Field Methods for Preserving Tissues

Written by Robb Brumfield and Donna Dittmann for a course in Museum Curatorial Methods, taught at Louisiana State University, Baton Rouge

TISSUE EVERY INDIVIDUAL!!!!

Uses of collections change over time. Over the approximately 25 years that the LSU Collection of Genetic Resources has existed, the genetic techniques du jour have changed dramatically. There is no reason to believe that this trend will not continue. We cannot predict what the future uses of this collection will be.

Specimen Handling

• As soon as an animal dies, DNA exonucleases in the cells begin digesting (i.e. destroying) the DNA. RNases begin digesting the RNA. Proteins begin denaturing. The value of the specimen for future genetic research is determined entirely by (1) The amount of time elapsed between death and tissue preservation; (2) The method of preservation; and (3) How long the tissue has been preserved. Thus, when preparing a specimen, the first thing you should do once the body is removed is sample the tissue and and place it in preservative (liquid nitrogen, buffer, EtOH).
• If you are mist-netting birds, trapping mammals, catching herps, or netting fish, keep them alive until they are to be prepared. For mist-netted birds, the potential downside of this is that feathers may be damaged while the bird is waiting to be prepped. If the animal is euthanized during capture, keep it out of the sun and get back to camp as soon as possible.
• The minimal acceptable amount of time between death and preserving the tissue will vary with temperature during field conditions. If feasible, carry some dry ice with you so that the fresh carcass can be frozen in the field.

Which Tissues to Preserve

• It would be great to preserve a sample of every organ, but practical considerations do not allow this. Minimally, you should preserve a sample of heart, liver, and muscle. If you wish, additional tissues such as kidneys, gonads, and brain can also be preserved. Make sure you make a note of this in the tissue catalog.
• Don't scrimp on tissue. The tube defines the amount of space that the tissue will take up in the collection, so you may as well fill it up. Fill the cryovial to about 5 mm below the threads of the cap. Do not overfill, because the cap might loosen when you drop the cryovial in the liquid nitrogen.
• Be sure to clean your tissue collection instruments between birds to minimize carryover contamination of the tissues! Rinsing the instruments with a 10% bleach solution followed by a water rinse is best. Use ethanol if bleach is unavailable. Use water if neither of those is available.

Tissue Preservatives

Liquid nitrogen (-196°C)

• Fresh, flash frozen tissues in liquid nitrogen represent the gold standard. If you are going to the extensive effort of getting all of the permits, transporting all of your equipment, collecting, preparing the specimens, and bringing them back here, toting a couple nitrogen tanks with you is not that much extra effort. Collections in buffer should only be made in those rare cases in which liquid nitrogen is unavailable.
• Always bring two tanks with you in case one of them fails.
• Airlines do not allow the transport of liquid nitrogen. Get to the airport early, because nitrogen tanks always seem suspicious! Prior to arriving at the airport add some water-filled 50 ml conical tubes to the tank. These will freeze and help keep the contents cool. Empty the tank of liquid nitrogen and add dry ice.
• CAVEATS: If you are in a situation where your tissues must be left behind in the country in which you were working (e.g. if export permits were not obtained), entrusting the tank to someone else is a huge risk. There are now several horror stories where entire tanks of tissues have been lost, because the person left in charge let the tanks run out of liquid nitrogen. In these situations, it is well worth changing your plane ticket and staying a few extra days if it looks like the permit is only going to be delayed for a short while. If you have to leave the tanks, explain the situation carefully to whoever will be in charge of them. If necessary, give them a financial incentive (minimally leave them plenty of cash to continue filling the tanks). Hope like hell they keep them filled!
• If you are in a situation where you might run out of liquid nitrogen in the field (or might have to leave your tanks behind), a second backup tissue could be preserved in small in buffer or ethanol. It is extra effort, but a nice insurance policy.
• Liquid nitrogen tanks are available in a variety of sizes to suit different needs; approximate holding time of the various size tanks is listed below. This is to be a rough guide as to the amount of time a tank will last and it is based on average use (nunc tubes or foil tissue containers). Heavy use (in and out many times daily) or storage of large tissues (including whole carcasses in foil) will lower the holding times significantly.

  	Size of Different Liquid Nitrogen Tanks
  	3DS**	5XTL	10XTL	18XTL	34XTL
  wt. full (lb)	15	24.6	31.5	59.0	94.0
  LN2 capacity (l)	3	5	10	18	34
  static holding time (days)	20	54	100	200	340

  *The actual holding time of a tank in use (being opened and closed repeatedly) will have a holding time of approx. 30% of the static holding time.

  **DS refers to Dry Shipper. This tank is made to transport materials. It has an inner core (filler) that absorbs the LN2. The inside of the tank and its contents are surrounded by cold nitrogen vapor from the saturated filler. This is an excellent means to transport tissue samples or for use in field for very short periods (less than 2 weeks). Holds approx. 30 1 ml tubes.

• In addition to tanks, Styrofoam containers may also be used for collection/ short term storage of tissues or carcasses. A large Styrofoam chest packed with dry ice and paper will hold specimens for a maximum of about one week, depending on how well it is packed, how often it is opened, and the ambient temperature.

DNA Buffers or Ethanol (95%)

• Preserving tissues in buffer or ethanol instead of liquid nitrogen reduces the value of the specimen considerably. It is well documented that DNA extracted from buffer or ethanol-preserved tissues has lower average molecular weight than liquid-nitrogen preserved tissue (i.e. it is more degraded). In addition, neither RNA nor proteins are preserved. In short, storage in DNA buffers and ethanol limit tissues to the PCR-based sequence analyses that are popular right now. Just as the biologists collecting specimens fifty years ago did not have the foresight to keep a tissue sample, it is impossible for us to predict future applications.
• For preserving DNA, the methods are (from best to worst):
  1. liquid nitrogen
  2. 70% - 95% EtOH
  3. 20% DMSO and Saturated Table Salt
• Ethanol preservation works by dehydration: water is removed from the cell and replaced by ethanol. Without water, few of the degradative biological processes responsible for decomposition can take place. Ethanol denatures nucleases.
• DMSO and saturated salt preservation works by osmotic dehydration. The mechanism is two fold: DMSO allows the salt to penetrate the tissue more readily while the salt draws the water out of the cell by osmosis.

RNA

• If you would like to preserve RNA, check out Ambion's RNAlater Tissue Collection RNA Stabilization Solution. This is a storage buffer into which field-collected samples can be placed.
• RNA is stable for 1 day at 37°C, 1 week at 25°C, 1 month at 4°C or indefinitely at -20°C. To extract RNA from frozen tissue, thaw it in the presence of Ambion's RNAlater-ICE Frozen Tissue Transition Solution.

Recipes for DNA Buffers