History:

Beginning in the 17th century, researchers and museums have been able to preserve

whole specimens by submersing and storing them in fluid chemicals. There are three

components to a fluid-preserved specimen:

 The fixed specimen: The specimen is prepared by “fixing” it, achieved by

injecting it with chemicals that stop the deterioration and decay process (known

as “autolysis”). The most common fixative is formaldehyde, or a formaldehyde

and water solution known as formalin. Some specimens may not be fixed before

being submersed in the fluid preserve.

 The fluid preserve: The preserve is commonly alcohol, either ethanol or isopropyl

alcohol.

 The container: Containers are typically glass jars or bottles sealed with a closure.

Types of closures may vary within a collection and often include lids with

gaskets. Large specimens may require the use of open glass tanks.

Although the fixative and fluid preservation process causes a chemical alteration of the

specimen and can lead to discoloration, shrinking, or swelling of the specimen, these

collections are able to last for hundreds of years.

Staff members working with these collections are not generally at risk of chemical injury

from the fluids unless a jar breaks. The greatest hazard of fluid-preserved collections is

the flammable nature of the chemicals. A build-up of fumes will increase the chances of

a fire.
Commonly Treated Items:

The most common animals to be preserved in fluid are aquatic invertebrates (mollusks,

crustaceans, etc.),Herpetology (reptiles and amphibians), and Ichthyology (fish). Some

entomology specimens may also be fluid preserved. Other materials may be stored in

fluids, such as plants, and minerals.

Detection:

Storage jars should be monitored for deterioration of closures (e.g. lids and gaskets)

that would lead to leakage of flammable chemical fumes into the storage area. Open

containers will continuously leak fumes into the environment, so the storage area should

be well ventilated.

Symptoms:

Exposure to formaldehyde and/or formalin can occur when initially immersing the

specimen, handling the containers, topping off the fluid in the containers, or transferring

the specimen to another solvent. Contact with lower concentrations may cause eye and

skin irritations, while higher concentrations can cause more serious symptoms like

pneumonia and pulmonary edema.

Response:

The following suggestions will help prevent breakage of the glass containers and the

possibility of chemical spills, and will promote protection of the specimens and museum

staff in the storage areas:

  Storage shelves should be flat and level, preferably with a lip on the edge to keep

containers from falling off and breaking.

 Do not overcrowd storage shelves.

 Ensure that all staff working with the specimens know proper handling

techniques.

 Use flammable materials signage in storage spaces. Set up appropriate fire

equipment (such as sprinklers and fire extinguishers).

To protect from contact with formalin if a container does break or during transportation

of the specimen from one solvent to another, always:

 Wear personal protection equipment (PPE) to protect clothes and the body, such

as disposable gloves, eye goggles, and when working with a large amount of

solvent, a lab coat.

 Install an eye wash station in storage areas.

 Keep storage areas well-ventilated.

 Keep formaldehyde spill kits accessible in storage areas. Kits are available for

purchase on-line.

Institutions that re-use ethanol as the fluid preserve through an ethanol recovery system

should follow guidelines set for the handling of hazardous wastes. The Environmental

Protection Agency website outlines hazardous waste regulations.

Case Study: The American Museum of Natural History recently completed the C. V.

Starr Natural Science Building that houses the Invertebrate Zoology alcohol-preserved
collections in a 4,000-square-foot compactor facility with an adjoining laboratory area.

The more than 700 cases in the unit allow for well-organized and secure storage in an

environment of stable temperature and humidity. The museum equipped the storage

area with several safety precautions that meet regulations set by the New York Fire

Department, including:

 Sprinkler system

 Storage of materials at least one foot away from a sprinkler head to allow

unimpeded water flow

 Supply of fire extinguishers that are inspected annually by an outside source

 Two-hour fire rated construction for the walls of the storage area

 One and a half hour fire rated construction for the doors of the storage area

 Automatic door closers on all doors

 The use of flammable storage refrigerators only for alcohol storage

 No smoking signage posted inside and outside the storage are

Preserving
vertebrates in alcohol
It's not every day that you run in to some recently deceased creature; when you do, you can turn it into an
informative specimen for your collection. The process described here works for mammals, reptiles
(including birds) and amphibians.

That said, for anything bigger than a cat I wouldn't be too hasty getting an aquarium full of alcohol. The
alcohol must penetrate all parts of the organism, and in big organisms decay may start before this can
happen. Bigger things are more suited to taxidermy. There's a reason you never see bears in jars.

