Protocol

Author

Amy Davidson

Definition

Chemically preserving punches (small sections) of fresh leaf tissue for microscopy

Overview

Small sections of leaf tissue can be chemically fixed to preserve cellular structures for observation under a microsope. FAA and FPA are effective chemical fixatives for preserving chromatin, nucleoli, and spindles. The cytoplasm is preserved as a stringy mass of coagulated material. Once fixation is complete, samples can be stored indefinitely.

Materials/Equipment

• Leaf punch of desired diameter
• Stick, or long, narrow implement to remove ‘punched’ leaf segment from leaf punch tool
• Small section of rubber (for placing on underside of leaf when punching)
• FAA or FPA
• Plastic storage vials with lids
• EtOH
• Beakers

Units, terms, definitions

Formalin-acetic acid-alcohol (FAA) and formalin-propionic acid-alcohol (FPA) are good general-purpose fixatives.

The so-called acid fixation image is produced as a result of stabilizing tissues with these types of fixatives.

EtOH – Ethanol

Procedure

1. Taking leaf sample
   • Using a leaf punch (or similar narrow metal cylinder with sharp rim), press firmly on upper surface of leaf, against a piece of rubber backing.
   • Twist punch gently to avoid damaging or disfiguring sample. Lift punch away from leaf, and use stick to ease punched sample out of the leaf punch.
2. Fixation
   • Immediately place punched sample into FAA or FPA solution.
   • Maximum 12 leaf punches in a single vial at any given time.
   • Leave leaf punches in FAA or FPA for minimum of 2 weeks and maximum of 2 months. (Any less than 2 weeks and leaf material will not be adequately fixed, any more than 2 months and leaf material may begin to deteriorate. Leaving samples in dark cool place is best.
   • NOTE: Label both the lids and bases of storage vials with an ‘ethanol-proof’ permanent marker
3. Dehydration of tissue sample post fixation
   • Wash tissues using the following an EtOH series, rinsing and leaving tissues for 15 minutes in new vials at each stage:
     70% EtOH, 80% EtOH, 90%, 95%, 100%, 100%, 100%.
4. Storing tissue
   • Store tissue samples in 70-80% EtOH.

Notes

• Some organelles, such as mitochondria, are dissolved in FAA and FPA fixatives.
• Some consider FPA to yield better preservation results than FAA, but effects may be tissue-specific.
• Tissue penetration is not as fast as some other fixatives such as acrolein.

References

Chemical Fixation: Formalin-Acid-Alcohol: FAA and FPA

Health, Safety & Hazardous Waste Disposal Considerations

Formaldehyde – toxic

Use gloves when handling formaldehyde and do not inhale.

Disposal of formaldehyde: Add milk powder to neutralise the acid. Needs to be disposed of in appropriate place (not down the sink) so check with local chemical authorities.

Disposal of Ethanol: slowly pipette down drain with running water to dilute.

Troubleshooting tips

• FAA becomes less effective with storage.
• The presence of alcohol can result in shrinkage of tissues. To reduce shrinkage you can vary the amount of acetic or propionic acid from 2 to 6%, which better preserves chromatin structure. Increasing the concentration of acetic or propionic acid induces tissue swelling, and counteracts alcohol shrinkage.