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DSMZ

DSMZ 1500 . ACANTHAMOEBA MEDIUM

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Equipment needed: Autoclave Filter
Final pH: 6.5
Select a strain (optional):

ml
1 Media for maintenance of infected amoeba cultures
Compound Amount Unit
Trypticase Soy Broth with Yeast Extract (TSY) 1000 ml
2 OR
Peptone-yeast-glucose medium (PYG) 1000 ml

Trypticase Soy Broth with Yeast Extract (TSY) #

1000
Compound Amount Unit
Trypticase soy broth 30 g
Yeast extract 10 g
Distilled water 1000 ml
1 Adjust pH to 7.3, autoclave at 121°C

Peptone-yeast-glucose medium (PYG) #

1000
Compound Amount Unit
Proteose peptone 20.000 g
Glucose 18.000 g
Yeast extract 2.000 g
Sodium citrate dihydrate 1.000 g
MgSO4 x 7 H2O 0.980 g
Na2HPO4 x 7 H2O 0.355 g
KH2PO4 0.340 g
Fe(NH4)2(SO4)2 x 6 H2O 0.020 g
Distilled water 1000.000 ml
1 Adjust pH to 6.5, autoclave at 110°C or filter-sterilize

Maintenance conditions #

1 Infected amoeba cultures can be maintained e.g. in 25 cm2 flasks (e.g. Nunc flasks, VWR, 734-2081, Thermo-Scientific, 156340) at 20-30°C in an incubator (no special requirements concerning CO2 or light).

Maintenance/Passage of infected amoeba cultures #

1 Infected Acanthamoeba cultures don't have to be transferred to fresh culture flasks as frequently as usually done for mammalian cell cultures. Amoeba cultures can be maintained in the same flask for several weeks or months. However, if the cultures reach a high cell density, medium should be exchanged. For that purpose amoebae are detached from the surface of the flask by knocking at the culture flask (no trypsin required, amoebae are only weakly adherent compared to most mammalian cells) and the medium (containing the majority of cells) is then completely removed. Fresh medium is added and the few remaining attached amoebae will continue to grow.
2 Alternatively, cultures can also be passaged regularly (e.g. weekly) by transfer of a small aliquot of a densely grown culture (after cell detachment, as described above) to a fresh flask containing medium.
3 If cultures contain few attached cells, but high amounts of cell debris, medium should be exchanged without prior cell detachment. Cultures usually recover fast.
4 Depending on the host cell strain, the bacteria may cause host cell lysis. To maintain such cultures, bacteria have to be transferred by adding culture supernatant (approximately 0.5 ml) to fresh culture flasks containing uninfected host cells (approximately semi-confluent culture) each 7-10 days.

Storage #

1000
1 Bacteria released from host cells can be stored in
Compound Amount Unit
Sucrose-Phosphate-Glutamate buffer 1000 ml
2 at -80°C for several months.
3 Infected cell cultures (supplemented with 10% DMSO) are best stored in liquid nitrogen.

Sucrose-Phosphate-Glutamate buffer #

1000
Compound Amount Unit
Sucrose 75.00 g
KH2PO4 0.52 g
Na2HPO4 x 2 H2O 1.53 g
Glutamic acid 0.75 g
Distilled water 1000.00 ml