TECHNIQUE
7 Lab Animal
Collection of Mouse Urine for
Bioassays
Joon Lin Chew, MSc, and
Kaw Yan Chua, PhD
Rodent bioassays often require
fresh, uncontaminated urine from a
large number of mice. The authors
describe a quick and simple method
to collect moderate to large
amounts of urine from individual
mice using disposable plastic trays.
The authors are in the Department of
Paediatrics, National University of
Singapore, Lower Kent Ridge Road,
Singapore 119074. Please send reprint
requests to Chua at the above address,
or email: paecky@nus.edu.sg.
48
Volume 32, No.
July/August 2003
In mucosal immunization studies, the inva-
sive methods required to obtain small
intestinal secretions hinder the detection of
specific secretory IgA antibodies in the
intestines. These invasive methods are not
feasible for routine analyses in which
samples must be obtained over a period of
several months. Because IgA plasma cells
generated after successful mucosal immu-
nization have been known to migrate to
such distant mucosal sites as the salivary
glands, bronchial tissues, and genitourinary
tract, secretory IgA antibodies originating
at these surfaces could serve as proxy mea-
sures for IgA production in the intestines1.
The collection of mouse urine for anti-
body detection using the enzyme-linked
immunosorbent assay (ELISA) requires
uncontaminated urine of a moderate vol-
ume (i.e., at least 100 µl per mouse).
Because immunization studies often
involve large numbers of animals, there is
a need for a quick and convenient method
to collect individual mouse urine samples
at designated points in time. We describe
an easy and quick method involving gentle
coercion to obtain fresh urine hygienically
from as many as 50 animals sequentially
using disposable plastic containers.
Materials and Methods
Mice
We used 50 female BALC/cJ albino
mice (The Jackson Laboratory, Bar
Harbor, ME) aged six weeks that had been
bred in the Laboratory Animals Centre
(Sembawang, Singapore). The mice lived
in the Animal Holding Unit, National
University of Singapore (NUS), in conven-
tional cages containing autoclaved paper
pellets, in groups of five to six mice per
cage, fed with meat-free mouse cubes
(Glen Forrest Stockfeeders, Glen Forrest,
Australia) and provided with water ad
libitum. The Animal Holding Unit main-
tains animals at room temperature of
~21 °C and a relative humidity of 40–50%.
The animal room has a controlled pho-
toperiod of 12 h on and 12 h off.
Laboratory technologists provided expert
care for the mice. These experiments were
done in accordance with the Institutional
Guidelines for Animal Care and Handling,
National University of Singapore.
Urine Collection
The plastic packaging from microtiter
plates (Costar 9018, Corning, NY) used in
previous ELISA experiments became a
holder for urine collection. After peeling
away the top of the microtiter packaging
and removing the plate, we collected the
plastic-bottom container, which measures
~135 mm
90 mm
20 mm deep. The
technician placed a mouse onto the plastic
packaging, and using the left hand to hold
the mouse’s tail, tapped its lower back (dor-
sal posterior) a few times with the fore and
middle fingers of the other hand, thereafter
applying equal pressure in a massaging
fashion at both sides of the lower back near
the tail with the thumb on one side and the
fore and middle fingers on the other side,
rubbing up and down (Fig. 1). Applying
pressure to the caudal area of the back of the
mouse is essential to the immediacy and
volume of the urination. Once the mouse
started urinating, the experimenter gently
pulled the skin at the lower back upward
with the massaging fingers to immobilize
the mouse and to prevent premature halt of
urination. The voided urine was aspirated
using a 200-µl Gilson Pipetman (Middle-
ton, WI; Fig. 2), transferred into a micro-
centrifuge tube, and stored at –80 °C until
assayed. A new disposable plastic container
was used with each mouse.
July/August 2003
FIGURE 1. Individual mouse urine collection method using a obtained is often contami-
disposable plastic container. The experimenter holds the
mouse by the tail and presses gently on its lower back.
Results
