I manage to cultivate L.pneumophila in BCYE agar plates, fut from the plates I innoculate a yeast extract liquid medium and after some days and see no growth at all. I cultivate the Medium+L.pneumophila at 35°C, with an oxygen concentration between 4.5 and 6 mg/l. However, I dont get the desired growth. Any experience with the growth of Legionella pneumophila in liquid medium?
Thanks.
Dear
I am going to recommend you some articles.
I hope it coulb be helpful.
Marius
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC84555/
https://www.ncbi.nlm.nih.gov/pubmed/20571163
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC273049/pdf/jcm00188-0169.pdf
Dear cynthia
Legionella need only rich medium to grow like nutrient agar or any rich media that use for gram negative bacteria
Cynthia,
remember, Legionella is auxothrophic for L-Cysteine (see BCYE and GVPC-media). You have to add some as a minimum condition, to get apperent growth.
Thanks for your messages.
Hanning, I use a medium with 10 g of yeast extract, 0.4 g of L-cysteine-HCl.H20,
0.25 g of ferric pyrophosphate in 1,000 ml of distilled water. Still I dont get Legionella´s growth. Do you have experience with Legionella´s growth in liquid medium?
Hello- Do not autoclave the broth, You have to filter sterilize it. Free radicals produced during autoclaving inhibit growth of the Legionella. The charcoal in the agar absorbs that, but is absent from the broth. Also, the amount of Legionella is important. Unlike E coli, just adding a colony to a flask will not promote growth. They are density dependent, so you have to add enough bacteria until the culture looks cloudy or has some visible turbidity. I have grown many, many of these cultures and your ingredient list looks fine. indeed, the iron can even be omitted but the cysteine is very important.
Hi, thank you very much for your answer. We do not autoclava the medium, we filter it sterilize as you mentioned. For the innoculation, we take several colonies, until we get a visible turbidity (McFarland ƛ 0.08), and from this we innoculate 2 ml in 30 ml Liquid Medium . The bottles are kept at 35 °C with enough oxygen concentration. After 3 days the Legionella pneumophila concentration decreases considerably. What Legionella specie do you use in liquid growth? is there any other suggestion you can give us?
Do you mean that you are agitating the cultures when you say they have enough oxygen? Certainly they need to be well mixed to maintain enough oxygen. I would try just growing the McFarland 0.0 directly, and not perform the 2 ml into 30 ml dilution. Make 30 ml with the visible turbidity. You may be making it too dilute to grow.
We keep the liquid medium in Erlenmeyer flasks, which are constantly agitated at 110 rpm. We have oxygen micro optical sensors in the Erlenmeyer flasks, at the begining just after the inoculation, the dissolved oxygen concentration decreases to 2 mg/L but after some hours it stabilizes at 4-6 mg/L. Which McFarland value will you suggest if we inoculate directly in the medium?
You could try a range of McFarland- we used a spectrophotometer and OD 600 of 0.3. I think it's about 0.5 McFarland? maybe closer to 1.0 McFarland?
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