Preserving sheep brains without formalin

Preserving sheep brains without formalin. We no longer use formalin (formaldehyde) for tissue specimens.  Which reagents or products do you recommend for fixing and preserving sheep brains?  We've had mediocre results with alcohols, and freezing did not work.

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Publication Date: 14 October 2015
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Preserving sheep brains without formalin

In Brief:

Fresh brain tissue is delicate, very soft, and can be easily damaged, even when being gently handled. Specimens are generally fixed to prevent tissue breakdown and to render them firm to allow for easy dissection. For long-term storage, they are kept in preservative solutions. The chemicals used to fix and preserve specimens can be hazardous and dangerous. Traditionally, 10% formalin and 70% ethanol have been the chemicals of choice, and are still in use today.

Formalin1 is toxic, carcinogenic, highly irritating and acts as a potent sensitizer. Formalin is usually used as a 10% solution made by combining 1 part formalin with 9 parts water. This is still the best fixative in use today. Formalin may be used in instances where colour is important, since alcohol dissolves most colours almost immediately. Formalin needs to be handled with great care in a fume cupboard or well-ventilated area. Science ASSIST does not recommend formalin for use in schools. [i]

Ethanol2 (Ethyl alcohol) usually comes in the 95% concentrated form. For long-term preservation and storage, it is usually diluted with distilled water to 70–75% strength. This is the lowest concentration at which preservation will be maintained. Alcohol is highly flammable, usually safe to handle, but can cause irritation to the skin in cases of prolonged contact.

Today there are many less hazardous chemicals available, as well as some chemical-free methods for fixing and preserving specimens.

Science ASSIST recommends you refer to the specific SDS for any chemicals being used and conduct a site-specific risk assessment to assess and control any risks. We have developed a Risk Assessment template for schools to use, see Risk Assessment Template. You will need to make sure that any fixative or preservatives are approved for use in your jurisdiction and educational sector and are disposed of appropriately following local guidelines.

We outline some methods below for you to try. As they require the use of a needle and syringe, we recommend you check with your school jurisdiction for any regulations on the use of needles.

Note: These preservative solutions should not be handled by students.

Chemical Methods:

(Note: % v/v is used for concentrations of solutions of liquids and is calculated as [(volume solute)/(volume of final solution)] x 100%.  For example, 80% v/v means that 100 mL of solution contains 80 mL of the solute.)

Method 1:  25% glycerol in 80% v/v ethanol

The Tasmanian Museum3 uses this method for preservation of its soft-bodied animal specimens.

  1. First prepare an 80% v/v ethanol solution: for 100 mL, measure 84 mL ethanol (95%) and make up to 100 mL with distilled water.
  2. For 100 mL glycerol/ethanol solution, measure 25 mL glycerol and make up to 100 mL with the 80% ethanol solution (or combine 1 part glycerol with 3 parts ethanol (80%)).
  3. Using a 10 mL syringe and 21 g x 38 mm needle, inject the solution into the brain tissue in several places—in both the left and right cerebral hemispheres. Injection of the brain helps penetrate the inner tissues before immersion in the preservative solution.
  4. Immerse the brain in the solution for storage in a clean glass container that has a tight-fitting lid to prevent any evaporation. The use of Parafilm or some silicone sealant can be used to provide a good seal.
  5. Store under conditions to prevent any deterioration i.e. a cool dry place with low-light levels and out of direct sunlight. As the solution is flammable, it should be stored in the flammable liquids cabinet.
  6. Leave for 2–3 days to fix the tissue.
  7. After 3 days, pour off the solution and refresh with a new batch.
  8. Leave for at least one month before use for dissection or as a display specimen.
  9. Collect any waste solution into a glass waste container, which can be properly sealed to avoid evaporative loss. Label appropriately and dispose via a chemical waste contractor.

It is beneficial to use at least 2–3 fresh changes of the solution. The more changes the better for fixing (retaining colour) and rendering the tissue firm and rubbery for dissection purposes.

Using the ratio of 25% glycerol in 80% ethanol has the benefit of no real shrinkage; the glycerol makes the brain tissue pliable and it is relatively safe to use. Glycerol4 has low toxicity and helps in preserving and reviving colour.

Method 2:  75% ethanol in 95% ethyl acetate solution (a variation on the Ethanol/Glycerol method)

  1. First prepare a 95% ethyl acetate solution: for 100 mL, measure 95 mL ethyl acetate and make up to 100 mL with distilled water.
  2. For 100 mL of ethanol/ethyl acetate solution, measure 80 mL of ethanol (95%) and make up to 100 mL with the 95% ethyl acetate solution, (or combine 4 parts ethanol (95%) with 1 part of the 95% ethyl acetate solution).
  3. Using a 10 mL syringe and 21 g x 38 mm needle carefully inject the solution deep into the brain tissue in several places—in both the left and right cerebral hemispheres, then leave the specimen in the solution for fixation for around 48 hours. This assists in hardening and reduces shrinkage.
  4. Drain and then preserve in the ethanol/glycerol mixture described above in Method 1. Store in a flammable liquids cabinet.
  5. Leave for at least one month before use for dissection or as a display specimen.
  6. Dispose of waste ethanol/ethyl acetate solution and ethanol/glycerol solution as in Method 1.

