I am culturing N. europaea ATCC25978 in minimal media with phenol red pH indicator, wrapped in foil at 30degC. I supplement the media with more sodium carbonate when the media turns yellow/acidifies. I usually sub culture once every two weeks (or when the culture stops turning yellow within 2 days after adding more carbonate), or start a new culture from a frozen stock (stored at -80degC in 5% DMSO). This was working just fine for about a year, and then suddenly, the cultures became non-viable. Even the frozen stocks would not grow again. I waited 9 weeks for a sign of viability - nada.
I had re-ordered the strain from the culture collection, and it looks like I may have lost viability after only two sub-cultures. I assess viability by the media acidifying, and confirm by measuring nitrite production. Are these cultures dead? Inactive? What could I do to revive/reactivate these cultures? And what could have happened that suddenly killed them off?
Hi Celeste,
Honestly, N. europaea is a very capricious organism to deal with in the lab. I don't know the exact components of your minimal medium, but there is a comprehensive medium for the growth and maintenance of N. europeaea. Usually, the recipe is included in the product passport when you purchase the cells from ATCC. When storing the cells, you can freeze them using any suitable cryoprotectant but it should contain about 10% inoculum of cells in this medium. The medium can be purchased but you could also make it in the lab if all ingredients are available. If you would like to prepare it yourself, here are the components, but please avoid autoclaving as it can lead to unwanted precipitates:
Nitrosomonas europaea medium Solution 1: (NH4)2SO4 (for 50 mM NH4+) ................................................. 4.95 g KH2PO4 ................................................................................... 0.62 g MgSO4 . 7H2O ......................................................................... 0.27 g CaCl2 . 2H2O ........................................................................... 0.04 g FeSO4 (30 mM in 50 mM EDTA at pH 7.0) ........................... 0.5 ml CuSO4 . 5H2O.......................................................................... 0.2 mg Distilled water ......................................................................... 1.2 L Filter sterilize.
Solution 2: KH2PO4 ................................................................................... 8.2 g NaH2PO4.................................................................................. 0.7 g Distilled water ..................................................................... 300.0 ml Bring to pH 8.0 with 10N NaOH. Filter sterilize.
Solution 3 (buffer): Na2CO3 anhydrous................................................................... 0.6 g Distilled water ....................................................................... 12.0 ml Filter sterilize.
Complete medium: Combine Solutions 1, 2, and 3. Dispense aseptically into desired aliquots.
Keep the culture on a shaker or with aeration. Cover the culture flask with black cloth bcz light can inhibit the ammonia oxidation. Check the consumption of substrate (ammonia) and product (nitrite)
Many thanks, V.J Rejish Kumar and Yaw Akosah, for your replies. I tried your suggestions, but my cultures were still nonviable. However, I was able to solve the problem eventually, and I just thought I'd update in case anyone else comes across this problem: the glassware.
Heterotrophic bacteria had been growing fine throughout this ordeal, but I noticed a footnote on ATCC website stated that Nitrosomonas was sensitive to impurities on glassware. There must have been some impurities (from other users? the washing process?), as I was able to culture Nitrosomonas within a week in sterile, virgin plastic. Now, I rinse each piece of glassware for media and culturing with distilled water several times before use, and I haven't had issues since.
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