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 taxon’s 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 re-identification 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.

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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 (Sörensen’s 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.

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Recipes

Bouin’s 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
 
Zenker’s 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 ...................................................................................................... 6 g
Formaldehyde (40 %) .................................................................................... 100 ml
Sea water or distilled water ............................................................................ 900 ml
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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 ------ 10% phosphate buffered formalin sediment tends to damage "soft" meiofauna during extended storage
Meiofauna General bulk processing without sediment

------

5% phosphate buffered formalin ------ 5% phosphate buffered formalin  
Bulk

Macrofauna

General bulk processing 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
Plankton General bulk processing MgCl2 isotonic to sea water (approx. 7%) 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* ETOH will dissolve gelatinous animals & long term storage in unbuffered formalin decalcifies animals
Annelida Hirudinea, Oligochaeta, Polychaeta MgCl2 isotonic to sea water (approx. 7%) 10% phosphate buffered formalin in sea water 30%, 50%, 70% ETOH 70%ETOH  
Arthropoda Decapoda & other larger crustaceans MgCl2 isotonic to sea water (approx. 7%) OR oil of cloves 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 MgCl2 isotonic to sea water (approx. 7%) 4-10% phosphate buffered formalin in sea water OR 70% ETOH (Ostracoda, Cumacea ------ 70%ETOH  
  Euphausiacea (Krill) MgCl2 isotonic to sea water (approx. 7%) 10% phosphate buffered formalin or 80% 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%) 4-10% phosphate buffered formalin in sea water OR 70% ETOH ------ 70%ETOH  
  Mysidacea MgCl2 isotonic to sea water (approx. 7%) 2-4% phosphate buffered formalin

------

2% phosphate buffered formalin OR 70-80% ETOH  
  Pycnogonida MgCl2 isotonic to sea water (approx. 7%) or 70%ETOH dropwise 10% phosphate buffered formalin 70% ETOH 70%ETOH  
Brachiopoda Brachiopoda MgCl2 isotonic to sea water (approx. 7%) 10% phosphate buffered formalin 30%, 50%,

70% ETOH

70%ETOH  
Bryozoa Bryozoa MgCl2 isotonic to sea water (approx. 7%) 5% phosphate buffered formalin or bleached in Clorox and dried 30%, 50%,

70% ETOH

70% ETOH or dry formalin will dissolve calcareous material over time
Chaetognatha Chaetognatha MgCl2 isotonic to sea water (approx. 7%) 8-10% phosphate buffered formalin-sea water

------

5% phosphate buffered formalin  
Cnidaria Anthozoa

(anemones&corals)

MgCl2 isotonic to sea water (approx. 7%) 6-10% phosphate buffered formalin 30%, 50%, 70% ETOH 70% ETOH  
  Hydroid polyps & Octocorallina MgCl2 isotonic to sea water (approx. 7%) 4% phosphate buffered formalin 30%, 50%, 70% ETOH 70% ETOH or dry  
  Hydromedusae MgCl2 isotonic to sea water (approx. 7%) 5% phosphate buffered formalin in sea water ------ 5% phosphate buffered formalin ETOH causes medusae to shrink
  Scyphozoa

(jellyfish)

MgCl2 isotonic to sea water (approx. 7%) 10% phosphate buffered formalin ------ 5% phosphate buffered formalin  
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General

Taxa

Specific Taxa

Relaxing Agent

Fixative

(solution)

Wash

(solution)

Final Solution

(preservative)

Dangers

  Siphonophora MgCl2 isotonic to sea water (approx. 7%) 5% phosphate buffered formalin & Zenker’s fluid* ------ 5% phosphate buffered formalin  
Ctenophora Ctenophora MgCl2 isotonic to sea water (approx. 7%) tri-chloracetic acid (1g) in sea water (99ml) OR p-toluene sulfonic acid (1g) in sea water (99ml)

------

sorting solution* ETOH will dissolve ctenophores
Echinodermata Crinoidea MgCl2 isotonic to sea water (approx. 7%) 90% ETOH (hold arms downward)

------

70%ETOH prolonged contact with formalin destroys the echinoderm skeleton
  Holothuroidea, Asteroidea,

Echinoidea

MgCl2 isotonic to sea water (approx. 7%) 70-75% ETOH

------

70%ETOH prolonged contact with formalin destroys the echinoderm skeleton
  Ophiuroidea MgCl2 isotonic to sea water (approx. 7%) 70-75% ETOH

