Methods Preservation Fixation

Specimen Processing Procedures
Museum Collection Management Terms and Invertebrate Specimen Processing Procedures: Methods of Fixation and Preservation
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Voucher Collections Definition of Lot Fixation and Preservation of Marine Invertebrates Recipes: Fixatives and Preservatives Processing Procedures for all major Marine Invertebrate Taxa
VOUCHER COLLECTIONS Voucher Series: This is a series of reference specimens for each reported taxon, including those taxa tentatively identified as "Genus A" or "species B". A voucher series should be established that includes a sufficient number of individual specimens to display all typical intra-specific variability encountered in that taxon for that study area. Whenever feasible, the series should include ovigerous and non-ovigerous adult females, mature males, juveniles and larval stages. If the taxon exhibits habitat-related phenotypic variability, representatives from each habitat should be included in the voucher series for that taxon. The voucher series for a taxon is as important to an ecological study as a taxons type series is to a taxonomic study.
Voucher Collection:
This is a taxonomic reference collection composed of one or more lots of specimens (i.e. a voucher series) of each reported taxon for a given ecological, biogeographic, physiological or other scientific study. The primary purpose of a voucher collection is to insure taxonomic consistency.
A voucher collection should be established early in the taxonomic phase of a project. The greatest number of voucher specimens will be derived from the first cruises or first collecting efforts. However, new specimens will continually be added to the various series as taxonomically important variations are encountered or as new taxa are identified. The voucher collection for each major taxon should be established by competent, professional taxonomists. Ideally the same taxonomists would be responsible for the identification of all non-voucher specimens. Taxonomic references used in the identification of the voucher series as well as the voucher specimens themselves must be available for use during the identification of the general (non-voucher) collections. All specimens in the general collection identified to any taxonomic level must exhibit taxonomically important characters identical to those characters exhibited by specimens in the corresponding voucher series. The reidentification of a voucher specimen/series by the taxonomic contractors will require that all corresponding specimens in the general collection be re-evaluated and, if necessary, re-identified to insure consistency between the names used for specimens in the voucher collections and those used in the general collections. Go To Top
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DEFINITION OF LOT A lot is a group of specimens associated at some taxonomic level and are from the same collection locality. As the identification of the specimens is refined, they are still associated as a sample, but are broken up into separate lots based on phylum, class, order, family, genus, or species associations of the sample. All specimens in a lot should be identified to the same Lowest Practical Taxonomic Level (LPTL).
FIXATION AND PRESERVATION OF MARINE INVERTEBRATES The fixation of biological specimens involves the coagulation of cell contents into insoluble substances with the purpose to prevent autolysis and the degradation of tissue. The optimal fixation of biological specimens is achieved on individual live, preferably relaxed, material. Bulk fixations usually yield mediocre results as various animal taxa must be fixed by variant means, due to the different chemical makeup of each animal group. A good fixation is generally achieved in a brief amount of time (hours to days) and as soon as the animal is collected. After a favorable fixation is attained, the next procedure is the transfer of the specimen(s) into the proper fluid for archival storage. General biological fixation/preservation procedures are discussed below. Specific fixation and processing for molecular analysis, TEM/SEM, histology, and histochemistry require specialized techniques that are not mentioned in this discussion. Formalin is generally the preferred fluid for fixation and is widely used. Formalin is often sold as 37-40% aqueous Formaldehyde. Formaldehyde is a gas produced by the oxidation of methyl alcohol, whereas 100% Formalin is a saturated solution of this gas in water. To make a solution of 10% Formalin, nine parts of water are added to one part of 40% (aqueous) Formaldehyde. Therefore, a 10% solution of Formalin is the equivalent of a 4% solution of Formaldehyde. Formalin/Formaldehyde solutions and vapors are hazardous. Avoid contact with skin and eyes, and use only in a well ventilated area or in a fume-hood. Formalin/Formaldehyde readily oxidizes into formic acid, an acidic solution in the pH 2.5 to 5 range. To prevent the dissolution of calcified tissue and other tissue damage, Formalin/Formaldehyde solutions are commonly buffered or neutralized. Since sea water has a pH of 8.2 and in solution with Formalin/Formaldehyde has approximate pH of 7, it is sometimes used as a buffer. However, the buffering capacity of sea water does not last over