ListServ Archive: Growth in Axenic Medium

Growing Dictyostelium in Axenic Medium

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We are experiencing problems in the last months with axenic cell growth. AX2 cells grow well up to 1 x 10E6 cells/ml and then stop growing or grow very slowly (depending on the batch of peptone). In addition, they accumulate fluorescent red-yellow particles that are neither degraded nor secreted even after 6 hours starvation in phosphate buffer. The problems are not solved by filtering instead of autoclaving the medium. It is possible that there are again problems with new Oxoid peptone batches as there were a few years ago. Do you have the same problems or have you found a solution to them? Do you have alternative peptones to suggest?
-Salvatore Bozzaro, University of Turin, Italy, 24 Oct 2001

  • I want to thank the many people who have answered my message concerning poor growth of AX2 cells and formation of red particles. Here a summary, that may be of help to other labs.
  • Dietmar Manstein suggested to switch to a modified HL medium (HL-C), originally proposed by C. Reymond, which allows growth up to 1-2 x 10E7 cells/ml with a generation time of 7-8 hours. Enthusiastic about this medium are also T. Soldati, R. Graef and Y. Lua. I report the recipe:
    HL-5C:
    • 5 g/liter Protease Peptone (Oxoid L85 or Merck 7229)
    • 2.5 g/liter Casein peptone (Merck 7216)
    • 2.5 g/liter Tryptone Peptone (Difco 0123-17-3)
    • 5 g/liter Yeast Extract (Difco 0127-17-9)
    • 10 g/liter D-glucose (Roth)
    • 1.2 g/liter KH2PO4 (anhydrous)
    • 0.35 g/liter Na2HPO4 (anhydrous)
    • add H20 to 1 liter and adjust pH to 6.5
    • autoclave for 23 min at 121°C and cool rapdily afterwards
    The Merck 7216 casein peptone is out of production, but a substitute is Merck 7213.

  • The Konstanzer (D. Lusche) and Berliner (R. Mutzel) use the classical AX2 medium, but have switched from Oxoid to Difco both for the peptone (Proteose Peptone Nr. 3) and the yeast extract.

  • H. Van der Wel also suggests Proteose Peptone Nr. 2 (Difco).

  • Media supplemented with vitamins, which have solved the problems of slow growth, are suggested by S. Saway (Cox'lab) and R. Insall.

  • According to G. Gerisch, who heard it from M. Titus, Oxoid has changed the production of Peptone in such a way that folic acid is destroyed

  • Saway uses Y. Maeda's HL5 version:
    • 10 g/L special peptone (Oxoid L72)
    • 7 g/L yeast extract (Oxoid L21)
    • 15 g/L glucose
    • 40 ng/ml vitamin B12
    • 80 g/ml folic acid

  • R. Insall adds to previously autoclaved axemic medium (prepared without sugar) a mix made of:
    per L:
    • 280 g glucose
    • 0.4 mg biotin
    • 0.1 mg cyanocobalamin
    • 4 mg folic acid
    • 8 mg lipoic acid
    • 10 mg riboflavin
    • 1 mg thiamine

  • Concerning the red particles, in T. Steck lab's there was the problem years ago but only when large flasks of cells (1-2 l) were grown (we find them also in 30-ml cultures). D. Fuller has found that using Difco yeast technological grade extract (Nr. 288620) helps reducing the fluorescent red particles, that they have also experienced in the past. As for the peptone, the Loomis lab has switched to Peptic peptone B (USB). G. Weeks also had a problem with red particles, which was solved by simply changing the peptone batch (Oxoid) (We were less happy: we tested three different Oxoid batches - and three yeast extract batches: none worked).

  • In summary, it appears that changing the Oxoid peptone with peptones from other companies or using a mixture of peptones (or supplementing with vitamins) helps solving the problems with cell growth. It is possible that also the red particles disappear.

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I was just reading the the pH optimum for axenic Dicty growth is around 5.5. I don't know the original source of this rumour - does anyone else? If it's true, why do we use medium at pH 6.2-6.5? Is there any good reason apart from the historical definition of HL5?
 -Robert Insall, University College London, UK, 1 Aug 1997

  • Where were you reading that the optimum is 5.5? That's hard to believe. If you put cells in starvation buffer at pH 5.5, they are not very happy.
    -Rick Firtel, UCSD, CA, 1 Aug 1997

  • I can't comment on the optimum pH, except to say that the pH of Dicty cultures rises considerably during growth, presumably because the peptone is converted to ammonia (and other amines) and most Dicty media are poorly buffered. Thus, our medium may start at pH 6.4, but growth stops at around pH 8.0. So it is likely that starting with a lower initial pH may give more growth before the culture hits an upper pH limit. This is particularly true when a stationary phase culture (approaching pH 8.0) is diluted into fresh medium - immediately raising the pH of the medium. In any event the pH of a culture will not stay at pH5.5 unless actively maintained there. The strongest buffer in most media is the peptone it self. A standard recipe uses 7 mM phosphate, which would have little buffering capacity below pH 7.0. MES or PIPES buffer is better and allow free calcium.
    -Martin Slade, Macquarie University, Australia, 5 Aug 1997