The method below also works for fish, insects and arthropods in general, although they will lose all
colouration. If you do wish to preserve fish or arthropods in ethanol, photograph your specimen fresh so
you'll know what it looked like.
This process will not work for soluble organisms (and aren't we all, somehow): snails, jellyfish, that sort
of thing. Anything that you think may dissolve in alcohol... it probably will. The preservation of very soft
things is something that has plagued scientists for a long time; the best way so far appears to be to
build breathtaking glass models portraying the delicate wonders as they were in life.

note on collecting
Don't handle animals with your bare hands while collecting. They may have died of disease, and it may
be transferable to you, or other animals. Get a plastic bag or a newspaper or anything that will form a
barrier between you and the dead animal. Whether you touch it directly or not, wash your hands with a
strong soap (washing up liquid for instance) afterward before touching anything else. Don't risk your
health.

alcohol
We're using 70% ethanol (usually labeled alcohol ketonatus); it's the percentage used for everything
ever in science. Less would permit rotting, and more would be a waste of precious alcohol.

You can get bottles of ethanol at drugstores. It will usually be methylated, meaning methanol has been
added so it becomes undrinkable. I recommend you buy 100% (or 98%) and then dilute it to 70% with
demineralised water (from the same store) to save cost; the high percentage and the 70% cost the same.

the container
It can be hard to find a perfectly sized glass jar. My modus operandi is to pay a visit to the supermarket;
there will often be a jar of asparagus or sundried tomatoes or mango chutney that is precisely the right
size. I'll use the contents for a refreshing salad or something and the container for some creepy science. If
you can't find the right jar or if you've ordered something perfect but you have to wait, just put your
specimen in any old container with the alcohol in. It'll keep; that is, after all, the idea.

into the alcohol

First, wash your animal with water to remove dirt. If the animal is very dirty and you have to get hands-
on, get some sturdy dishwashing gloves and get to work. You can also use soap if there's a lot of grease.
Worry not, however; generally, a rinse under the tap is enough.

After that it's as simple as putting the specimen in the jar and filling the jar up with 70% ethanol. Turn
and shake it until there's no more bubbles anywhere, and your specimen is ready for eternity.

positioning
This is a subject of some creativity. For smaller animals I tie nylon string into a noose around the neck,
attached to the top of the jar. I make a little crossbar out of a skewer that I wedge into the mouth of the
jar, as if I were propping it open, so that I can tie stuff to it.

For things that require more elaborate tying, use your imagination. I've used transparent plastic plates to
tie things to that didn't want to look pretty on their own; things like bat wings or frog's legs will test your
mettle. If you're stuck, email me and I'll happily think along with you.
A pheasant (Phasianus colchicus) that died in its egg because a child had shaken it. The farmer on whose
land the crime took place caught the culprit red-handed and gave the egg to me, that I could preserve it. It
required several alcohol changes, I imagine due to the yolk which kept colouring the fluid yellow.

after some time

Over time, the liquid will acquire some colour or murkiness. Leave it to colour up for a while, then take
out your specimen, rinse it under the tap, replace the alcohol and put the specimen back in. This may have
to be repeated a few times, especially for larger creatures (who may also have appreciable amounts of
food in their stomachs and feces in their colons that will pollute your fluid). It's the soluble materials
dissolving out of your animal; they will run out in the end, and the alcohol will stay clear. Luckily alcohol
is not the most expensive thing in the world.
A preserved mouse (Mus musculus), from a trap in my friend's 400 year old mouse-infested house. Some
yellowing of the fluid is perceptible; I'll leave it for another couple of weeks and then replace the alcohol.
The mouse is suspended by a little nylon noose. Incidentally, this jar is terrible as the alcohol keeps
slowly evaporating from it. I prefer twisty tops or proper sealine.

 us materials signage

Preservation Of Ctenophores.
The ctenophore, Mnemiopsis leidyi, is a widely distributed species in North American and European

coastal waters. The species is a significant planktonic predator feeding on prey such as copepods, fish

eggs and fish larvae. Mnemiopsis leidyi are self-fertilizing hermaphrodites with each individual

producing several thousand offspring and blooms of this ctenophore occur at regular intervals (Purcell

et al., 2001). In such situations M. leidyi may seriously influence zooplankton communities and cause

long-lasting perturbations of the ecosystem; the most prominent example is perhaps the recent rapid

changes that took place after its introduction into the Black Sea causing a complete collapse of the

pelagic ecosystem and serious declines in anchovy and other fish populations (Shiganova, 1998).

