Revision as of 17:01, 13 May 2019

Statement of Purpose

These links and documents contain information about best practices for tissue sample collection

Introduction

Contributors

Major editor: Breda Zimkus. Original content generated during The American Society of Ichthyologists and Herpetologists (ASIH) Annual Joint Meeting - 2016, during an iDigBio sponsored workshop by the following individuals participating in the "Field to Database" Group of the aforementioned workshop: Breda Zimkus, Cesar Aguilar, Ben Frable, Meredith Mahoney, Zachary Randall, and David Wernecke.

Preparation

• Sterilization of all dissection equipment (including surface if not using disposable for surface)
• Weighing paper, scraps of thermal paper (left over from printing tags), parafilm, paper towels may be used as disposable surface covers, though none are sterile. Can use on top of cutting board.
• Gloves
• Field catalog to write additional notes (see Preparation)

Vials

One area of immense variability is with the number of vial types used in natural history collections (Zimkus and Ford 2014). Potential vial issues are the same for both mechanical freezers and cryovats, although more extreme for cryovats because of their colder temperatures and quicker temperature change. Samples stored using LN₂ must have vials that are rated for use with cryogenic temperatures, preferably made of polypropylene with screw-top caps. Glass vials are problematic when frozen because of their fragility when handling and vulnerability to cracking when stressed, and their use with LN₂ is unacceptable because leakage into the vials can lead to the vessels exploding. In addition, pop-off lids are not recommended because this vial type can easily open on its own. Vials can have threads located internally or externally on the vial opening; both vial types exhibit advantages and disadvantages. Externally-threaded closures promoteb more sterile conditions because internal threading can allow contaminants to enter if the cap is placed on an unclean surface when removed, but these vials can be susceptible to cracks and loss of air-tightness, which can lead to sample dessication or oxidation (Corthals and DeSalle 2005, Corthals 2006). Internally-threaded vials might allow increased storage capacity, depending on the vial selected, but users also suggest that material can be trapped within the threads of this vial type (Johnson 1999). Some manufacturers suggest that vial caps that incorporate a silicone gasket or O-ring (internally or externally threaded) are ideal for vapor-phase LN2 freezing because the seal is enhanced, but care must be taken if the vial cap is over-tightened because the gasket can become distended. In addition, the presence of gaskets or O-ring might improve the initial performance of seals but can be problematic when used with some alcohols (e.g., ethanol) because some gasket material, such as silicone, is vapor permeable. When working with liquid-phase nitrogen cold storage, extra caution must be taken because the accidental entrapment of liquefied nitrogen inside the vial leads to pressure build up and, upon removal, rapid vaporization of the liquid can result in leakage or even explosion. As a precaution, vial manufacturers recommend that samples not be immersed in LN₂ unless they have secondary containment. Heat-sealing vials into flexible polyethylene tubing is recommended for safe storage in the liquid-phase environment.

Labeling and organizing vials

• Can pre-label vials (maybe include specimen tags inside)
• Alcohol-proof pen
• Scribe
• Paper inside vials not ideal because of possible contamination

- Pencil for inside labels, or rinse ink before inserting

• Organizing vials numerically in boxes is preferable to storage in bags (in case writing comes off vials)
• Tissue samples should be taken as soon after euthanization as possible

Vial Considerations

• Internally threaded fit more/box (100 vials/box versus 81 vials/box) but if overfilled can be difficult to open
• Silicone o-ring is vapor permeable (Stuart and Hogue, 2012)

Tissue Collection Methods

The methods currently used to preserve genetic material during initial sampling vary widely owing to the sampling location, tissue type, and intended research use (Dessauer and Hafner 1984, Prendini et al. 2002, Bus´ and Allen 2014). A comprehensive treatise of methods to preserve genetic samples is discussed in Nagy (2010). Some general recommendations include:

• Fill two sets of tissue vials in the field, one for current project and one for institutional collection (rather than subsampling one vial later).
• Label vial to indicate tissue type, one tissue type per vial

- Epigenetic studies require same tissue type for all samples in study (i.e. all muscle)

• Cut/slice up tissue sample, small size allows permeation by preservative
• Fresh preservative may be needed if in field for extended period of time
• Don’t over-fill vial with tissue, or tissue and preservative (expansion, can interfere with vial threads, etc.)
• NGS methods need reasonable sample size to yield lots of DNA, ~500 ng (my notes say 500 ng, but is that the tissue or the desired amount of DNA? I’m guessing DNA)
• Collect sample as soon after euthanasia as possible

In a survey of genetic resource collections associated with natural history museums, samples were found to be initially preserved in a variety of different ways (Zimkus and Ford 2014).

Flash-frozen

Ethanol

Ethanol at concentrations of 95% to 99% is commonly used by researchers in the collection of zoological samples but is generally not used for the preservation of plant material. Ethanol can be considered expensive when compared to alternative preservatives and can be difficult to transport, especially at high concentrations, because of its flammable classification (Williams 2007).

DMSO

DMSO is used as a preservative in many different aqueous solutions. One of the most commonly used formulations is a salt-saturated solution of DMSO (20% DMSO, 0.25 M ethylenediaminetetraacetic acid [EDTA], sodium chloride [NaCl] saturated, pH 7.5; Seutin et al. 1991, Nagy 2010), which is a cost-effective method for preserving and transporting genetic samples from remote locations, but the long-term effects on sample quality are still unknown (Williams 2007).

RNAlater

RNAlater solution and a similar product, AllprotectH Tissue Reagent (Qiagen, Valencia, CA), are used in sample collecting for the stabilization of DNA, RNA, and protein in tissues. Although these solutions minimize the need to immediately process and/or freeze tissue samples, material preserved in RNAlater and AllprotectH buffers cannot be stored at room temperature indefinitely if genetic samples are to remain viable.

=

Tissue Preservation Methods

• Immediate freezing without preservative
• Alcohols

- 95% ethanol recommended; above 95% is technically dried (to remove the residual water which at this point can hardly be removed by distillation) with low boiling benzines so not recommended

- 1:10 tissue:ethanol seems to be a good ratio, ethanol may need to be replaced after ~24 hrs to maintain concentration.

• RNAlater

- Fill vials no more than 80% because will expand upon freezing

- Only good for 10 days? at room temperature without freezing

Allprotect

• DMSO

- DMSO solution has the advantage of being stable at room temperature, non-toxic and non-flammable. It can be carried on airplanes without a permit. However, it is photosensitive and can easily penetrate skin.

• FTA cards/swabs
• Non-iodized salt (e.g. Kosher salt)

Preserving Tissue Quality

• Minimize exposure to heat, light, moisture, and other chemicals
• Maintain cold chain if samples are frozen

Discipline Specific Information

Herpetology

• Liver
• Toe clips
• Muscle
• Tail clipping (salamanders, tadpoles)

Ichthyology

• Take tissue clips from right side (as for all destructive samples)
• Fin
• Muscle (hypaxial)
• Gill
• Whole animal (e.g. larval fish)
• Eye (especially from larvae), from right side

References

Nagy, Z.T. A hands-on overview of tissue preservation methods for molecular genetic analyses. Gamble, T. Collecting and Preserving Genetic Material for Herpetological Collection