Legionella colonies can take up to 2 weeks to appear.

First grow in Buffered charcoal yeast extract (BCYE) broth, followed by streaking on BCYE agar. You can also refer to ATCC culture manual and EN 12353 standard.

Growth Conditions as per ATCC:

Medium: ATCC Medium 1099: CYE (Charcoal Yeast Extract) Buffered Medium Temperature: 37°C Atmosphere: 95% Air, 5% CO2

BCYEA with L-cysteine and iron source (as iron pyrophosphate most commonly).

It takes at least 7 days to grow.

If L-cysteine is not added to the media or there is something wrong with it, Legionella will not grow at all. If you used saline or anything with NaCl for the dilution or rehydration of your ATCC vial, Legionella will not grow.

Povilas Kavaliauskas Thank you so much. I used 1X PBS to rehydrate. It makes sense now.

Povilas Kavaliauskas For making dilution, which one is the best? As PBS has NaCl.

Povilas Kavaliauskas is TSB with 10% glycerol good broth for making the Legionella stock?

Mohammad Siddiqur Rahman Khan you might want to check with your repository the best way to rehydrate your sample. I generally use sterile deionized water since the lyophilization media already has a bunch of stuff inside.

We do a lot of work on Legionella both in vitro and invivo and we are always avoiding any sodium since it is interfering with growth.

For making dilutions of Legionella, I would suggest using deionized water. It works well in my lab without loss of virulence in Legionella.

For storing stocks, we use mikrobank beads from Hardy Diagnostics or YEB (legionella media without charcoal and agar) supplemented with L-cysteine and iron pyrophosphate, 20% glycerol. We scrape colonies and suspend them in a freezing media (1 ml per tube). then just dropping tubes to -80.

Good luck,

P.

Povilas Kavaliauskas thank you so much.

Povilas Kavaliauskas Hi, here I'm again looking for your expert opinion.

I want to prepare Ferric pyrophosphate (25%) but it's not water-soluble. Which one is the best to use to prepare the solution? I'm going to use it for legionella media preparation. Thanks in advance.

Mohammad Siddiqur Rahman Khan Hi,

Actually, Iron pyrophosphate is soluble in water, although it is pain in the ass to dissolve it. It is important to note, that it is not recommended to make it as a stock solution and then re-add it to the media since it is unstable and prone to oxidation. Moreover, Fe+2 is stable in BCYEA due to its complex with L-cysteine, therefore both additives are best added fresh each time.

The way we do is to weigh the calculated amount of FePP and L-cysteine (we usually prepare 500 ml of BCYEA media) into separate tubes and dissolve them in 5 mL of DI sterile water. Then right before adding them to the media, we mix those 2 solutions together in 20 mL syringe with 0.22 um membrane filter (the color will change temporarily) and filter directly FePP/L-cysteine into the sterile media called down to 55C.

Sometimes FePP is acting up so gentle warming in a microwave (5 s cycle) helps to dissolve it without loss of activity.

Give it a swirl and pour it to the plates. If protected from light, the media is quite stable. Alternatively, you can use commercial vials of Legionella supplement (Oxoid™ Legionella BCYE Supplement (thermofisher.com).

Best,

Povilas

Povilas Kavaliauskas Thank you so much. I'll give it another try. It mixed well in 1 N HCl. But may it's not ideal to use HCl.

I'm trying to make "Modified gonococcal-ferric cysteine broth (without sodium chloride) supplemented with 1% yeast extract". Is there any chance you use broth-based media if you have the SOP for that preparation? I got one from this paper. DOI: 10.1128/jcm.14.6.623-627.1981

Mohammad Siddiqur Rahman Khan yes, you might want to be careful of using HCl, although it might still work.

No, unfortunately, I have never used this media.

I tried to make the ferric pyrophosphate solution using your technique. but still, it's not soluble in water. Could you please share the catalog number of the ferric pyrophosphate that you use in your lab? Thanks in advance.

Povilas Kavaliauskas I tried to make the ferric pyrophosphate solution using your technique. but still, it's not soluble in water. Could you please share the catalog number of the ferric pyrophosphate that you use in your lab? Thanks in advance.