The idea behind this is to “set” the tissue before preserving while the glycerol keeps it from getting too hard. If the brain tissue still ends up too soft, then before putting into the preserving solution, place in a solution of 20 drops of ethylene glycol to 100 ml of ethanol/ethyl acetate for 24 hrs.

      7.  Collect any waste ethylene glycol/ethanol/ethyl acetate solution into a glass container, which can be properly sealed to avoid evaporative loss. Label appropriately for disposal via a chemical waste contractor.

Ethyl acetate5 is used as a fixative and is classified as hazardous and flammable. It is a colorless liquid and has a characteristic sweet smell (similar to pear drops).

Ethylene glycol6 assists with hardening tissue. It is classified as hazardous and is toxic if ingested.  

Method 3: 75% v/v Ethanol Solution

  1. For 100 mL of solution: measure 80 mL of ethanol (95%) and make up to 100 mL with distilled water, (or combine 4 parts ethanol (95%) with 1 part distilled water). Note do not use denatured alcohol if using as a preservative. Some denaturants can have adverse effects on specimens7.
  2. Using a 10 mL syringe and 21 g x 38 mm needle carefully inject the ethanol solution deep into the brain tissue in several places—in both the left and right cerebral hemispheres.
  3. Immerse the brain in the ethanol solution for storage in a clean glass container that has a tight-fitting lid to prevent any evaporation. The use of Parafilm or some silicone sealant can be used to provide a good seal.
  4. Leave for 2–3 days to fix the tissue.
  5. After 3 days, pour off the solution and refresh with a new batch.
  6. Leave for at least one month before use for dissection or as a display specimen.
  7. Store under conditions to prevent any deterioration i.e. a cool dry place with low-light levels and out of direct sunlight. Store in a flammable liquids cabinet.
  8. Collect any waste solution into a glass waste container, which can be properly sealed to avoid evaporative loss. Label appropriately and dispose via a chemical waste contractor.

This is a good fixative and preservative that will not overly dehydrate the tissue. Concentrations higher than this are not recommended as they can excessively dehydrate the tissue.

Note: While waiting for pick up, store and segregate waste chemicals safely in approved store rooms or chemical storage cabinets.

Method 4: 2-phenoxyethanol

Another alternative is 2-phenoxyethanol (synonyms: phenoxetol, phenoxytol), which is non-flammable compared with 70% ethanol, is less volatile and of lower toxicity when compared with formaldehyde. See the following article which discusses the relative hazards of 2-phenoxyethanol compared with formaldehyde and ethanol: http://www.academia.edu/9751098/Phenoxetol_as_a_formaldehyde-removing_ag... . See the following link for a recipe: https://web.archive.org/web/20170219043724/http://www.rtg.wa.edu.au/solu...(link changed to an archived copy on the Internet Archive's Wayback Machine July 2017). Note that this is a storage solution only and was introduced because it is non-flammable. This removed the requirement in WA to store specimens that were preserved in 70% ethanol in a flammable liquids cabinet8.

Safety

When working with preserved specimens it is important to:

  • work in a fume cupboard or well-ventilated area;
  • wear appropriate PPE (i.e., safety glasses, nitrile gloves, laboratory coat, closed-in shoes);
  • refer to the specific SDS for the chemicals being used and prepare a site-specific risk assessment;
  • rinse preserved specimens with running water or preferably soak overnight in water before using for dissection;
  • store all flammable preserving solutions in a flammable-liquids cabinet;
  • dispose of all waste solutions via a chemical waste contractor;
  • following dissection, all of the brain tissue should be wrapped in newspaper, double bagged and frozen for later disposal via a waste contractor;
  • attach and dispose of syringe needles safely. Attach sheathed needles only to the syringe and never re-sheath needles as this is how most needle-stick injuries occur.

Dispose of used syringe needles in an approved sharps container—positioned at the point of use. Seal and dispose of a full sharps container at a sharps collection facility, sharps disposal bin via a State Health recommended facility or a facility recommended by your local council. More information on sharps and disposal can be found on the Science ASSIST website Sharps container disposal, and at the following website.

Safe Work Australia National Code of Practice for the Control of Work-related Exposure to Hepatitis and HIV (Blood-borne) Viruses [NOHSC: 2010(2003)] http://www.safeworkaustralia.gov.au/sites/swa/about/publications/Documen...

Alternatives

1) Purchase of preserved specimens

Purchasing preserved brains from a reputable biological supplier is another option available to you. Specimens are initially fixed with a formaldehyde solution, then displaced first with water and lastly preserved with a glycol solution, producing a low-fume specimen, which will not decay over time. No refrigeration is required.

Safety Note–Specimens should be washed in water before use and the dissection should be performed in a well-ventilated laboratory.

Wear appropriate personal protective equipment (PPE).

Obtain and read the safety data sheet (SDS) from the supplier and prepare a site-specific risk assessment.

