Molecular Techniques and Methods

FAA Fixation of Plant Tissue

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Although FAA is a very harsh fixative and not balanced to cellular pH or osmoticum, it penetrates rapidly. Also, plant tissues can be stored in FAA almost indefinitely. FAA (or FPA) is stable and has good hardening action, and most material may be stored in it for years. These properties make it suitable for large or impervious objects such as woody twigs and stems, tough herbaceous stems, and old roots. The high concentration of alcohol is likely to produce shrinkage of succulent materials and also precipitation of the proteins, which gives a coarse appearance to the cytoplasm. However, it is possible to develop a formula for some apparently tender subjects and even for filamentous algae. A balanced formula can be worked out by varying the acetic acid, which has a swelling action on protoplasm, from 2 to 6% by volume. The acetic acid also preserves nuclear structure. The formaldehyde and alcohol have a shrinking effect and should be held at the indicated concentrations. When making trials of variations from fundamental formulas, fix a trial lot in the formula to be tested and a check lot in the standard formula. Process the two lots simultaneously and identically and compare the cellular detail in both lots.




MATERIALS AND SOLUTIONS

FAA (Formalin-Acetic-Alcohol) (100 ml)
Ethyl alcohol ------------------------------- 50 ml
Glacial acetic acid -------------------------- 5 ml
Formaldehyde (37-40%) ------------------- 10 ml
Distilled H2O ------------------------------- 35 ml


FPA (Formalin-Propionic-Alcohol) (100 ml)
Ethyl alcohol ------------------------------- 50 ml
Propionic acid ------------------------------ 5 ml
Formaldehyde (37-40%) ------------------- 10 ml
Distilled H2O ------------------------------- 35 ml




PROCEDURES

1. Fix pieces of thin leaf for 6-12 hours. Longer fixation may make sectioning difficult because the vascular tissue may become much harder than the protoplasmic constituents.

  • Thick leaves or pieces of small stem require at least 24 hours. Woody twigs should be kept in FAA at least a week before continuing the processing for embedding.

  • Materials do not need to be washed after FAA. The ingredients of this fluid are soluble in the dehydrating agents and are thus removed before infiltration is begun.

    2. An extensively used formula consists of FAA containing mercuric chloride (HgCl2) to saturation (1 g/10 ml FAA). This fluid penetrates and hardens tissues rapidly. It preserves bacterial zoogloea in plant tissues, thus being useful in pathological studies.

  • The alcohol may be increased to 70%. Prolonged storage in fluids containing mercuric chloride is undesirable. The tissues should be transferred after 48 hours, or at most a week, to a fresh solution of the original formula which does not contain mercuric chloride. After four or five changes of the latter solution, most tissues may be stored indefinitely in the last change. Mercuric chloride is a highly poisonous substance, and fixatives containing it should be mixed and used in a hood.




    NOTES

    Classification of Fixatives

    (a) Acid fixation image preserves particularly well the chromosomes, nucleoli, and spindle mechanism. Nucleoplasm and mitochondria are dissolved.

    (b) Basic fixation image fluids preserve mitochondria, nucleoplasm, and in some instances nucleoli and vacuoles. Chromatin and the spindle mechanism are dissolved.

    (c) Coagulant fixatives cause the protoplast to form into a netlike structure and are the most numerous types. Some of the common coagulant fixatives are ethanol, methanol, chromium trioxide, picric acid, mercuric chloride, and acetone.

    (d) Noncoagulant fixatives are exemplified by formaldehyde, glutaraldehyde, acrolein, osmium tetroxide, acetic acid, and potassium dichromate. Some noncoagulant fixatives can render tissue resistant to coagulation by coagulating substances during postfixation and subsequent processes, thus permitting design of dual fixation systems.

    (e) Additive fixatives such as aldehydes crosslink with structures in the cell, namely proteins, and thus become a corporeal part of the the cell structure. For some histochernical purposes such as the use of the Schiff reagent this may not be desirable.

  • No single substance has been found to meet the requirements for successful fixation of all specimens. Fixatives usually consist of several ingredients in such proportions that there is a balance between the respective shrinking and swelling actions of the ingredients.




    KIT INFORMATION




    REFERENCES

  • Berlyn GP, Miksche JP (1976) Botanical Microtechnique and Cytochemistry, Ames, Iowa, Iowa State University Press.

  • Please send your comment on this protocol to "editor@MolecularInfo.com".



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