Hello together,
I am trying to reactivate A. salmonicida ssp. salmonicida from a bead based kryo system for bacteria. I initially tried to move the bead directly to the Tryptic Soy Agar (TSA, at 20°C) and Tryptic Soy Broth (TSB, 100ml, 20°C, 180rpm), but I had no growth. I used TSB from SIGMA-ALDRICH and used the recommended 30g / 1L.
Having no succeess with TSA/B, I used Columbia Blood Agar to reactivate them. That turned out to work, so the bacteria are still viable...
After that I tried to move them from the blood Agar to the TSA / TSB, again I have poor growth (TSB growth stagnated at a OD600 of 1) compared to a Blood Agar control.
Does anyone of you has experience with growing A. salmonicida on TSA/TSB, should I use double strength media to achieve satisfying growth? I need it to collect a defined number of cells for DNA isolation.
Thank you
Manuel
Hello Manuel,
After inoculating your beads into TSB, Incubate at 20°C for (48-72 hours). The growth you have on this tube are not fully resuscitated. So, follow that by one or two more successive subculture using fresh TSB and incubate (24-72 hours) then measure the OD & Continue your experiment.
All the best👍
Some animal-associated bacteria have trouble growing on media that do not contain animal products like peptone or beef extract. I suspect it'll grow on a simple Nutrient Agar https://en.wikipedia.org/wiki/Nutrient_agar
Double-strength media are likely to result in scanty growth due to the osmotic pressure.
You can try any general purpose media broth supplemented with serum for primary recovery followed by an agar-based general media (with/without serum).
Hello
Mohamed Kishk, Jakub Grzesiak and Jameel M. Abduljalil thank you for your answers.
I did another passage on solid media, TSA seems not to be a good media in my case, I am successful with Columbia Agar without blood and Columbia Blood Agar and picked a early growing colony of the second passage to a higher concenctratet TSB.
It works now.
Thanks for your help!
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