World Oddities Expo - April 15, and Sunday, April 16 2023 Pennsylvania Convention Center

Peculiarities Under Glass:

an in Depth Look at Wet Specimen Preservation

Introduction

Join Dr. Mark Miller President of the Philadelphia Herpetological Society and moderator of the Facebook group Wet Specimen Preservation Techniques for an amazing talk on the art and science of preserving zoological specimens in jars. He will bring some interesting specimens and explain what really goes into making these important educational displays that could last a century or more. Whether you are a collector, preservation enthusiast, or just a biology nerd, this is sure to be an interesting and insightful presentation followed by a Q & A session. All ages welcome.

Why preserve specimens?

Museums and scientists use preserved specimens to compare species, study morphology, or even look for early evidence of pathogens. Doctors and medical students use anatomical specimens to expand their understanding of anatomy. Some people like the artistic component of Nature -- there are many varied reasons and all should be appreciated.

Cleared & Stained frog wet specimen embedded in epoxy by the author.

This is my basic outline of wet specimen preservation followed by a list of some common supplies:

Inject 10% buffered formalin into all organs, brain, major muscle groups, etc. Inject well into the abdomen. Try to get as much in without too much distortion of specimen. Then soak in the same formalin for a couple weeks to effect fixation. The larger the specimen, the longer it needs to soak. It will stiffen up when complete.

Next soak in distilled water for no more than one day to remove excess formalin.

Finally store in 70% alcohol (ethanol or isopropyl) in a sealed jar with a non-metallic lid to avoid rusting.

Formalin requires personal protection equipment [PPE] like a respirator with 6005 cartridge, gloves, etc. Work in an area away from food, pets, children.

Alternatively you can try to “fix” with high concentration ethanol but failure is more likely until you gain experience in injection.

Formalin may be reused a few times but needs to be disposed according to your community hazardous materials drop off location when finished. Label appropriately. Include the contaminated distilled water. Never discard down the drain, or in the environment. Also do not attempt neutralization of discarded formalin. Avoid buying non-buffered formalin.

If you have supplies available, do not freeze. If you need time to assemble what you need, then you may freeze the specimen.

Here are some common supplies (you will need some but not all)

formalin: https://amzn.to/3ifV4nq

https://amzn.to/3eQ1Vmy

https://amzn.to/3jOpuOa

https://amzn.to/300KzNi

5 gal Bucket lid: https://amzn.to/38jJLHU

70% Isopropyl Alcohol: https://amzn.to/3iUEsCJ

70% Iso Alcohol https://amzn.to/3gZcfbv

99.9% Isopropyl Alcohol https://amzn.to/2Z7YKQD

20g needle/syringe https://amzn.to/2RUQlwk

18g needles https://amzn.to/3r3Dfg3

100ml Large Syringe https://amzn.to/3syPIbJ

Qorpak jars https://amzn.to/3d4fSv9

Mini screw top vials https://amzn.to/3sAt0QM

Empty Floater Pens https://amzn.to/3AheVMB

Cork sealing wax https://amzn.to/3vgdLP5

Specimen Labels: https://amzn.to/3nSrqYb

Measuring cylinders: https://amzn.to/2Zn8hUs

Stainless Steel Positioning Pins https://amzn.to/38c3Vnx

20 Pcs Dissection Tool Kit https://amzn.to/3nhbT4A

Moisture Absorbing beads (blue to pink) https://amzn.to/2NZeqmH

Silica Gel Desiccant Beads (orange to green) https://amzn.to/3e1oVAK

Re-openable jar sealant: https://amzn.to/3jQ9XNJ

Clear, White, Black Silicone https://amzn.to/3bOg56H

Bottle sealing wax https://amzn.to/3eHJuSw

Mason jar lids: https://amzn.to/3zrY20k

Trypsin: https://amzn.to/3bcwaUg

Red dye: https://amzn.to/3xYIaCN

1 Gal Vegetable Glycerin https://amzn.to/2FiL5zt

Thymol https://amzn.to/33IFjQy

Reference Book: Fluid Preservation by Simmons https://amzn.to/34HgQfb

Reptile collecting wets https://amzn.to/3AaOWp7

Safety equipment and PPE:

Respirators https://amzn.to/38TWUGK

Goggles https://amzn.to/2BiRGbl

Respirator cartridge 6005 only https://amzn.to/3eoXq0O

4mil thick Nitrile gloves https://amzn.to/2Zl5SbF

Nitrile 4mil https://amzn.to/3dWUuf8

Extended cuff https://amzn.to/3biYxAI

Disposable Aprons: https://amzn.to/32hb43I

Red bags: https://amzn.to/338cHzX

Puppy Pads for work surface: https://amzn.to/2ZE49je

Plastic Sheeting https://amzn.to/3bQa0Hf

Fire Extinguisher https://amzn.to/3q1SXqC

Formaldehyde Test Kit https://amzn.to/2XAHItC

Formaldehyde Spot Check https://amzn.to/3sm8EdA

Formalin Spill Kit (one liter capacity) https://amzn.to/3sjZDml

(larger kits) https://amzn.to/35xSeWA

Here is a human part the author preserved in 1982. A teenage boy was using a snow blower and the snow kept clogging the machine. He grew tired of turning it off to clean out the chute and while unclogging with the engine on, the blade caught two fingers. Initially we thought he just lost the tip of his fingers but as you can see entire tendons were pulled out from the tendon sheath and from the muscle belly that runs the length of the forearm. I was working as a paramedic at the time. Since they were not replantable the parents gave me permission to make an educational specimen.

