PMID- 1779304
OWN - NLM
STAT- MEDLINE
DCOM- 19920309
LR  - 20131121
IS  - 0022-3395 (Print)
IS  - 0022-3395 (Linking)
VI  - 77
IP  - 6
DP  - 1991 Dec
TI  - Simultaneous purification of merozoites and schizonts of Eimeria tenella 
      (Apicomplexa) by Percoll flotation and assessment of cell viability with a double 
      fluorescent dye assay.
PG  - 989-93
AB  - The asynchronous development of Eimeria tenella in orally infected chickens makes 
      it possible to purify second generation merozoites (meros) and shizonts from a 
      single mucosal homogenate. After centrifugation in 30% Percoll in 
      phosphate-buffered saline (Percoll-PBS), debris, villi, and schizonts float, 
      whereas meros and erythrocytes are pelleted. Erythrocytes are lysed by a mild 
      hypotonic shock; meros are filtered through a cotton wool plug and collected by 
      centrifugation. The 30% Percoll-PBS supernatant fraction is diluted to 25% 
      Percoll-PBS and centrifuged to sediment mature schizonts. By repeated slow-speed 
      centrifugation, schizonts are separated from nuclei and small-sized debris. In 
      less than 3 hr, 8.8 +/- 2.3 x 10(8) meros and 7.2 +/- 3.9 x 10(6) schizonts are 
      collected from 10 infected chickens. Contamination with host material is 2% for 
      meros but variable for schizonts. For the assessment of cell viability, ethidium 
      bromide (EB) and acridine orange (AO) have been used as markers for dead and 
      living cells, respectively, in a single step method. More than 95% of the 
      schizonts and meros accumulate AO and no EB, whereas lysed erythrocytes and all 
      cells hosting a schizont are permeable to EB. After incubation of meros and 
      schizonts in synthetic media with [5,6- 3H]uracil, label accumulates in the 
      perchloric acid-soluble and -insoluble fractions, indicating transport, salvage, 
      and incorporation of the pyrimidine precursor in nucleic acids. If stored on ice, 
      meros and schizonts retain metabolic activity for at least 5 hr, but metabolism 
      declines rapidly during incubation at 41 C.
FAU - Geysen, J
AU  - Geysen J
AD  - Janssen Research Foundation, Department of Comparative Biochemistry, Beerse, 
      Belgium.
FAU - Ausma, J
AU  - Ausma J
FAU - vanden Bossche, H
AU  - vanden Bossche H
LA  - eng
PT  - Journal Article
PL  - United States
TA  - J Parasitol
JT  - The Journal of parasitology
JID - 7803124
RN  - 0 (Fluorescent Dyes)
RN  - EN464416SI (Ethidium)
RN  - F30N4O6XVV (Acridine Orange)
SB  - IM
MH  - Acridine Orange
MH  - Animals
MH  - Centrifugation, Density Gradient
MH  - Chickens/*parasitology
MH  - Coccidiosis/parasitology/*veterinary
MH  - Eimeria tenella/growth & development/*isolation & purification
MH  - Ethidium
MH  - *Fluorescent Dyes
MH  - Microscopy, Fluorescence
MH  - Poultry Diseases/*parasitology
EDAT- 1991/12/01 00:00
MHDA- 1991/12/01 00:01
CRDT- 1991/12/01 00:00
PHST- 1991/12/01 00:00 [pubmed]
PHST- 1991/12/01 00:01 [medline]
PHST- 1991/12/01 00:00 [entrez]
PST - ppublish
SO  - J Parasitol. 1991 Dec;77(6):989-93.