  • A comment from a non-axenic slimey. We routinely grow nc-4h in sussman's medium [with pH 6.3 sodium/potassium phosphate buffer @ 0.01M (close to "m/60 sorensen's buffer)]. I don't see why this buffer component should not be a standard part of any recipe - even the axenic-supporting media. A long time ago, because i worried about changes the feed-bacteria might make in growth conditions, I measured the pH of "my" medium at various times during suspension growth, all the way to slug formation on the walls of the growth flask. The pH stayed at 6.3-6.4. so - what's the problem of pH maintenance??
    -Jared Rifkin, Queens College, NY, 5 Aug 1997

  • I too prefer Dicty grown on real food. Yes, the pH change growing on bacteria is much less than in axenic medium. I think this is because bacteria are a balanced source of all the components the cells need. In contrast, axenic medium has a highly unbalanced amino acid composition - high in proline and glycine (components of collagen) and low in the essential hydrophobic amino acids. Do the cells spit out the unrequired amino acids or metabolise them to release amines? Regarding the buffer. What is the concentration of free calcium in your 10 mM phosphate?
    -Martin Slade, Macquarie University, Australia, 5 Aug 1997

  • Don't know- never measured it.
    -Jared Rifkin, Queens College, NY, 8 Aug 1997

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Does anyone have tried to set the dicty concentration with OD at 600 nm (or another wavelength)? I´m trying to make some growth curves and the hemocytometer I think is not exact with low concentrations of dicty?
 -Juan J. Vicente, IIB, Madrid, Spain, 21 Dec 2004

  • My impression is that counting is the only way. You can count in triplicate or more to get good #s (and error bars) at low concentration.
    -Janet Smith, Boston Biomedical Research Institute, 21 Dec 2004

  • The sizes of dicty cells vary so much that ODs also give problems.
    -Alan Kimmel, NIH, 21 Dec 2004


  • The hemocytometer is perfectly reliable, but you have to use a freshly cleaned chamber and coverglass, and you may have to count a few full chambers if the concentrations are low. One should count at least 100 or more cells for statistically meaningful results. OD readings have never worked satisfactorily, that's why there are few, if any, coulter-counter protocols for determining cell density. Part of the problem is that there is a wide range of cell sizes, depending on strain, density, medium, etc. It means that it is almost impossible to generate a reliable calibration curve. -Jakob Franke, Columbia University, 21 Dec 2004

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Is there a simple way to determine the approximate concentration of Dicty cells over time while they are still growing in HL-5 media? I would like to take many time points to observe the growth rate. Using a hemocytometer seems cumbersome and inaccurate. Is there a way to directly measure the rate of oxygen consumption or production of waste material?
 -Derek James, Concordia University, Montreal, Canada, 2 Sep 1997

  • I do not think it is cumbersome and inaccurate to use a hemocytometer. If you feel it is cumbersome, there is no way to know growth rate. There are some methods to measure cell growth, which are not so cumbersome (but more cumbersome than hemocytometer) and are accurate. MTT assay is one of popular methods, which measures the activity of a mitochondrian enzyme that is thought to corresponds to cell number. Alamar Blue (Alamar Biosciences) is a useful tool for measurement for cell number (cell growth), that measures the metabolism of cells. You can find some other methods in reagent catalogs. Best wishes.
    -Yuzuru Kubohara, Gunma University, Japan, 3 Sep 1997

  • While the haemocytometer might seem cumbersome and inaccurate it is really neither. In the Dicty world all of us count using this ancient device. In our lab we the technique we uses is as follows: In a 1000 ml flask containing 250 ml HL-5 we inoculate to a density of between 5X10e5 to 1X10e6. We then count every two or three hours until the density reaches stationary when plotted on log paper. Use three log paper if you start at 10e6 and four log if you start with 10e5. To count, initially we do not diluted but simply draw off 10 microliters) for each side of the haemocytometer (20 total). The count is multiplied by 10e4 [10,000]. We count every cell in the large grid. The large grid consists of all of the squares. As you can tell if you plate at 10e5 then you should count about 100 cells. If you count above 300 or below 30 you should adjust your dilutions accordingly. Count both large grids and then average the two counts. As I said earlier, this technique is ancient but very accurate. Other techniques are possible. Just remember measuring O2 consumption for bacteria is easier than doing it for Dicty. Also, you chould consider that whatever measure you use should be established in the literature. If you want to try an alternative measure, such as O2, then you should verify it with an established procedure, so either way the haemocytometer will still need to be employed.
    -Janice Stites, DR Soll Laboratory, University of Iowa, 3 Sep 1997

  • The most accurate method to determine concentration of any cell population in any medium is to count the cells using a hemocytometer. The most common derivative method is to measure absorbance (usually at 550 nm) - see the Beer-Lambert Law for explanation. BUT- this requires a conversion (cell count vs abs) curve which is almost invariably made from hemocytometer counts at various dilutions. other derivative methods (metabolite concentration, oxygen uptake, etc.) are far more variable and prone to too much variation. There are no short cuts to good science!
    -Jared Rifkin, Queens College, NY, 3 Sep 1997

  • The hemocytometer is not ancient- it is merely older that many of the scientists now functioning. On a related subject- there was good science, relevant science, and a great scientific literature before 1983!!
    -Jared Rifkin, Queens College, NY, 3 Sep 1997

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Does anyone know how long that dicty cells attached to the dish in HL5 can stay alive at 4°C?
 -Minghang Zhang, University of Texas, Houston, TX, 12 Jan 2001

  • A couple of weeks (liquid culture on plate/TC flask).
    -Eugenio L. de Hostos, Exelixis Inc. South San Francisco, 12 Jan 2001

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