time. Calcium Carbonate (CaCO3) or Borax (Sodium Borate) in supersaturated solutions with Formalin/Formaldehyde are commonly used buffers for short term formalin fixation. They should not be used to buffer Formalin/Formaldehyde solutions used for long-term storage since their effects do not last over time and Borax may cause lysis of tissue and clearing of pigments. For long-term Formalin/Formaldehyde storage, a phosphate buffer is preferred. A suitable phosphate buffer (Srensens Buffer) is prepared using four grams of monobasic sodium phosphate monohydrate (NaH2PO4 H2O) and six grams of dibasic sodium phosphate anhydrate (Na2HPO4) per liter of a solution of 10% Formalin. The archival preservation fluid that has been used the longest and is generally preferred is alcohol. The standard is 70-75% ethyl alcohol or ethanol, however, 40-50% isopropyl alcohol or isopropanol is used on some animal taxa. Formalin may be used for some meiofauna and plankton specimens, especially gelatinous forms. "Sorting solution" (1.5 parts propylene phenoxetol, 5.0 parts propylene glycol, 10.0 parts full strength Formaldehyde, & 83.5 parts distilled water) has been used successfully for the long-tem storage of some taxa, but larger specimens may deteriorate over time. The following table describes recommended fixation and preservation procedures for various invertebrate animal taxa and bulk samples. Specific lengths of time that a specimen is placed in a relaxation, fixation, or wash fluid is a matter of preference and greatly is determined by the size of the specimen. For specific animal taxa, it is recommended that more research on methods of relaxing, fixing and preserving specimens be conducted before specimens are collected and processed. Recipes for various fluids of fixation and preservation (indicated with an asterisk) are shown below. Go To Top
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Recipes Bouins fluid Picric acid, saturated aqueous solution ............................................................. 75 ml Formaldehyde (40 %) ..................................................................................... 25 ml Glacial acetic acid ........................................................................................... 5 ml "Sorting solution" Propylene phenoxetol ..................................................................................... 0.5 ml Propylene glycol ............................................................................................. 4.5 ml Distilled water or sea water ............................................................................ 95 ml Zenkers fluid Mercuric chloride ........................................................................................... 5 g Glacial Acetic acid ......................................................................................... 5 ml Potassium dichromate .................................................................................... 2 g Sodium sulphate ............................................................................................ 1 g Distilled water ............................................................................................... 100 ml 10% Phosphate Buffered Formalin NaH2PO4 H2O ............................................................................................ 4 g Na2HPO4 ...................................................................................................... 6g Formaldehyde (40 %) .................................................................................... 100 ml Sea water or distilled water ............................................................................ 900 ml Go To Top
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General Taxa
Specific Taxa
Relaxing Agent
Fixative
(solution)
Wash
(solution)
Final Solution
(preservative)
Dangers
Meiofauna
General bulk processing with sediment
------
10% phosphate buffered formalin
------
sediment tends to damage "soft" meiofauna during extended storage
Meiofauna
General bulk processing without sediment General bulk processing
------
------
Bulk Macrofauna
MgCl2 isotonic to sea water (approx. 7%) MgCl2 isotonic to sea water (approx. 7%)
5-10% phosphate buffered formalin
30%, 50%, 70% ETOH
70-80% ETOH
remove CRT, ECH, and shelled MOL from formalin solution ASAP ETOH will dissolve gelatinous animals & long term storage in unbuffered formalin decalcifies animals
Plankton
General bulk processing
10% phosphate buffered formalin in sea water
if long term storage in ETOH use 30%, 50%, 70% ETOH, otherwise no wash
70% ETOH or 5% phosphate buffered formalin or sorting solution*
Annelida
Hirudinea, Oligochaeta, Polychaeta
MgCl2 isotonic to sea water (approx. 7%) MgCl2 isotonic to sea water (approx. 7%) OR oil of cloves
30%, 50%, 70% ETOH
70%ETOH
Arthropoda
Decapoda & other larger crustaceans
5-10% phosphate buffered formalin in sea water OR 75% ETOH
50%, 70% ETOH
70%ETOH
legs will separate from the body if spm is over-fixed (too much time in formalin solution)
Branchiopoda, Ostracoda, Copepoda, Cumacea, Tanaidacea, Amphopoda Euphausiacea (Krill)
MgCl2 isotonic to sea water (approx. 7%)
4-10% phosphate buffered formalin in sea water OR 70% ETOH (Ostracoda, Cumacea 10% phosphate buffered formalin or 80% ETOH
------
70%ETOH
50%, 70% ETOH
70%ETOH
