Media and buffers

Media

HL5 Axenic Medium
Maltose-HL5 Medium
MES-HL5 Medium
FM Defined Medium
SM
SM plates
SM/2 plates
SM/5 plates
LP agar
LP/2 agar
2% agar plates

Buffers and Solutions

Bonner's salts
Sor (Sörensen's buffer)
SorC (Sor with 50 µM Ca++)
DB (Development Buffer)
KK2
PDF (Development buffer)
LPS (Lower Pad Solution)
UPS (Upper Pad Solution)
1mM cAMP solution
2 x HBS

• HL5 (Axenic Medium) (1 liter) ^{Stock Center Recipe}
  
  
  • 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
  • 0.05 g dihydrostreptomycin-sulfate
  
  Bring to a volume of 1 liter with distilled water
  Adjust pH with HCl to pH 6.4 - 6.7
  Autoclave for 20 minutes to sterilize
  
  
Reference : Watts and Ashworth (1970)

Notes:

1. HL5 medium must be stored at 4°C, but one bottle can be kept at room temperature for regular use.
2. To insure consistent growth rates, use medium warmed to 22°C.
3. Dihydrostreptomycin-sulfate is stored in the refrigerator. Warm up to room temperature before opening the bottle.
4. Antibiotics are added to reduce bacterial contamination. Instead of Dihydrostreptomycin-sulfate, which is added as a powder before autoclaving, antibiotics such as ampicillin (100 μg/ml) and/or streptomycin sulfate (300 μg/ml) may be added after sterilization; stock solutions: 100 mg/ml ampicillin and 300 mg/ml streptomycin sulfate in distilled water, filter sterilize and freeze in small aliquots; use 1/1000 dilution in HL5.
5. Recommendations: proteose peptone 2 (Difco #212120); thiotone E peptone (BBL #212302); yeast extract (Oxoid #LP0021). 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).

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• Maltose HL5 (1 liter)^{Stock Center Recipe}
  
  
  • 20 g maltose
  • 5 g yeast extract
  • 5 g proteose peptone
  • 5 g thiotone E peptone
  • 0.67 g Na₂HPO₄.7H₂O
  • 0.34 g KH₂PO₄
  • 0.05 g dihydrostreptomycin-sulfate

pH should be 6.4-6.5

Notes:

Refer to 'Notes' for HL5 (see above).

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• MES-HL5 (1 liter)^{Stock Center Recipe}
  
  
  • 10 g glucose
  • 5 g yeast extract
  • 5 g proteose peptone
  • 5 g thiotone E peptone
  • 1.3 g MES
  • 0.05 g dihydrostreptomycin-sulfate

Adjust the pH to 7.1 with NaOH (important!)
Autoclave for 20 minutes

Notes:

1. This medium is used for phosphate labeling.
2. Refer to 'Notes' for HL5 (see above).

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• FM Defined Medium^{Stock Center Recipe}

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• SM broth (1 liter)^{Stock Center Recipe}



• 10 g glucose
• 10 g proteose peptone
• 1 g yeast extract
• 1 g MgSO₄·7H₂0 (or 0.5 g MgSO₄)
• 1.9 g KH₂PO₄
• 0.6 g K₂HPO₄


Adjust pH to 6.0 - 6.4 with KOH

Reference: Sussmann (1966) in Methods in Cell Physiology, Vol. 2. Edited by D. Prescott, Academic Press, NY pp. 397-410.

• SM plates^{Stock Center Recipe}
  
  
  • Add 20 g bacto agar to 1 liter SM broth.
  • Adjust the pH to 6.5 ± 0.1; if too low, use 1 N KOH, if too high, use 1 N HCl
  • Autoclave for 30 minutes
  • Let cool for about 30 minutes or longer in a 55°C water bath and pour plates
  
  Notes:
  
  1. Pour thick plates (35 ml per 100 mm Petri dish); 1 liter should yield around 30 plates.
  2. Dry plates overnight at room temperature and then store at 4°C. If the plates are too dry the bacteria won't grow well.
  