Nothing is a more powerful reminder of machine safety than a specimen like this.

Fixed with 10% NBF and stored in 70% ethanol. Never reopened since it was made.

Available for inspection at the WOE lecture.

Q&A

What is formalin?

Formalin [German trade name] is a solution in water of the colorless gas formaldehyde (CH2O) or (HCHO). A saturated solution contains about 40% by volume — or 37% by weight — of the gas, plus a small amount of a stabilizer, usually 10-12% methanol; this prevents polymerization.

We use Neutral Buffered Formalin (NBF) to prevent acidification due to formaldehyde’s tendency to be oxidized to formic acid. The buffer solution also enhances formation of monomeric formaldehyde (methylene hydrate), as a fixation reagent.

For fixing specimens the standard fixative is 10% neutral buffered formalin (NBF). To make a fixative for this we need a 10% solution of this stock formalin i.e. 1 part of the stock formalin (37%) with 9 parts distilled water. This makes an unbuffered formalin solution, which will have a pH of 3-4. If used unbuffered the acidity can react with haemoglobin in the tissues to produce dark brown acid - formaldehyde haematin precipitates, which discolor or cloud the specimen. To adjust the 10% formalin solution to a neutral pH you would need to mix in quantities of a buffer, traditionally sodium phosphate as follows: 100ml Formalin (37-40% stock solution) 900ml Water 4g/L NaH2PO4 (monobasic) 6.5g/L Na2HPO4 (dibasic/anhydrous.)

How do you more safely store chemicals at home?

A locked and labeled metal cabinet in a climate controlled room would be a good start.

How long do you leave a specimen in 10% buffered formalin after injection?

Tiny things like a tadpole or hairless mouse could take just a couple days, while bigger specimens take a couple weeks or more. Some hefty things like newborn goats or horses could take a couple months to be sure of fixation.

How do you know it is properly “fixed” after injection and soak?

The specimen becomes rubbery then stiff. After a little experience you will be able to determine easily. While learning, just allow generous time in the formalin and feel it periodically.

Axolotl is preserved with 10% formalin and stored in 70% ethanol. Collection of Mark F. Miller

How long does it take to be able to tell if your specimen has been fixed properly?

Depends on the density/mass of the specimen. The first indication is that it becomes very stiff.

If unsure of fixation, just allow extra time in formalin.

Can you put something other than your specimen in a jar to prop it up the way you want it?

Sure, I use clear marbles frequently to position specimens. After fixation, I remove the marbles and the specimen (usually) retains the position. Stainless steel insect pins are also ok. Specimens can also be mounted to glass or plastic panels.

How do we remove excess formalin in the wet specimen prior to transfer to isopropyl alcohol?

Just soak in distilled water for up to a day. Excess formalin will be washed out into the water. Dispose of the water as hazardous material, same as used formalin.

How much color fade would one expect in a wet specimen? Why do some animals fade a lot and some only a little?

The chemicals that make the pigments in animals are delicate and both formalin and alcohol tend to fade them. The most common losses are warm reds and yellows. Most blacks and grays will persist.

If you are working with a snake or lizard, etc. I have noticed that sometimes there is shedding skin. Is there a good way to either prevent this or a good way to get off all the shed so your finished specimen looks cleaner?

Sometimes rubbing reptile scales with your fingers or a microfiber cloth can remove shedding skin but if it wasn't ready to shed, you might have to live with it.

Can you "over" fix a specimen by leaving it in formalin for too long? Also, how do you know when you've injected them with enough?

Yes, you can leave it in formalin too long. But that time is considerable. The buffers in the formalin don't last forever. When the buffer ages, the solution becomes acidic and may damage specimens. 10 months to a year is definitely too long. Some delicate specimens might find 6 months too long.

I fixed some fetal kittens, not sure how far they are in term but they are formed and palm sized but no hair. How do I prevent wrinkling and shrinking when I transition them to isopropyl?

Alcohol is very dehydrating. You can often mitigate wrinkles by slowly increasing the alcohol concentration. Start low, say 30%, then 50%, then finally 70%. About a day in each concentration. Use an online "dilution calculator" to determine parts of alcohol to water. A graduated cylinder helps here.