Because of the high feeding rates of the species (Purcell and Arai, 2001; Purcell et al., 2001; Waggett and

Sullivan, 2006), the spread of M. leidyi to waters outside its historical range is currently of great concern

due to the possible impacts on pelagic ecosystems in general and fish populations in particular (Purcell

and Arai, 2001). In 2006, M. leidyi was observed in the North Sea, Kattegat and western parts of the

Baltic Sea (Hansson, 2006; Javidpour et al., 2006) and by 2007 it was widely distributed in inner Danish

waters and the Baltic Sea (Tendall et al., 2007; Kube et al., 2007). The potential threat of this invasive

ctenophore makes close monitoring of its patterns of distribution and abundance imperative. However,

routine sampling and monitoring of M. leidyi abundance has been seriously hampered because no

method for preservation has been available. Biomass estimates of ctenophores have therefore

commonly been restricted to measuring the total live volumes from plankton tows. Estimates of density

and size distribution, which are essential for calculation of clearance rate and predation impact, have

required immediate and labour intensive examination of fresh material. The commonly used fixatives,

buffered formaldehyde and ethanol, both cause gelatinous plankton such as ctenophores to disintegrate

making species identification, counting and measuring practically impossible (e.g. Hosia and Ba˚mstedt,

2007; Kube et al., 2007). Only one study describes a method in which the size distribution and density of

preserved doi:10.1093/plankt/fbp030, available online at www.plankt.oxfordjournals.org # The Author

2009. Published by Oxford University Press. All rights reserved. For permissions, please email:

journals.permissions@oxfordjournals.org JOURNAL OF PLANKTON RESEARCH j VOLUME 31 j NUMBER 8 j

PAGES 917–920 j 2009 by guest on October 21, 2015 http://plankt.oxfordjournals.org/ Downloaded

from (5% formalin), and therefore deteriorated, specimens of M. leidyi were estimated using the size of

tentacle bulbs of preserved animals (Purcell, 1988). Preservation of larval stages of M. leidyi with 5%

acid Lugol’s solution was reported by Costello et al. (2006), but the efficiency of this preservative was

not discussed in detail. Here we describe a simple but efficient method to preserve ctenophores using

acidic Lugol’s solution. Lugol’s solution is a recommended fixative for phytoplankton, acidic Lugol’s

solution being the most gentle fixative for delicate organisms with the exception of organisms having

calcium carbonate in their cell wall structures (Edler, 1979). Acidic Lugol’s solution may also be used for

fixation of microzooplankton (Stoecker et al., 1994). Adult M. leidyi for this study were collected in

Femern Belt (Western Baltic Sea) in the beginning of December 2008, using a standard Bongo net with a

mesh size of 500 mm. Salinity was 14 psu and water temperature was 68C at the time of collection. Live

specimens were kept cold and aerated during transport to the laboratory. Subsamples were collected

for analysis of the effect of different fixation methods on the average length of the animals. The

following acidic Lugol fixation strengths (final concentrations) were tested: 0% (reference), 1, 2, 5 and

10%. Acid Lugol’s solution was made by dissolving 100 g KI in 1 L of distilled water. Fifty grams of

crystalline iodine (I2) were dissolved in 100 mL glacial acetic acid, and the two solutions were mixed

(Throndsen, 1978). Precipitates were later removed and the solution was stored in a dark bottle.

Following fixation for approximately 24 h, a number of M. leidyi were measured to quantify shrinkage in

different concentrations of fixative. Ctenophore body length was expressed as oral–aboral length

(Sullivan and Gifford, 2007). Reference specimens were measured prior to fixation. When preserved

with 1% acidic Lugol’s solution, the animals disintegrated very quickly. Shaking of the fixed sample

resulted in a “soup” of slime with no trace of M. leidyi other than fragments like comb-plates and parts
of the digestive system. However, with increasing strength of the acidic Lugol’s solution, preservation of

the animals was markedly improved. Thus, at a final concentration of 2%, the animals stayed intact and

were quite stable even after preservation for 105 days. After this time, about 80% of the specimens

remained intact and identification was possible. With further increase in the strength of the Lugol’s

solution, the individuals became increasingly tanned, hardened and the mechanical strength of the

gelatinous bodies of the animals improved (Fig. 1). At 5% solution, it was possible to manipulate the

animals with a pair of tweezers without Fig. 1. Photographs of fixed Mnemiopsis leidyi after 24 h of

fixation in petri dishes, as well as specimens manipulated with a pair of tweezers. Upper panel, from left

to right: unfixed, 1, 2, 5 and 10% acidic Lugol’s solution; a 5 by 5 mm grid is shown to indicate size of

specimens. Lower panel, from left to right: 2 and 10% acidic Lugol’s solution. JOURNAL OF PLANKTON

RESEARCH j VOLUME 31 j NUMBER 8 j PAGES 917–920 j 2009 918 by guest on October 21, 2015

http://plankt.oxfordjournals.org/ Downloaded from any damage (Fig. 1). After 24 h at 10% Lugol’s

solution, the shrinkage effect was significant with preserved animals resembling a raisin-like structure.

