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Collection, preservation of samples
Preservation of entire animals, skins or fluid-preserved soft tissues for longterm storage in collections: see chapter "preservation for collections"
Table of content (including links
to other
files)
General
considerations
Preservation
methods:
cooling, refrigeration, drying, desiccants, chemical
preservation:
general considerations
Transport
of
biological samples
Permits for
collection
and transport of biological samples
Tissue
samples
Blood and
other
liquids
Faeces,
stomach content, other samples
General considerations
The term sample may either mean a specimen (animal, blood
sample,
other) or, used in the statistical sense, a sub-collection or
sub-set of
units. Aim in general is collection of samples representative of
the study
population. Wobeser (1994 b) provides some information about
planning of
sample collection, choice of samples, statistics, basic types of
bias (selection,
measurement, confounding), dealing with examining laboratories and
other
matters which need to be considered.
General rules
Wobeser (1994 b) recommends that the collector of samples of any
kind
first contacts the person(s) who do the analyses and establish the
type
and number of samples or specimens and the precise methods of
collection
and preservation required. For example, freezing may be a good
method to
preserve specimens for certain tests, but it ruins tissue for
detailed
histological examination. Fixation in 10% neutral buffered
formalin is
suitable for most histological examination, but from such samples
no living
agents for identification of diseases can be isolated. Cooling
specimens
with dry ice may also inactivate certain pathogens. The type of
container
used may invalidate the results of some toxicologic analyses. The
sampling
plan may be determined by a hypothesis to be tested
For assessing causes of deaths, it may be necessary to take
samples
not only from an animal or its remains, but also from parts of the
environment
(water, plants) (Wobeser, 1994 b)
In the field, there may be limited access to materials and
equipment
necessary, so preliminary preservation with more simple methods
may be
necessary.
Some general information about preservation
methods
Cooling, refrigeration
Refrigeration at 4ēC is an excellent way to preserve biological
materials over short periods. Insulated containers with wet or dry
ice
can be taken into the field. Containers for dry ice (CO2) should
not be
completely airtight because sublimation of dry ice produces
expanding gas
which, with increasing pressure, may cause a minor explosion
(Wobeser et
al., 1980).
Freezing at about - 10ēC is a good method for preserving specimens
for skeletal preparation, study skins or mounting, certain
pathologic specimens
and legal evidence. Laboratory freezers with - 70-80ēC are best.
Freezing of entire specimens for later necropsy should be avoided,
particularly if histopathology is required, because it causes
numerous
artifacts. Immediate necropsy and preservation of samples is
better (see
sample collection plan and methods below). Storage for short
periods of
time requires no special treatment. If specimens are to be frozen
for more
than a few days, desiccation must be prevented by wrapping the
specimen
and / or storage in waterproof containers (Wobeser, Spraker, 1980;
Wobeser
et al., 1980).
Removal of the skin with insulating fur before cooling or freezing
may help to cool carcasses down more quickly (Schoon, lecture
manuscript).
Drying:
For certain samples, for instance for stomach contents and faeces,
air-drying is recommended.
Desiccants - keeping materials dry
Dry samples may be protected from humidity by addition of silica
gel
which can be purchesed loose, in sachets or capsules. Silica gel
absorbs
water. When saturated with adsorbed humidity, it can repeatedly be
regenerated
by heating at 100 - 120°C. Sachets or capsules may be less
tolerant
of heat.
Non-indicating (white) silica gel is non-toxic and non-flammable,
but
it may cause some skin problems and allergic reactions, contact
with skin
and eyes and inhalation of dust particularly by people with
bronchitis
or asthma should be avoided. Self-indicating silica gels are mixed
with
some indicator substance and change their colour after adsorbing a
certain
amount of moisture. The usual indicator substance earlier has been
cobalt
chloride which is blue when dry and turns pink when humid. But it
is toxic,
and inhalation of dust of silica gel with cobalt chloride causes
cancer
hazard. Therefore, use of protective gloves and a dusk mask is
recommended
for handling it, and it must be disposed of as hazardous waste
(GeeJay
Chemicals Ltd. website). Meanwhile, silica gel with other
indicators (orange
or yellow) is available, but it changes colour more quickly, and
certain
yellow indicators such as Phenolphthalein might be even more
hazardous
for health (DES-CASE Europe sarl website). White silica gel
without indicator
added does not change visible when saturated, the amount of
humidity adsorbed
can only be determined by weight increase if initially the dry
amount had
been weighed. Self-indicating silica gel can be regenerated in the
same
way, during regeneration it returns to original colour; but
eventually
the crystals will lose their colour (GeeJay Chemicals Ltd.
website, http://www.geejaychemicals.co.uk/silicagel.htm).
Rice also adsorbs considerable amounts of humidity, and in
places
where silica gel is not available, it may be used to keep material
dry.
Of course, some animals might want to eat it (one of us: A.
Nekaris). Rice
may also be regenerated with heat for desiccation; one of us (E.
Curio)
recommends drying of humid rice for instance by frying it in a
pan, if
no oven is available in the field, until steam becomes visible.
Rice also
does not change externally when humid, but weight control may
allow determination
how much moisture has already been absorbed.
