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 4ºC 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 - 10ºC is a good method for preserving specimens for skeletal preparation, study skins or mounting, certain pathologic specimens and legal evidence. Laboratory freezers with - 70-80ºC 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

Labelling, protocol

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
 
 

Tissue

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).
 
 
 

Blood and other liquids

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).
 
 

Back to the top of this page
 

In: Loris and potto conservation database: field methods

Last amendment: 30November 2002