Size distributions of the samples fixed at strengths of acidic Lugol’s solutions from 2 to 10% were easy to

obtain, and the number of individuals per subsample was remarkably constant between the different

subsamples indicating no loss of animals as a result of the fixation during the first 24 h (Table I). After

105 days, some specimens deteriorated, and precise measurement of length was possible for only 53–

80% of the preserved animals. The average measured length of M. leidyi decreased with increasing

strength of the acidic Lugol’s solution. For example, in 10% acid Lugol’s solution, individuals shrank to

45% of their original size, indicating that a correction factor of approximately 2.2 must be used to

estimate the length of live specimens from specimens preserved in 10% solution. The degree of

shrinkage increased with time and after 105 days the length of preserved specimens was only about half

that of specimens preserved for 24 h. This is largely consistent with the results reported by Sullivan and

Gifford (2009) for larval stages of M. leidyi preserved with acidic Lugol’s solution. We used the data
shown in Table I to generate a relationship between the fixative strength and the conversion factor (Fig.

2). The shrinking observed for M. leidyi is similar to shrinking rates found for phytoplankton preserved in

acidic Lugol’s solutions (Montagnes et al., 1994; Stoecker et al., 1994). This study and the accompanying

paper by Sullivan and Gifford (2009) demonstrate that, contrary to what has often been stated, it indeed

is possible to preserve M. leidyi. Depending on the desired stability of preserved specimens, there will

be a trade-off between the increasing stability of the fixed animals and a reduced possibility of

observing details in/on the animal. For quantitative studies with ctenophore populations dominated by

M. leidyi, strong fixation can be used routinely. In more complex situations with mixed populations of

different species of ctenophores, the relative abundance of each species may be obtained from lightly

preserved (2%) and/or live samples, provided that preservation of other ctenophore species is possible

with this technique; more research is needed to answer this question. However, the shrinkage observed

may depend on salinity (Sullivan and Gifford, 2009) and probably is species specific. More studies are

needed to elucidate these issues before specific recommendations for preservation can be given.

However, for most purposes, it appears that preservation in 2% acidic Lugol’s solution gives good results

with the quality of specimens remaining stable for up to 3 months (Table I; Sullivan and Gifford, 2009).

Specimens kept for 3 months could easily be identified with morphological characters remaining

distinct. If tanning makes identification difficult, the colour can be removed by adding small amounts of

sodium thiosulphate. The amount of Fig. 2. Linear relationships between final concentration of acidic

Lugol’s solution in 14 psu sea water and the conversion factor necessary to estimate oral–aboral length

of Mnemiopsis leidyi prior to preservation from oral–aboral length of preserved specimens. Closed

circles are preservation for 24 h. Open circles are preservation for 105 days. Each point is the mean of at

least 47 observations. See text for further details. Table I: Mean oral–aboral length (mean+SD) of

Mnemiopsis leidyi after fixation for 24 h and 105 days, respectively, in 14 psu sea water with different

concentrations of acidic Lugol’s solutions Final concentration (%) Length after 24 h (mm) Length after
105 days (mm) Un-preserved sample 6+6 (48) – 1 Not determineda Not determineda 2 4.6+3.6 (88)

2.6+0.9 (71) 5 3.3+2.2 (89) 2.0+0.8 (47) 10 2.7+1.5 (91) 1.5+0.6 (73) A subsample of 100 mL was analysed

at each concentration except for un-preserved animals where only a 50 mL subsample was used. Figures

in brackets indicate the number of observations. a Length and number of animals could not be

determined because animals disintegrated at this concentration. K. ENGELL-SØRENSEN ET AL j

PRESERVATION OF THE INVASIVE CTENOPHORE MNEMIOPSIS LEIDYI 919 by guest on October 21, 2015

http://plankt.oxfordjournals.org/ Downloaded from sodium thiosulphate can be varied to achieve the

desired bleaching effect. A simple method for preservation of ctenophores, as we describe here, has

several advantages apart from those that are obvious. For example, when performing ichthyoplankton

surveys, various medusae, including lobate ctenophores, are often much more numerous in the samples

than fish eggs and fish larvae. If the ctenophores disintegrate during fixation of samples, this will result

in potential drawbacks such as failure to estimate the predation impact that ctenophores might have on

ichthyoplankton and making it difficult to find ichthyoplankton in the sample. Lastly, it should be

mentioned that acidic Lugol’s solution is less harmful to humans when compared with aldehyde-based

fixative.