Yes I also recommend one of the well described axenic media: PYG (Chang) or Jenssen medium for the growth of Ac. But why not use agar-agar plates seeded with bacteria? It is not axenic but such a culture reflects "reality" certainly more than the axenic cultures wher things like "cannibalism" seem to happen: cysts are phagocytosed by the trophozoites. All together the xenic culture will be easier...

Hi

I saw that in the medium that you have used there isn't the glucose. I normally use for the growth of Acanthamoebae the PYG (Peptone, Yeast Extract and Glucose) and I have always a very good growth.

Yes I also recommend one of the well described axenic media: PYG (Chang) or Jenssen medium for the growth of Ac. But why not use agar-agar plates seeded with bacteria? It is not axenic but such a culture reflects "reality" certainly more than the axenic cultures wher things like "cannibalism" seem to happen: cysts are phagocytosed by the trophozoites. All together the xenic culture will be easier...

Hi Gaby, 

in our lab we grow Acanthamoeba cultures in PYG (Proteose peptone, Yeast extract, Glucose) medium, as you described it. We incubate at 30°C, in incubator (without light), and without shaking. We manage to get confluent monolayers in about 4 days. I advise you to change the medium every 4 or 5 days, and maybe try to grow them without shaking, as they are adherent cells. 

Also, did you use a treatment like trypsin or saponin to detach amoebae? they don't deal pretty well with this kind of treatment. For detachment of amoebal cells, we usually simply tap the flasks.

did you check their morphology by microscopy before your subculture step? As Acanthamoeba encyst when starved or stressed, it takes more times for the culture to grow if you start from encysted amoebae.

Finally, you didn't mention the presence of glucose in your culture medium. I believe it is necessary to add glucose to your medium for optimal growth of Acanthamoeba in axenic liquid culture. You can check the composition of PYG medium on internet (ATCC medium712)

Hope that'll help!

Good luck for your work

Vincent

Hi Gaby,

I have very good experiences with PYG medium according to Jensen (Jensen, T., W.G. Barnes, and D. Meyers, Axenic Cultivation of Large Populations of Acanthamoeba castellanii (JBM). J Parasitol, 1970. 56(5): p. 904-906) for fast cultivation of acanthamoebae. I obtain a monolayer after 2 days cultivation by doing incubation of 1ml culture in 5ml fresh medium in a T25 flask (24°C).

But maybe your problem could be, that in 2ml culture are not enough cells for getting a stable culture in a T100 flask. I experienced that acanthamoebae "like" to be very close to each other at the start of incubation. So try to increase the cell number for inoculation of T100 flask.

Hi Gaby,

We do it as Vincent, but I wanted to warn you that if you use environmental samples, not all Acanthamoeba are able to grow in axenic medium. In fact, at some point   possibility of considering their ability to grow as a virulence factor was suggested. Those from keratitis usually grow well.

Hi Gaby!

We also growth the Acanthamoeba in PYG. We usually add the glucose right before inoculating the amoeba. As Vincent Delafont, we dont shake them at all. We let them growth in monolayers. If they don't attach due to the shaking, I dont think they will growth well. We are using flasks that are tissue-treated. Besides the glucose and the shaking, I think that all the others steps you are doing are ok.

I hope you manage to solve your problem. Good luck!

Sarah

I also use PYG medium for axenic culture of Acanthamoeba Neff strain. The culture is incubated on a rotary shaker with an acentric rotation of 3 cm (120 rpm/

min) at 28 C. Sometimes we lower rpm value to 110 since amoeba cells are very gentle and can be disrupted at higher values. But i've noticed that one of AK strain in my collection prefer satic culturing without shaking.

Hi,

according to my expertise, the PYG medium always works for culturing most of the Acanthamoeba strains, but I have never used the shaking culture. 30°C seems to me a bit high, once I did a growth curve and I obtained generation time ca. 10 hours, optimal conditions 25°C and 0,5% CO2; you can obtain your monolayer within 1-2 days. With most of the amoeba I would not go higher than 25°C. The other thing is, that it may sound funny but some amoeba don´t like to grow alone, so I would not dilute the culture too much. For the subculturing I always use a sterile cell scraper, you don´t have to be afraid of destroying the cells.

Good luck !

Silvia

Dear Gaby,

I could suggest you to try also with plates of Non Nutrient Agar (NNA) and a layer of E.coli. Acantamoeba grows very well on this culture medium and you can observe the trophozoites and their characteristic cysts.

Hi Gaby,

It sounds like you have them growing well in T25's.  I concur on the glucose (or Dextrose) previously mentioned.  I have always supplemented medium with Calf Serum and Bovine hemin as well, and also use Liver Digest, per Balamuth's medium.  See John and Howard 1996, and John 1993.

Also, for agitated culture, check out this article, though it deals with Naegleria.  I also concur with comments regarding the required attachment of the Acanthamoeba.  You may want to simply move up to a T75 or T150 if you need larger volumes. 

Agitated Mass Cultivation of Naegleria fowleri

R. R. Weik and D. T. John

The Journal of Parasitology

Vol. 63, No. 5 (Oct., 1977), pp. 868-871

Best of luck!

Marsha

In summary, you just need to stop shaking the flasks ( because Acanthamoeba need to be attached to the surface in order to feed) and add some low molecular weight sugar (glucose, or dextrose) and your culture shall grow well 

Hey Gaby,

Use bactoagar medium with E.coli. I have successfully cultured it already. E. coli is necessary for it's growth.

AB

Hi Gaby,

I recommend you stop shaking Acanthamoeba cultures and try to grow with this conditions: 10 g yeast extract, 5 g D-glucose, 10 g protease peptone, 5 g NaCl, 3.57 mg Na2HPO4, 3.45 mg KH2PO4. In our lab, we supplement this medium with 2% Bactocasitone and our cultures grow well.

Hope it helps!

Irene