This document provides a protocol for safely collecting pooled biological samples from wild bat roosting sites in 3 sentences: It outlines supplies needed, methods for placing tarps under tree-roosting and cave-dwelling bat colonies to collect urine and feces samples before the bats leave at dawn, and guidance for collecting and recording the samples while minimizing risks to field workers. Examples of bat feces are also provided to help with identification and differentiation from other animal waste.

1.
PROTOCOL
Bat Population Sampling Methods
v. July 9, 2013
Prepared by
Jonathan Epstein, EcoHealth Alliance,
David Wolking, UC Davis,
Kshitiz Shrestha, Narayan Lamichane,
Ankita Poudal, and Manisha Bista,
Center for Molecular Dynamics Nepal,
and the PREDICT One Health Consortium
Objectives: To safely collect pooled biological samples from wild bat roosting
sites. (See separate detailed protocols for bat and rodent capture, wild animal
capture, personal protective equipment, and data collection).
USAID Disclaimer
This document was made possible by the generous support of the American people through the
United States Agency for International Development (USAID) Emerging Pandemic Threats
PREDICT. The contents are the responsibility of the authors and do not necessarily reflect the
views of USAID or the United States Government.
Suggested Citation Form: PREDICT One Health Consortium 2013. Protocol for Bat Population
Sampling Methods.
http://www.vetmed.ucdavis.edu/ohi/predict/PREDICT_Publications.cfm#Protocols

2. USAID PREDICT Protocol for Bat Population Sampling Methods 2
CONTENTS
SECTION 1. SUPPLIES
SECTION 2. METHOD AND PROTOCOL
SECTION 3. FECES GUIDE
APPENDIX. Example data sheet for use in bat population sampling from the
PREDICT Nepal team

3. USAID PREDICT Protocol for Bat Population Sampling Methods 3
SECTION 1. SUPPLIES
1. Polyethylene sheets (long sheets of clear plastic for non-cave bats, white
plastic for cave dwelling bats for better contrast) - often available locally,
can also purchase rolls.
2. Stones (or weights to hold tarp corners down)
3. Pipette
4. Sterile pipette tips to pool urine
5. Sterile polyester swabs with plastic shafts
6. Cryo-safe vials or tubes [larger cryovials (4-5mL that combined can hold
up to 750mL for pooled urine]: we will be using 5-6 tubes/plastic sheets.
7. Cryo-safe vials (up to 2mL) to hold actual samples for testing.
8. LN2 dryshipper
9. PPE [Hats, nitrile gloves (2 pair at a time), respirator, goggles]
SECTION 2. METHOD AND PROTOCOL
For tree roosting bats (fruit bats):
1. Reconnaissance: observe the bat roost and conduct a count during the
day and a flyout count (count bats as they leave the roost at sunset) the
evening of sampling to obtain an estimate of roost size. Record a GPS
location of the roost. If more than one tree, record the approximate size of
the roost and track the length of the roost using GPS.
2. Timing: For morning sample collection, go to the field site to set the tarps
around 4AM (before bats return to the roost), and collect any samples
from the tarps around 6AM (adjust according to actual bat activity1
- goal is
to set the tarps as the bats are returning to their roost and getting settled
as urine and feces are best collected prior to departure for foraging and at
arrival back to the roost site). This timing will also help maximize bat
excrement collection and minimize bird excreta collection.
3. Placing traps: Place cut up plastic polyethylene sheeting2
(2x3m sections)
and position underneath the roost to best align with the bat location above
(either linearly if aligned linearly in trees, or more blocked - depends on
bat tree roosting positions. Add stones (or weights) to hold tarp in
position.
1
Behavior
may
vary
regionally
and
with
species.
A
night
of
observation
may
be
required
to
assess
the
timing
of
return.
In
some
cases,
bats
may
return
earlier
in
the
night,
in
which
case
tarps
could
be
laid
at
2AM
and
samples
collected
between
5-­‐6
AM
once
the
majority
of
bats
are
back.
2
If
reusing
sheets,
rinse
them
with
water
and
allow
to
dry
in
the
sun
to
disinfect
before
next
use,
or,
use
new
sheets
for
each
sampling
event.