Figure 1 illustrates the urine collection
method, with a mouse resting on the dis-
posable plastic container and the experi-
menter’s fingers placed at the lower back of
the mouse. The time required to collect
urine from 50 mice sequentially was ~2 h,
whereas the time for successful urine
induction in a single mouse ranged from a
few seconds to 2 min, yielding 50–500 µl of
urine. If a mouse did not excrete enough
urine for immunoassay, the experimenter
returned it to its cage and repeated urine
collection 1 h later. We were able to collect
ample urine biweekly from all the mice for
a period of 20 weeks. None of the urine
samples was contaminated with feces or
food debris.
Discussion
In mucosal immunization studies, the
analyses of systemic antibodies and secre-
tory IgA responses at mucosal surfaces are
essential. Secretory IgA plays an important
role in the protection of the host from for-
eign molecules and microorganisms to
which the mucosal surfaces are exposed.
Lab Animal Volume 32, No. 7
Many murine immuniza-
tion studies have used
serum, feces, saliva, and
urine samples for detec-
tion of IgA antibodies
using ELISA, a common
and sensitive method for
antibody detection.
The most common
technique for collecting
rodent urine using meta-
bolic cages is not practical
for such studies because of
the need to use many cages
when large numbers of
animals are involved and
individual mouse urine
samples are needed.
Furthermore, the duration
of urine collection is
uncertain and the urine
nated with feed and feces.
Although the single-ani-
mal method (SAM) and multiple-animal
method (MAM) described by Kurien and
Scofield2, using clear plastic wrap, does not
involve coercion, it is also time-consuming
with SAM and space-consuming with
MAM, when samples from a large number
of animals are needed in a day. Previously
reported urine collection methods for
FIGURE 2. Using a micropipette, an experimenter aspirates excreted mouse urine and
transfers it into a microcentrifuge tube for storage.
TECHNIQUE
individual mice3,4 and rats5 require mate-
rials such as glass cages, conical
polypropylene centrifuge tubes, beakers,
tape, and wire cloths that may be inconve-
nient for urine collection from a large
number of animals in a short time.
In IgA antibody kinetics study, it is nec-
essary to make weekly or biweekly urine
collections from each mouse and ELISAs
require at least 100 µl of urine per mouse.
Various other methods have proved useful
for mouse and rat urine collection in bio-
chemical, urological, and metabolic stud-
ies2,6–8. However, those assays require only
a few drops of urine.
This study describes a simple way to
obtain 50–500 µl of urine quickly and
hygienically from as many as 50 animals
in ~2 h. We sampled urine biweekly for
20 weeks. This method originated from
our observation that, when the mice were
taken out of their cages for immunization
or blood-drawing purposes, they re-
sponded by urinating or defecating.
Therefore, in this study we collected urine
before bleeding each mouse biweekly.
Most of the mice in this study urinated
within a few seconds with gentle coercion.
For ELISA tests, the crucial requirements
for mouse urine collection are the ability
to obtain relatively pure urine without
contamination with feces or feed and the
49
 July/August 2003

ease and convenience of collection for

large numbers of animals. Because this
method requires only a clean surface to
collect urine, our use of disposable plastic
packaging from ELISA plates (Fig. 1)
made it inexpensive, space-saving, and
convenient. Alternative equally useful
collection vessels include the plastic
weighing boats, glass plates, and Petri
dishes that are commonly found in most
laboratories. This method also ensures
that mice are not in further contact with
the excreted urine, because it is immedi-
ately aspirated and stored. The urine can
also be collected on ice or immediately
stored in the refrigerator.

Acknowledgments
We would like to thank A*STAR
(R-178-000-010-303), Republic of
Singapore for funding as well as Lim Lay
Hong and Lim Wei Seng for reading the
manuscript.

Received 3/11/03; accepted 4/10/03.

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