Can used formalin be filtered or reused for another specimen? And if so, how many times or in what time frame is it okay to reuse it?

Sure you can reuse it a few times depending on the moisture content of the specimens. Only need to filter if using for injection. You can tell its losing potency when fixation takes longer than expected. Specimens become stiff when fixed. I would use formalin for up to a year.

I have tried to do a few lizards, and many of them end up detaching their tails. Any tips or tricks to help with this?

Just handle carefully. You can also glue the tail back on with superglue gel or E6000 but a few yrs in alcohol might release the glue if the jar is handled roughly.

How long should I leave a 1500g ball python in formalin for? What is the proper way to inject the formalin? I did every inch in the belly, and I cut horizontal slits in the belly as well every few inches to try and help the fluid exchange.

A snake that size I'd probably leave in 10% formalin for three weeks.

Note: Fixation time is an art and science. There are few hard and fast rules when it comes to fixation.

In reality, we routinely overestimate the soak time in formalin. Safer that way.

But it also depends on how well we inject, if we inject. Injection starts fixation from the inside as soaking does from the outside.

I would use weight as a better measure of specimen density than size (volume.)

If you keep a log of the weight of everything you process, and compare over time, you can get a pretty good idea of fixation time.

The scientific reason is a little more complex and relates more towards thickness though:

Penetration rate can be expressed as d = K√t, where d is the depth of penetration, K is the coefficient of diffusion (specific for each fixative), and t is the time. In practical terms this means that the coefficient of diffusion (K) is the distance in millimeters that the fixative has diffused into the tissue in one hour. For 10% formalin K = 0.78. This means that your formalin fixative should not be expected to penetrate more than say 1 mm in an hour.

Medawar PB. The rate of penetration of fixatives. J Royal Micros Soc 1941;61;46-57.

Is there an amount of time after which a frozen specimen is no longer good? How long is too long for a specimen to be frozen?

Sometimes frozen specimens become "freezer burned" but generally that is just cosmetic and the specimen can still be processed.

For long term freezing, place the specimen in a plastic container submerged in water. This will prevent freezer damage. It's even better than vacuum packing. Some specimens can wait years in the freezer to be processed as wets.

Does alcohol volume to specimen(s) in a jar matter? Like, can two animals be in the same jar and completely submerged and it be okay?

The alcohol volume to specimen volume is not critical. You don't want the specimens packed in like sardines, but any reasonable amount is OK.

The formalin to specimen ratio, however IS important.

I would suggest at least 3-4 parts of formalin to 1 part specimen. Some people even advise more formalin.

I have a snake with two holes in it; I think it was hit by a car -- do i need to do anything special about that?

The holes could be closed with fishing line if desired.

Can I put anything in the bottom of a wet specimen longterm? Like river rock, aquarium rock, marbles, etc?

Yes, certainly. Anything that will not dissolve or stain the alcohol can be used for artistic effect.

Is it possible to make specimens from alcohol soft again? Reverse fixation?

For most fixed specimens, I'd probably say the cross proteins that stiffen them can not be reversed. But some experts that do restoration of valuable antique wet specimens do employ some chemical tricks that may allow a degree of repositioning but much depends on the type of specimen and the resources you have available. For most ordinary specimens, it is not worth the effort. They may also be damaged in the attempt.

Another common question is about temperature and our solutions...

Moderate swings in temperature are tolerated but extreme temps could be a problem. At temperatures below about 15°C, alcohols tend to layer (particularly isopropyl), formaldehyde begins to precipitate out as paraformaldehyde (indicated by a whitish cloudiness in the fluid or the formation of paraformaldehyde needles), dissolved lipids will congeal, and some compressible gaskets will lose seal and therefore permit some evaporation.

At warmer temperatures, the processes of deterioration proceed faster (e.g., extraction of lipids and proteins in alcohol) and evaporation rates are higher. Once the specimen is fixed the storage should be in a stable environment away from direct sunlight.

How can I ensure a specimen (say, small piglet) has a specific stance, like sitting? Can I fix it beforehand? Are there some things I can’t use to keep a position in formalin? (Styrofoam, plastic, wood, metal, etc?)

You can use most any non rusting metal like stainless steel but I usually use nylon zip ties, or fishing line. Occasionally I use Titanium wire for some mounts where the wire would be retained within the specimen or holding it to a support. Ti wire will never degrade. You can use Styrofoam to make a cradle or armature of sorts. You will need to weigh down the foam or float the specimen face down in the formalin. Formalin does NOT dissolve common plastics.

A 22 year old Charles Darwin preserved this octopus while exploring the Galapagos Islands in 1832. He used ethanol alcohol.

“I took several specimens of an Octopus, which possessed a most marvellous power of changing its colours; equaling any chamaelion, & evidently accommodating the changes to the colour of the ground which it passed over. – yellowish green, dark brown & red were the prevailing colours: this fact appears to be new, as far as I can find out.”