4. USAID PREDICT Protocol for Bat Population Sampling Methods 4
4. Label the tarp sections (Tarp A, Tarp B, etc.) and place in the same spot if
conducting multiple sampling over several days – the idea is to get same
bats at same locations, as bats will often roost in the same place over
time.
5. GPS bat roosting location, including tarp locations, and take pictures for
species identification.
6. Urine sample collection: Using sterile pipette tips, pipette urine from the
tarps into the 4 or 5mL cryovials, mix, and then aliquot them evenly into
the pre-filled 2mL cryovials with Lysis buffer and VTM. Be sure to mark
which tubes came from which tarps so that location within the roosting site
can be tracked back post-testing. The optimal sample volume is 2mL
urine.
7. Fecal sample collection3
: Use the sterile swab and swab wrapper like a
spatula to collect feces and place into 2mL cryovials with Lysis buffer and
VTM.
For cave roosting bats (insectivorous bats):
1. Timing: For morning sample collection, go to the field site to set the tarps
around 5AM (before bats return to the roost), and collect any samples
from the tarps around 8AM (adjust according to actual bat activity - goal is
to set the tarps as the bats are returning to their roost and getting settled
as urine and feces are best collected prior to departure for foraging and at
arrival back to the roost site).
2. Place white cut-up plastic polyethylene sheeting (2x3m sections) and
position underneath the roost to best align with the bat location above (we
use white tarp in caves for high contrast and greater visibility of feces in
the dark). Add stones (or weights) to hold tarps in position.
3. Label the tarp sections (Tarp A, Tarp B, etc.) and place in the same spot if
conducting multiple sampling over several days – the idea is to sample the
same bats at the same locations, as bats will often roost in the same place
over time.
4. GPS bat roosting location, including tarp locations, and take pictures for
species identification.
3
It
is
easier
to
visibly
tell
whether
a
urine
sample
is
separate
from
feces,
but
much
more
difficult
to
know
if
a
fecal
sample
is
not
mixed
with
urine.

5. USAID PREDICT Protocol for Bat Population Sampling Methods 5
5. Sample collection: Place ~2-3 pellets of feces4
into the 4mL (or 5mL) pre-
filled cryovials with Lysis buffer and VTM (collect about 5-6
cryotubes/tarp). If sampling urine from cave dwelling fruit bats (e.g.
Rousettus spp.), use sterile pipette tips, and pipette urine from the tarps
into the 4 or 5mL cryovials, mix, and then aliquot them into the pre-filled
2mL cryovials with Lysis buffer and VTM. Be sure to mark which tubes
came from which tarps so that location within the roosting site can be
tracked back post testing.
6. Determine if the cave is mixed species or single species roosting site.
Count the bats to get an estimate of the roost size, get species information
(to lowest taxonomic descriptor possible), and collect any additional
information required by the field data sheets (see example data sheet in
Appendix A).
7. If evening, conduct a fly-out count of bats leaving the roosting site to
estimate numbers actively foraging.
4
With
insectivorous
bats
in
caves,
we
do
not
expect
to
find
urine
for
collection.
Focus
instead
on
fecal
pellet
collection
with
this
group
of
bats.
The
exception
is
Rousettus
spp.,
cave-­‐dwelling
fruit
bats,
which
are
large
enough
that
you
may
find
enough
urine
on
tarps
to
sample.

6. USAID PREDICT Protocol for Bat Population Sampling Methods 6
SECTION 3. GUIDE TO ANIMAL FECES
Below are photos of feces of large fruit bats and micro-bats.
Figure 1. Greenish-black feces of mega-bat (fruit bat)
Figure 2. Brown-black rice pellet looking feces is micro-bat feces (insectivorous)

7. USAID PREDICT Protocol for Bat Population Sampling Methods 7
APPENDIX
Example data sheet for bat population sampling used by the
PREDICT Nepal team.
ANIMALS – BAT POPULATION SAMPLING (ADD BARCODE STICKER/LABEL TO TOP
RIGHT CORNER)
Group (Roost) ID:
Tarp Number: Tarp location:
Tarp Latitude: Tarp Longitude:
Height of tree/roosting site:
Type of tree/roosting site:
Taxonomic Descriptors: Mixed species Single species
Species & Scientific Name (if known)
Identification Certainty: Actual Estimate Unknown
Bat roost size (No. of bats):
Bat fly out count (No. of bats leaving roost):
Site Map/Notes:

8. USAID PREDICT Protocol for Bat Population Sampling Methods 8