  

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

Adjust pH to 6.5 ± 0.1 and pour plates as described for SM plates

• SM/5 plates (1 liter) ^{Stock Center Recipe}
  
  
  • 2 g glucose
  • 2 g bacto peptone
  • 0.2 g yeast extract
  • 0.1 g MgSO₄ (or 0.2 g MgSO₄.7H₂O)
  • 1.9 g KH₂PO₄
  • 1.0 g K₂HPO₄
  • 15 g agar

Adjust pH to 6.5 ± 0.1 and pour plates as described for SM plates

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• LP agar (1 liter) (also called 0.1LP agar)^{Stock Center Recipe}
  
  
  • 1 g lactose
  • 1 g bacto peptone
  • 15 g agar
  
  Autoclave 20 minutes.

• LP/2 agar (1 liter) (also called 0.1LP/2 agar)
  
  
  • 0.5 g lactose
  • 0.5 g bacto peptone
  • 15 g agar
  
  Autoclave 20 minutes.

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• 2% agar plates (1 liter) (also called non-nutrient agar)
  
  
  Add 20 g liter^-1 bacto agar to distilled water and autoclave. Pour approximately 20 ml per 100 mm plate.

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• Bonner's salts (1 liter) ^{Stock Center Recipe}
  
  
  • 0.6 g NaCl
  • 0.75 g KCl
  • 0.3 g CaCl₂
  
  
  Autoclave for long-term storage.
  
  Reference: Bonner (1947)

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• Sor (Sörensen's buffer; 4 liters)^{Stock Center Recipe}
  
  
  • 8.0 g KH₂PO₄
  • 1.16 g Na₂HPO₄ (or 2.2 g Na₂HPO₄.7H₂O)

pH should be 6.0 ± 0.1

Reference: Gerisch, G; Lüderitz, O; Ruschmann, E. Antikörper fördern die Phagozytose von Bakterien durch Amöben. Zsch Naturforschung. 1967;22b:109. (PMID: 4384821).

• SorC (Sor with 50 µM Ca++)
  
  
  • Add 4 ml 50 mM CaCl₂ (or 0.2 ml 1 M CaCl₂) to 4 liters Sor, and autoclave.

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• DB (Development Buffer)
  
  
  • 5 mM Na₂HPO₄
  • 5 mM KH₂PO₄
  • 1 mM CaCl₂
  • 2 mM MgCl₂
  
  
  Adjust pH to 6.5.


Notes:

1. Prepare the phosphate solution as 25mM (5x), adjust the pH to 6.5 and autoclave.
2. Make 10x CaCl₂ (10 mM) and MgCl₂ (20 mM) solutions each separately and autoclave.
3. To make 1 liter of DB, mix 600 ml of distilled, autoclaved water with 200 ml of 5x phosphate solution and 100 ml of 10x CaCl₂ and 10x MgCl₂ solution, respectively.
4. 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 and dirt. Therefore this buffer may be more physiological.

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• 10x KK2 buffer (1 liter)
  
  
  • 22 g KH₂PO₄ (monobasic)
  • 7.0 g K₂HPO₄ (dibasic)
  
  
  Notes:
  
  1. Autoclave for long-term storage.
  2. Use at 1x concentration ( dilute 1:10 with deinodized water).
  
  
  
• KK2 plates
  
  
  • To 1 liter of 1x KK2, add 15 g agar
  
  
  Notes:
  
  1. Pour about 20ml/plate.
  2. Develop 2-4x10⁶ cells/cm², ie up to 2x10⁸ cells/plate.

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

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• LPS (Lower Pad Solution) plates (1 liter)



• 1.14 g Na₂HPO₄ (anhydrous)
• 4.35 g KH₂PO₄
• 0.5 g KCl
• 0.5 g MgCl₂
• 18 g agar


Autoclave.
Pour 20 ml/plate.

Reference: Sussman 1987

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• UPS (Upper Pad Solution)



• 1 M KH₂PO₄

Reference: Sussman 1987

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• 1mM cAMP solution
  
  
  • Dissolve 330mg cAMP (cat. no. A9501; Sigma-Aldrich) in 100 ml DB
  • Adjust pH to 6.5 with NaOH and filter-sterilize
  • Store in 5 ml aliquots at -20°C

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• 2 x HBS (250 ml)^{Stock Center Recipe}
  
  
  • 4 g NaCl
  • 0.18 g KCl
  • 0.05 g Na₂HPO₄ (or 0.094 g Na₂HPO₄.7H₂O)
  • 0.5 g glucose
  • 2.5 g HEPES

Notes:

1. Adjust pH to 7.0-7.1 with NaOH. Very important!
2. Filter sterilize.
3. Keep frozen in 50 ml aliquots

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