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General Taxa
Specific Taxa
Relaxing Agent
Fixative
(solution)
Wash
(solution)
Final Solution
(preservative)
Dangers
Mictacea
MgCl2 isotonic to sea water (approx. 7%) MgCl2 isotonic to sea water (approx. 7%) MgCl2 isotonic to sea water (approx. 7%) or 70% ETOH dropwise MgCl2 isotonic to sea water (approx. 7%) MgCl2 isotonic to sea water (approx. 7%) MgCl2 isotonic to sea water (approx. 7%) MgCl2 isotonic to sea water (approx. 7%) MgCl2 isotonic to sea water (approx. 7%)
4-10% phosphate buffered formalin in sea water OR 70% ETOH 2-4% phosphate buffered formalin
------
70%ETOH
Mysidacea
------
2% phosphate buffered formalin OR 7080% ETOH 70%ETOH
Pycnogonida
70% ETOH
Brachiopoda
Brachiopoda
30%, 50%, 70% ETOH
70%ETOH
Bryozoa
Bryozoa
5% phosphate buffered formalin or bleached in Clorox and dried 8-10% phosphate buffered formalin-sea water 6-10% phosphate buffered formalin
30%, 50%, 70% ETOH
70% ETOH or dry
formalin will dissolve calcareous material over time
Chaetognatha
Chaetognatha
------
Cnidaria
Anthozoa (anemones&corals)
30%, 50%, 70% ETOH
70% ETOH
Hydroid polyps & Octocorallina
30%, 50%, 70% ETOH
70% ETOH or dry
Hydromedusae
5% phosphate buffered formalin in sea water 10% phosphate buffered formalin
------
ETOH causes medusae to shrink
Scyphozoa (jellyfish)
------
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General Taxa
Specific Taxa
Relaxing Agent
Fixative
(solution)
Wash
(solution)
Final Solution
(preservative)
Dangers
Siphonophora
5% phosphate buffered formalin & Zenkers fluid*
------
Ctenophora
Ctenophora
tri-chloracetic acid (1g) in sea water (99ml) OR p-toluene sulfonic acid (1g) in sea water (99ml) 90% ETOH (hold arms downward)
------
sorting solution*
ETOH will dissolve ctenophores
Echinodermata
Crinoidea
------
70%ETOH
prolonged contact with formalin destroys the echinoderm skeleton prolonged contact with formalin destroys the echinoderm skeleton prolonged contact with formalin destroys the echinoderm skeleton
Holothuroidea, Asteroidea, Echinoidea
70-75% ETOH
------
70%ETOH
Ophiuroidea
70-75% ETOH
------
70%ETOH
Echiura
Echiura
MgCl2 isotonic to sea water (approx. 7%) MgCl2 isotonic to sea water (approx. 7%) MgCl2 isotonic to sea water (approx. 7%) MgCl2 isotonic to sea water (approx. 7%)
70% ETOH
70%ETOH
Entoprocta
Entoprocta
------
Gastrotricha
Gastrotricha
------
Gnathostomulida
Gnathostomulida
------
5% phosphate buffered formalin or 70% ETOH
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General Taxa
Specific Taxa
Relaxing Agent
Fixative
(solution)
Wash
(solution)
Final Solution
(preservative)
Dangers
Hemichordata
Enteropneusta
let sit in sea water for a few days to empty intestine then MgCl2 isotonic to sea Water (approx. 7%) MgCl2 isotonic to sea water (approx. 7%) MgCl2 isotonic to sea water (approx. 7%) MgCl2 isotonic to sea water (approx. 7%)
Bouins fluid* or 5-10% phosphate buffered formalin
------
Pterobranchia
------
Kinorhyncha
Kinorhyncha
70% ETOH
70%ETOH
Mollusca
Bivalvia
10% phosphate buffered formalin OR 70% ETOH
30%, 50%, 70% ETOH
70%ETOH
Cephalopoda
if dead fix directly OR MgCl2 isotonic to sea water (approx. 7%)
6-10% phosphate buffered formalin (size dependant) OR 70% ETOH
drain mantle cavity of fluids; then 70-75% ETOH or 50% isopropyl alcohol 30%, 50%, 70% ETOH
70-75% ETOH or 50% isopropyl alcohol
Gastropoda
MgCl2 isotonic to sea water (approx. 7%) MgCl2 isotonic to sea water (approx. 7%) ------
70%ETOH
Monoplacophora/
30%, 50%, 70% ETOH
70%ETOH
Polyplacophora
Nematoda
Nematoda
5% formalin-sea water 4-10% phosphate buffered formalin
------
5% phosphate buffered formalin 3-5% phosphate buffered formalin or 70% ETOH
Avoid using ETOH
Nematomorpha
Nematomorpha (Nectonema sp.)
------
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General Taxa
Specific Taxa
Relaxing Agent
Fixative
(solution)
Wash
(solution)
Final Solution
(preservative)
Dangers
Nemertea
Nemertea
------
Phoronida
Phoronida
30%, 50%, 70% ETOH
70% ETOH
Platyhelminthes
Turbellaria
10% phosphate buffered formalin in sea water and freeze in flat container. Float or coax specimen onto filter paper and place filter paper on frozen mix.
70% ETOH
70%ETOH
Pogonophora
Pogonophora
------
10% phosphate buffered formalin 10% formalinsea water buffered by methenamine
70% ETOH
70%ETOH
Porifera
Porifera
------
70-80% ETOH change twice
70-80% ETOH
long term storage in formalin will macerate tissue and impede ID
Priapulida
Priapulida
------
5% phosphate buffered formalin or 70% ETOH
"Protozoa"
"Protozoa"
10% phosphate buffered formalin or Bouins fluid* or 2.5% glutaraldehyde 5-10% phosphate buffered formalin
------
5% phosphate buffered formalin or dry (some Sarcomastigophora)
Rotifera
Rotifera
MgCl2 isotonic to sea water (approx. 7%) rock dwelling spms in 10% ETOH & sand dwelling spms in MgCl2 isotonic to sea water (approx. 7%)
70% ETOH
70%ETOH
Sipuncula
Sipuncula
10% formalin in 70%ETOH or 5% formalin
70% ETOH
70%ETOH
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General Taxa
Specific Taxa
Relaxing Agent
Fixative
(solution)
Wash
(solution)
Final Solution
(preservative)
Dangers
Tardigrada
Tardigrada
------
3-7% phosphate buffered formalin or 70-80% ETOH
Urochordata
Appendicularia (=Larvacea)
------
Ascidiacea
------
70% ETOH
Thaliacea
------
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Specimen Processing Procedures