------

70%ETOH prolonged contact with formalin destroys the echinoderm skeleton
Echiura Echiura MgCl2 isotonic to sea water (approx. 7%) 5% phosphate buffered formalin 70% ETOH 70%ETOH  
Entoprocta Entoprocta MgCl2 isotonic to sea water (approx. 7%) 5% phosphate buffered formalin in sea water

------

5% phosphate buffered formalin  
Gastrotricha Gastrotricha MgCl2 isotonic to sea water (approx. 7%) 5-10% phosphate buffered formalin

------

5-10% phosphate buffered formalin  
Gnathostomulida Gnathostomulida MgCl2 isotonic to sea water (approx. 7%) 5-10% phosphate buffered formalin

------

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%) Bouin’s fluid* or 5-10% phosphate buffered formalin

------

Bouin’s fluid* or 5-10% phosphate buffered formalin  
  Pterobranchia MgCl2 isotonic to sea water (approx. 7%) Bouin’s fluid* or 5-10% phosphate buffered formalin

------

Bouin’s fluid* or 5-10% phosphate buffered formalin  
Kinorhyncha Kinorhyncha MgCl2 isotonic to sea water (approx. 7%) 10% phosphate buffered formalin 70% ETOH 70%ETOH  
Mollusca Bivalvia MgCl2 isotonic to sea water (approx. 7%) 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 70-75% ETOH or 50% isopropyl alcohol  
  Gastropoda MgCl2 isotonic to sea water (approx. 7%) 10% phosphate buffered formalin 30%, 50%, 70% ETOH 70%ETOH  
  Monoplacophora/

Polyplacophora

MgCl2 isotonic to sea water (approx. 7%) 10% phosphate buffered formalin 30%, 50%, 70% ETOH 70%ETOH  
Nematoda Nematoda

------

5% formalin-sea water

------

5% phosphate buffered formalin Avoid using ETOH
Nematomorpha Nematomorpha

(Nectonema sp.)

MgCl2 isotonic to sea water (approx. 7%) 4-10% phosphate buffered formalin

------

3-5% phosphate buffered formalin or 70% ETOH  
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General

Taxa

Specific Taxa

Relaxing Agent

Fixative

(solution)

Wash

(solution)

Final Solution

(preservative)

Dangers

Nemertea Nemertea MgCl2 isotonic to sea water (approx. 7%) 5-10% phosphate buffered formalin

------

5-10% phosphate buffered formalin  
Phoronida Phoronida MgCl2 isotonic to sea water (approx. 7%) 3-7% phosphate buffered formalin 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 70% ETOH 70%ETOH  
Porifera Porifera ------ 10% formalin-sea water buffered by methenamine 70-80% ETOH change twice 70-80% ETOH long term storage in formalin will macerate tissue and impede ID
Priapulida Priapulida MgCl2 isotonic to sea water (approx. 7%) 5-10% phosphate buffered formalin

------

5% phosphate buffered formalin or 70% ETOH  
"Protozoa" "Protozoa" MgCl2 isotonic to sea water (approx. 7%) 10% phosphate buffered formalin or Bouin’s fluid* or 2.5% glutaraldehyde ------ 5% phosphate buffered formalin or dry (some Sarcomastigophora)  
Rotifera Rotifera MgCl2 isotonic to sea water (approx. 7%) 5-10% phosphate buffered formalin 70% ETOH 70%ETOH  
Sipuncula Sipuncula rock dwelling spms in 10% ETOH & sand dwelling spms in MgCl2 isotonic to sea water (approx. 7%) 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 MgCl2 isotonic to sea water (approx. 7%) 3-7% phosphate buffered formalin

------

3-7% phosphate buffered formalin or 70-80% ETOH  
Urochordata Appendicularia (=Larvacea) MgCl2 isotonic to sea water (approx. 7%) 10% phosphate buffered formalin

------

10% phosphate buffered formalin  
  Ascidiacea MgCl2 isotonic to sea water (approx. 7%) 10% phosphate buffered formalin ------ 70% ETOH  
  Thaliacea MgCl2 isotonic to sea water (approx. 7%) 10% phosphate buffered formalin ------ 10% phosphate buffered formalin  
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