2) Freeze-dried specimens

Freeze-dried brains. They are preserved without chemicals or fluids, can be used for external study and dissection. The specimens are rehydrated with dilute (20%) alcohol solution and need to be rinsed before use. This is also a technique taxidermists use to preserve specimens. However, this is an expensive procedure and would only be cost effective with a bulk order of specimens.

Contact your local museum or taxidermist for more information.

3) Rubber/plastic models

Rubber/plastic brain models to examine the brain. These can be sourced from Biological suppliers.

4) Freezing

Fresh offal is always best for dissection purposes, but if you want to store fresh sheep brains in the freezer for later dissection, they are best dissected semi-frozen to avoid the mush factor! Freezing can cause loss of integrity, colour and some shrinkage. The formation of ice crystals can also damage the tissue.

Using a 0.9% saline solution9 may assist the cells and tissue structure of the brain to maintain their normal state during the freezing process.

Soak the brain tissue overnight in the 0.9% saline solution and inject parts of the brain tissue (described in Method 1) before freezing separately in sealed zip-lock bags.

Additional Information:

Fixation and Preservation

When preparing specimens for preservation, they are generally put through a multi-stage process.

  • Fixation to prevent autolysis and microbial breakdown
  • Water wash to remove excess fixative
  • Preservation for long-term storage

Labels

Containers should be labelled to state type of specimen and type and date of preservative. Most museums put the jar label inside the jar, not on the outside or on the lid. This will lessen the likelihood of the specimen and label being separated. It is important to use paper intended for long-term preservation in fluids. There are a several papers that will do, including laundry tag paper. See Resistall labels and specimen tags: http://www.universityproducts.com/cart.php?m=product_list&c=241. Soft lead pencil can be used to write on the paper and there are certain inks or ink pens that can be used as well. Any inks used should be of archival quality, resistant to fading and smearing, and be insoluble in the preservative solution. Suitable inks and ink pens can be found in some art or office supply stores and museum supply companies. It is recommended to allow the ink to completely dry before placing the label into the storage solution. Ordinary ballpoint pens should not be used for labelling as they generally dissolve in most preservative solutions.

See the Science ASSIST School science suppliers list for local museum supply companies for similar products.

More information on fixation and preservation can be found on the Science ASSIST website link: preserved specimens.

The following links have more information on fixation and preservation of wet collections.

http://conservation.myspecies.info/node/33

Fluid Preservation: A Comprehensive Reference

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References:

1 Chemwatch Gold 2013 Safety Data Sheet: 10% formalin. https://jr.chemwatch.net/chemwatch.web (Subscription required) (Accessed October 2015)

2 Chemwatch Gold 2013 Safety Data Sheet: Ethyl Alcohol. https://jr.chemwatch.net/chemwatch.web (Subscription required) (Accessed October 2015)

3 Gordon, Tammy. 2015. Natural Science Collections Officer, Tasmanian Queen Victoria Museum, Launceston. Personal communication

4 Chemwatch Gold 2013 Safety Data Sheet: Glycerol. https://jr.chemwatch.net/chemwatch.web (Subscription required) (Accessed October 2015)

5 Chemwatch Gold 2013 Safety Data Sheet: Ethyl Acetate. https://jr.chemwatch.net/chemwatch.web (Subscription required) (Accessed October 2015)

6 Chemwatch Gold 2013 Safety Data Sheet: Ethylene Glycol. https://jr.chemwatch.net/chemwatch.web (Subscription required) (Accessed October 2015)

7’’Standards in the care of wet collections’, NHM Conservation Centre website, http://conservation.myspecies.info/node/33 (July 2014)

8 Kempton, Ruth. 2014. Team Leader, Regional Laboratory Technicians, WA Department of Education. Personal communication

9 Dungey, Barbara. 2006. The Laboratory: a science reference and preparation manual for schools (Rev. ed), National Library of Australia: Traralgon, Vic.

‘National Code of Practice for the Control of Work-related Exposure to Hepatitis and HIV (Blood-borne) Viruses [NOHSC: 2010(2003)]’, Safe Work Australia website http://www.safeworkaustralia.gov.au/sites/swa/about/publications/pages/c... (1 January 1993)

Simmons, John E. 2014. Fluid Preservation: A Comprehensive Reference. Rowman & Littlefield. https://books.google.com.au/books?id=_WqYAwAAQBAJ&pg=PA47&lpg=PA47&dq=sa...

Tandon, A; Bhatnagar, R; Pokhrel, Rishi and Solanke, Kirti. 2014. ‘Phenoxetol as a formaldehyde-removing agent for long-term preservation:out experience’ Eur. J. Anat 18 (4) : 267-272. Academia.edu website, http://www.academia.edu/9751098/Phenoxetol_as_a_formaldehyde-removing_agent_for_long-term_preservation_our_experience (Click on ‘Read paper’ at the base of the screen to open article)

[i] ‘List of recommended chemicals for science in Australian schools’, Science ASSIST website http://assist.asta.edu.au/resource/3052/list-recommended-chemicals-science-australian-schools?search-id=34587a4 (October 2015)

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