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http://invertebrates.si.edu/USAP2/usapspec.html (1 of 10)16/08/2010 08:39:56 p.m.

Specimen Processing Procedures

http://invertebrates.si.edu/USAP2/usapspec.html (2 of 10)16/08/2010 08:39:56 p.m.

Specimen Processing Procedures

http://invertebrates.si.edu/USAP2/usapspec.html (3 of 10)16/08/2010 08:39:56 p.m.

Specimen Processing Procedures

General bulk processing with sediment

10% phosphate buffered formalin

10% phosphate buffered formalin

sediment tends to damage "soft" meiofauna during extended storage

General bulk processing without sediment General bulk processing

5% phosphate buffered formalin

5% phosphate buffered formalin

5-10% phosphate buffered formalin

30%, 50%, 70% ETOH

General bulk processing

10% phosphate buffered formalin in sea water

70% ETOH or 5% phosphate buffered formalin or sorting solution*

Hirudinea, Oligochaeta, Polychaeta

10% phosphate buffered formalin in sea water

30%, 50%, 70% ETOH

Decapoda & other larger crustaceans

5-10% phosphate buffered formalin in sea water OR 75% ETOH

50%, 70% ETOH

Branchiopoda, Ostracoda, Copepoda, Cumacea, Tanaidacea, Amphopoda Euphausiacea (Krill)

MgCl2 isotonic to sea water (approx. 7%)

MgCl2 isotonic to sea water (approx. 7%)

50%, 70% ETOH

http://invertebrates.si.edu/USAP2/usapspec.html (4 of 10)16/08/2010 08:39:56 p.m.

Specimen Processing Procedures

2% phosphate buffered formalin OR 7080% ETOH 70%ETOH

10% phosphate buffered formalin

10% phosphate buffered formalin

30%, 50%, 70% ETOH

30%, 50%, 70% ETOH

70% ETOH or dry

formalin will dissolve calcareous material over time

5% phosphate buffered formalin

30%, 50%, 70% ETOH

Hydroid polyps & Octocorallina

4% phosphate buffered formalin

30%, 50%, 70% ETOH

70% ETOH or dry

http://invertebrates.si.edu/USAP2/usapspec.html (5 of 10)16/08/2010 08:39:56 p.m.

Specimen Processing Procedures

5% phosphate buffered formalin in sea water 10% phosphate buffered formalin

5% phosphate buffered formalin

ETOH causes medusae to shrink

5% phosphate buffered formalin

5% phosphate buffered formalin & Zenkers fluid*

5% phosphate buffered formalin

ETOH will dissolve ctenophores

Holothuroidea, Asteroidea, Echinoidea

MgCl2 isotonic to sea water (approx. 7%)

http://invertebrates.si.edu/USAP2/usapspec.html (6 of 10)16/08/2010 08:39:56 p.m.

Specimen Processing Procedures

5% phosphate buffered formalin

5% phosphate buffered formalin in sea water

5% phosphate buffered formalin

5-10% phosphate buffered formalin

5-10% phosphate buffered formalin