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Storage of Amoeba

Page update 23/8/02

Many amoeba can be easily stored as they create their own protective casing around themselves in the form of the cyst.  Others of course do not and these tend to be less easy to maintain both in long term culture and as frozen stocks.  The availability of liquid nitrogen for rapid freezing has greatly helped in the storage of amoeba and most can be stored satisfactorily using a combination of rapid freezing with liquid nitrogen and storage at below -20oC.

This method (John et al, 1994), is for the long-term storage of amoebal strains at -70oC. 

1)            Take 1ml of dense culture of amoebae and spin down.

 

2)            Remove supernatant and take up in 100Ál of freezing media

 

3)            Acclimatise for 30 minutes at room temperature.

 

4)            Place in -20oC freezer for 1 hour.

 

4)            Place in -70oC freezer.

 

Freezing Medium

Working Concentration                       500mls
12% dimethylsulphoxide                         60mls
20% Heat Inactivated calf serum*          100mls
Axenic medium (or other)                       340mls

 

* Heat inactivate by warming to 55 oC for 1 hour.

 

References:-

Gupta, S. & Das, S. R. (1999) Stock cultures of free-living amebas: Effect of temperature on viability and pathogenicity., J.Parasitol. 85, 137-139.

John, D.T., Eddy, P.L. & John, R.A. (1994) Cryopreservation of pathogenic free-living amoebae. Folia Parasitol 41; 110-114.

 

 
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