Chemical preservation:
Formalin preservation ca be used for specimens for
histological
examination and for fluid-preserved specimens. It should not be
used to
preserve specimens for preparation of skeletal material, for
microbial
examination or for preparation of study skins and mounted
specimens. Formalin
discolours fur and, after a longish immersion, softens the bones
(one of
us: C. Groves).
Preservation in alcohol is useful for longterm storage of
tisseus
already preserved in formalin because it does not harden the
tissue excessively
(one of us: A. Schlichting). A whole animal can also be preserved
in a
container of alcohol (70-90%). Removal of the intestines prior to
storage
of the animal in alcohol is recommended (Rabinowitz et al., 2000).
Necessary / useful equipment: in preparation
Transport of biological samples
No leakage should occur when fresh or frozen samples are shipped,
containers
must be safe (Munson, 2000).
In case of transport by carriers, hazardous goods regulations, for
instance for chemicals or dry ice, must be considered to avoid
delay or
refusal by the carrier (Wobeser, 1994)
For transport of formalin-fixed material (after fixation over 10
weeks),
most of the formalin can be removed and tissues can be wrapped in
towelling
soaked in formalin for shipping. The tissues soaked in formalin
can then
be placed in leakproof containers for shipping (Munson, 2000).
Permits for collection and transport of
biological
samples
Before taking samples with invasive methods (including blood
samples
or plugging of hair from an animal), laws on animal
experimentation of
the state where the samples are taken must be considered. Hairs
for instance
can also be collected non-invasively intead of being plugged.
Before shipping samples, regulations, permits and necessary
measures
for disease control must be considered. For biological samples
taken from
endangered species, international shipment may require special
permits
such as CITES export permits from the country of origin and CITES
import
permits and disease control permits for samples from primates (AZA
Prosimian
Taxon Advisory Group, 2002). Munson (2000) recommends to contact
the examining
laboratory before shipping
Checklist: Tissue samples
fixed
for histology + other samples: in preparation
In carcasses of rare species, preservation of organs in situ
for anatomical studies may be useful, with only small samples
taken out
for examination, causing as little disturbance as possible. If a
necropsy
is only done for detection of health problems, enough tissue for
examinations
including bacteriology should be taken (one of us: K. Petry),
samples including
abnormal areas and surrounding normal areas; Munson (2002)
recommends samples
no thicker than 1 cm (for good fixation), but long and wide enough
to represent
different areas of a tissue and possible abnormalities. In small
animals,
entire organs instead of samples may be collected. Mechanical
damaging
of samples, for instance by compessing them with forceps, must be
avoided.
Tissue samples for DNA analysis: for genetic
evaluation,
the AZA Prosimian Taxon Advisory Group (2002) recommends storage
of samples
of heart, skeletal muscle and liver (about 10 g, if available),
stored
in plastic bags and frozen. Preservation in the field when
freezing is
not possible: in alcohol (one of us: Ch. Roos).
For detection of some viral, bacterial and blood parasite DNA or RNA or antibodies against some pathogens, small amounts of whole blood can be collected from the freshly opened body cavity by dripping it on a thick Whatman-type filter paper (see equipment) or by touching to organs or muscles with the filter paper. The blood spots can then be dried at room temperature and stored in plastic bags in a dry place. Adding a small silica gel pack to each plastic bag is recommended (Munson, 2000).
For serology, serum (clear fluid, yellow, in autolysed animals red-tinged) or plasma should be separated for the blood cells, divided into at least two sterile tubes and then refrigerated or frozen at -20° or -70° if possible until transport to a laboratory. Serum, plasma or blood from the heart of a carcass can be collected in vials during necropsy and left undisturbed for approximately 30 min to encourage clot formation, then centrifuged at approximately 2000 X G for 20 min. When a centrifuge is not available, serum can be obtained by letting the clot or blood cells settle. If blood is obtained from a live animal or a dead animal whose blood has not yet clotted, whole blood can be removed into a blood tube and stored with the tube inverted (rubber stopper down) until it clots; then the tube can be cautiously turned and the stopper can be removed with the blot clot attached, leaving the serum in the tube (Munson, 2000).
For DNA analysis from blood cells for genetic studies when
refrigeration
is not available, Munson (2000) recommends preservation
with "Easy
Blood". This method can also be used to preserve DNA
for longer
periods of time if refrigerated or frozen
Faeces, stomach content,
other
samples
Samples for food analysis
If a carcass is found in the wild, collection of the content of
the
entire alimentary tract for food analysis may be useful.
Examination of
the stomach content alone may not be sufficient for this purpose;
in galagos
and pottos, after gum-eating it seems that gums are retained in
the stomach
only for few minutes, so usually no trace of gum is found there,
but gum
may be found in the caecum (Hladik 1979, partly quoting
Charles-Dominique
1971 and 1974). Contents of the digestive tract can be preserved
in 5%
formalin or 30-40% alcohol (Rabinowitz et al., 2000), or the whole
alimentary
tract may be preserved in formalin, after injection of formalin
into the
stomach, for later analysis (one of us: A. Nekaris).
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In:
Loris and potto conservation database: field methods
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Last
amendment: 30November 2002
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