Media and buffers

Media and buffers

Media

Buffers and Solutions


Use deionized (or distilled) water to prepare all media and solutions.


  • HL5 (Axenic Medium) (1 liter)
    • 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

    • 14.3 g bacto peptone [Oxoid L37 (Fisher Sci. LP0037B)]
    • 7.15 g yeast extract [Sigma Y-1625 or Difco 0127-17-9]
    • 18 g Maltose monohydrate [Sigma M-5895]
    • 0.641 g Na2HPO4∗2H2O [Merck 1.06580]
    • 0.490 g KH2PO4 [Fluka 60220]

  • pH should be 6.6-6.7

    Notes:
    If adding antibiotics to prevent bacterial contamination, refer to 'Notes' for HL5 (see above).

    This recipe was provided by Anna Marchetti / P. Cosson lab.

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  • Maltose HL5 (1 liter)

    • 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)

    • 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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  • SM broth (1 liter)Stock Center Recipe


    • 10 g glucose
    • 10 g proteose peptone
    • 1 g yeast extract
    • 1 g MgSO4∗7H2O (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
    • 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∗7H2O
    • 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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  • GYP agar (Glucose, Yeast, Peptone, 1 liter) Stock Center Recipe

    • 1 g glucose
    • 2 g bacto peptone
    • 0.25 g yeast extract
    • 5.1 g Na2HPO4∗7 H2O
    • 4.2 g KH2PO4
    • 20 g agar

    Autoclave 20 minutes.
    Reference: Fey et al. (1995).


  • LPB agar (buffered Lactose/Peptone, 1 liter) Stock Center Recipe

    • 1 g lactose
    • 1 g bacto peptone
    • 5.1 g Na2HPO4∗7 H2O
    • 4.2 g KH2PO4
    • 20 g agar

    Autoclave 20 minutes.
    Reference: Fey et al. (1995).


  • LP agar (1 liter) (also called 0.1LP agar)

    • 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)Stock Center Recipe

    • 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 33 mg 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)

    • 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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