (anhydrous)
  • 4.35 g KH₂PO₄
  • 0.5 g KCl
  • 0.5 g MgCl₂
  • 18.0 g agar
  
  
  Autoclave.
  Pour 20 ml/plate.

• PDF (Development buffer) (1 liter)
  
  • 1.5 g KCl
  • 1.6 g K₂HPO₄
  • 1.8 g KH₂PO₄
  
  
  Bring to a volume of 1 liter. pH with KOH to pH 6.4.
  Autoclave to sterilize.
  
  After autoclaving add:
  
  • 1.0 ml 1 M CaCl₂
  • 2.5 ml 1 M MgSO₄

• Upper Pad Solution (UPS)   Sussman 1987
  
• 1 M KH₂PO₄

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