Biology Reference
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with PCR after centrifugation at 2000 × g for 10 min.
64
For concentration
of bacteria after ultrafiltration, centrifugal forces of 3300-4200 × g are com-
monly used.
8,22,62,65
When deceleration conditions are mentioned, brakes
are not applied.
Thus, no standard conditions for centrifugation can be given and con-
ditions should be chosen based on the organisms studied and downstream
processing. For example, higher forces are likely to pellet more bacteria and
parasites but may decrease viability and damage surface antigens important
for IMS. If PCR-based detection is used, these factors are not important
while the effect on PCR inhibitors needs to be considered.
4.4.2. Density gradient separation
Density gradient separation is mainly used for purification of parasites
from feces but may be applicable also to isolation of parasites from water
concentrates, and some examples are given next. The techniques are based
on gradients formed by centrifugation of solutions of known gravity that
either float parasites and pellet debris, or more rarely, float lighter debris and
pellet the parasites. Commonly used solutions are Percoll, cesium chloride,
sucrose, and zinc sulfate.
Taghi Kilani and Sekla (1987) placed a
Cryptosporidium
oocyst suspen-
sion on top of a discontinuous gradient consisting of three layers of CsCl
(1.40, 1.10, and 1.05gmL
−1
).
66
After centrifugation for 1h at 16,000×g
at 4°C, a distinct band of very clean oocysts was observed and could be
collected. Stine et al. purified
E. intestinalis
spores by placing a spore sus-
pension on top of a Percoll-sucrose solution with a density of 1.2 g L
−1
(cre-
ated by diluting Percoll with 2.5 M sucrose) and centrifuged for 10 min at
1500 × g.
63
The top layer and interface were collected and washed once in
PBS. This purification technique was applied to tap water samples concen-
trated by Envirocheck filtration and recoveries compared to centrifugation
alone. Recoveries were very similar, but this extra purification step may be
useful for turbid surface water samples. Percoll-sucrose gradients have also
been used after filtration of surface water from reservoirs fed from wells or
springs to separate protozoa from denser particular matter.
64
Both CsCl and
Percoll are fairly costly, and potassium bromide has been investigated as a
cheaper alternative.
67
A fecal suspension containing
Cryptosporidium
oocysts
was placed on top of three layers of KBr (28%, 16%, and 6%) in Tris-EDTA.
After centrifugation at 3000 × g or 16,000 × g for 1 h at 4 °C, the oocysts
were visible as a white band in the gradient that could be collected using a
Pastuer pipette.
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