Calf Foetus

Calf Foetus: A student has brought in a calf foetus stored in methylated spirits. Can you suggest what to transfer it into for preservation? Thank you.

 

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Publication Date: 01 December 2015
Asked By: rhouse
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Calf Foetus

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In Brief

Source of animal

We recommend you check with your school jurisdiction for regulations regarding the use of dead animals or animal body parts that may not have been sourced from a certified abattoir, butcher or science supply company. More information on this can be found on the Science ASSIST website link: Dissection materials.

Preservative solutions for foetal tissue

Foetal tissue is very delicate and is usually preserved in a weak formalin solution (5%), or 75% to 80% ethanol containing 25% glycerol to maintain colour and softness of specimens.  Normally preparation includes injection of preservative into the flesh in several places and into the body cavities to avoid decay of the internal structures.

Recommended Solutions:

The following are recommended as suitable and safe long-term preservative solutions. Depending on the size of the foetus, it may 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.

  • 25% glycerol in 80% v/v ethanol (The Tasmanian Museum1 uses this method for preservation of its soft-bodied animal specimens.)
  • 75% v/v Ethanol Solution
  • 2-phenoxyethanol

Detailed methods for all three solutions are provided below, as well as instructions for transferring from one preservative to another.

Replacement of the old preservative solution

When transferring any specimen from one preservative to another, it is recommended to go through a stepped procedure which includes:

  • decanting the existing preservative solution;
  • rinsing the specimen several times with water to remove the old preservative (This is important to avoid the possibility of combining incompatible chemicals, which may create a hazardous chemical reaction or new substance that may damage the specimen.);
  • replacement with a new preservative solution (Depending on the solution, it may require putting the specimen through several increasing concentrations to avoid any osmotic issues.)

Additional information

Fixation and preservation

Specimens are generally fixed to prevent tissue breakdown and to render them firm. 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.

Formalin2 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]

Ethanol3 (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.

Detailed method for replacing the methylated spirits with new preservative solution

There is not a lot of difference between methylated spirits4 and ethanol, except that it makes the specimen quite brittle and it may have rendered the skin of the foetus transparent, since alcohol destroys most colours almost immediately.

Before transferring the calf foetus to a new preservative solution, it is recommended that the work be done in a fume cupboard with appropriate PPE. You should also not pour any preservative chemicals down the sink and at all times care must be taken to avoid damaging the specimen in the process.

  1. In the fume cupboard or well-ventilated area, decant the methylated spirit solution into a glass container, which can be properly sealed to avoid evaporative loss. Label as 'Caution Flammable liquid - Do Not Use -' for disposal via a chemical waste contractor. Whilst waiting for pick up, store and segregate the waste chemicals safely in approved store rooms or chemical storage cabinets.
  2. Wash the calf foetus several times by soaking for 30 minutes in tap water to remove the old methylated spirits solution.
  3. Place the foetus into the final preservative solution in a clean glass container that has a tight-fitting lid to prevent any evaporation. Use either 25% glycerol in 80% v/v ethanol or one of the alternatives, see methods below.  The use of Parafilm or some silicone sealant can be used to provide a good seal.
  4. Label the container to state type of specimen, 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. The Australian Museum5 currently uses Resistall paper, which they source from the US, but other types of papers have been used in the past, including laundry tag paper: 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.
  5. Store under conditions to prevent any deterioration i.e. a cool dry place in low-light levels and out of direct sunlight.

You will need to take precautions to avoid chemical exposure by contact with skin and eyes, inhalation, and ingestion.  It is recommended to work in a fume cupboard and wear appropriate PPE. At all times care must be taken to avoid damaging the specimen in the process. Care is also required if needles are used. Never re-sheath a needle and dispose of used syringe needles in an approved sharps container

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 fixatives, preservatives and the use of needles are approved for use in your jurisdiction and educational sector and are disposed of appropriately following local guidelines.

Methods for suitable preservative solutions

(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

  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 21g x 38mm needle inject the solution into the body cavity and also into the leg muscles (depending on the size of the foetus). This helps penetrate the inner tissues before immersion in the preservative solution.
  4. Immerse the foetus 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 in 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 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 for display.

Using the ratio of 25% glycerol in 80% ethanol has the benefit of no real shrinkage; the glycerol makes the tissue pliable and it is relatively safe to use. Glycerol6 has low toxicity and helps in preserving and reviving color.

Method 2: 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 as a preservative. Some denaturants can have adverse effects on specimens7.
  2. Using a 10 mL syringe and 21g x 38mm needle, carefully inject the ethanol solution into the body cavity and leg muscles.
  3. Immerse the foetus 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 in 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: whilst waiting for pick up, store and segregate waste chemicals safely in approved store rooms or chemical storage cabinets.

Method 3: 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

  • Refer to the specific SDS for the all chemicals being used and prepare a site-specific risk assessment.
  • Wear appropriate PPE (i.e. safety glasses, gloves, laboratory coat, closed-in shoes).
  • Work in a fume cupboard or well-ventilated area.
  • Store all flammable preserving solutions in a flammable liquids cabinet.
  • Dispose of all waste solutions via a waste contractor.
  • Attach and dispose of syringe 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...

Links to further information

See the Science ASSIST School science suppliers list for local museum supply companies for suitable labels, storage containers and other similar products.

More information on fixation and preservation can be found on the Science ASSIST website links: Preserving sheep brains without formalin

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

References:

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

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

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

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

5 McGrouther, Mark. 2015. Fish Collection Manager, Australian Museum Research Institute, Australian Museum, Sydney. Personal communication

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

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 (December 2015)

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