This training video will show the
basic steps for concentrating and
preserving eDNA water samples by
centrifugation and alcohol addition,
in accordance with the U.S. Fish and
Wildlife Service Region 3 invasive carp,
eDNA Programs Quality Assurance
Project Plan, or QAPP.
For the most comprehensive information
on how to decontaminate gear,
please refer to section B2 of the QAPP.
Prior to receiving samples,
make sure that the eDNA trailer or
designated processing space has been
thoroughly cleaned and decontaminated
and set up for sample processing.
Centrifuges should be turned on to allow
refrigeration to cool the centrifuge
compartments to 4 degrees Celsius.
Upon receiving samples at
the processing trailer,
50 milliliter tubes should be removed from
the transport cooler with clean gloves.
Caps should be checked and tightened.
Sample tubes need to be dried before
being placed into centrifuge inserts.
Using a clean paper towel and then placed
into the green centrifuge inserts.
Replace the paper towels with new
sheets as they become saturated.
It can be beneficial to have a stack
of clean paper towel sheets at the
ready prior to receipt of samples.
The same pair of gloves may be
used for the drying process.
Tubes from a single sample may
be split among separate inserts.
However, it is crucial that all the
tubes from a single sample are placed
in the same centrifuge to help ensure
samples remain together and in the
same step throughout processing.
Field blanks should be handled in
the same manner as a regular sample
throughout this entire process.
Dry and load tubes into inserts
until a full centrifuge batch or
four complete inserts are prepared.
Load all four inserts into a centrifuge,
making sure the tubes are distributed
evenly on opposite sides to
properly balance the centrifuge.
With the centrifuge loaded,
set the centrifuge to spin for
30 minutes at the max speed,
which depends on your insert type.
Press start to begin spinning,
change your gloves and begin preparing
the next set of samples and inserts.
Once samples have been centrifuged
for 30 minutes,
remove the batch of samples from the centrifuge.
After this first spin,
the eDNA will be on the bottom of the tube.
With a clean pair of gloves for each
set of tubes comprising 1 sample,
carefully remove cap and gently pour
off water into a wastewater container.
Gloves should be changed between each
sample and always change gloves when
potential contamination is suspected.
For example, the same pair of gloves can
be used for all five tubes of sample 5.
Then a new pair of gloves should be
used to handle the tubes in sample 6.
It is important to try to pour off as
much water as possible without disturbing
the pellet at the bottom of the tube.
If it looks like a full decant
isn't going to be possible,
section B2 of the QAPP has detailed
instructions on how to proceed.
After pouring off the excess water,
add 20 milliliters of either
95% non denatured ethanol,
or 70 to 99% isopropyl to each tube.
Field blanks should be handled in the
same manner as a regular sample.
Replace the cap and swirl the tube to
ensure the preservative coats the pellet.
Put the tube back in the insert and continue.
The process up to this point must
be completed within 24 hours of
individual sample collection.
At this point, if you are
hand delivering samples,
you can place the samples into
clean storage or shipping racks.
If you plan to ship the samples using FedEx,
continue with the processes in this video.
However, it is important to note that
if a full decant of the water was not
possible due to the pellet being loose,
you must continue with the following steps
for all samples that had issues to ensure
an adequate preservation level.
Once all tubes in a batch have been
decanted and ethanol has been added,
return the tubes and inserts to the
centrifuge and centrifuge preserve
samples for an additional 10 minutes.
After the 10 minute spin is complete, again,
remove tubes and inserts from the centrifuge.
Decant as much excess alcohol as
possible without disturbing the pellet,
so that only the conical end of the
tube has any alcohol left in it.
At this point, if you had
trouble decanting the water,
add an additional 5 to 10 milliliters of
alcohol to ensure proper preservation,
and leave the excess in the tube.
Recap each tube and place in a
clean storage or shipping rack.
Once again, gloves should
be changed between samples.
After you have completed
decanting alcohol from a batch
of samples and the tubes are now
in a storage or shipping rack,
record the time centrifuging was
completed for that batch and the
initials of the designated processing
QA/QC in the field maps app.