Method for Excystment of Amoebae To Put Into Axenic Culture

by

Jennifer Dee

1. Wash cells off plate in 2ml 10mM Sodium Phosphate Buffer pH 6.0 (Gorman et. al. 1977, Mol Gen Genet 151, 253-259). Place in 12 - 15ml tube and pellet cells gently (1500 rpm, 1.5 min). 2. Remove supernatant containing bacteria and resuspend pellet in 2ml buffer. 3. Pellet as above. 4. Decant supernatant and resuspend in a further 2ml of buffer. 5. Pellet as above. 6. Decant supernatant and resuspend in 2ml of buffer. 7. Take a 200ul sample for haemocytometer count and triton test (see below). Put the remainder of the amoebal suspension to shake at 26oC for 1 hour. 8. After 1 hour, take a further 200ul sample and carry out another triton test. If at this point the majority of the cells are stained, continue. If more than a small proportion of the cells are unstained (i.e. cysts) then continue to incubate the suspensions with shaking, checking the state of the cells at hourly intervals (use a haemocytometer count - if a high % of flagellates present then it is time to do another triton test; they often take 3 to 6 hours to excyst) 9. Pellet cells as above, decant supernatant and resuspend in 2ml SDM. 10. Take a 200ul sample for cell count and triton test. 11. Adjust amoebal density to 1 x 106 cells/ml, and incubate on shaker at 30oC - not more than 1.5ml per 12 - 15ml tube. Add pen and strep for first couple of subcultures. 12. Count after 24 hours to give a more accurate count of viable cell numbers. 13. Count after 3 to 4 days and subculture before cells reach 1 x 107 cells/ml. NB. Once you are confident that you can recognise active (excysted) amoebae it is not always necessary to carry out Triton tests every time you sample the amoebal suspension. Triton Test (Dee et al., 1997) Microbiology 143, 1059-1069
Take 200l sample of cell suspension. Add 2l of 2.5% triton-X100 (v/v) and vortex 10sec. Add 10l 0.4% trypan blue (w/v; Sigma) and mix. Leave 5 mins and examine cells in haemocytometer. Cysts do not stain and remain white, active cells stain blue.


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Last modified: Tuesday, December 22, 1998