Factors affecting sample preparation

The first consideration for preparation is understanding your sample as a hydrated object that needs to be modified for the previously mentioned problems. This would include most samples, biological or otherwise.

Definition:
Fixation is a process which is used to preserve (fix) the structure of freshly killed material in a state that most closely resembles the structure and/or composition of the original living state

If too slow – crosslinking of proteins, carbohydrates and lipids is not optimal. Enzymatic activity is not halted. Cytoplasmic degradation may occur. Chemical components may migrate or be leached. Cell may collapse or swell. Organelles may change their morphology.

Factors affecting quality of chemical fixation

Tissue:
Size of sample
Reactivity of tissues to fixatives or buffers
Penetration of fixative

Reagents:
pH of buffers and fixatives
Temperature during fixation
Osmolarity and tonicity
Concentration
method of application of fixatives (immersion vs. perfusion)
Affects of added substances (e.g. tannic acid, UA, KMgO4)

Tissue Variables:
Tissue block must be small
– in lab we traditionally make the blocks 1mm or less.
This varies with different samples depending on tissue density, permeability, structures that impede fixative penetration (cell walls, extracellular matrix, insect cuticle, etc..)

Good preservation by immersion is limited to 2-3 cell layers.

OsO4 penetrates ~0.25 mm in most tissues, so the block should not exceed 0.5mm

Glutaraldehyde penetrates further, but then the tissue must be trimmed further for OsO4, and is not recommended. Formaldehyde is smaller and penetrates faster, but preservation is bad and is not recommended. Although, some workers have used mixtures of FA/GA.

Some damage is done cutting the tissue. Outer regions are also subject to leaching, loss of structural components.
 
 
Sample should be accessible to the fixative
– if there are large vacuoles, intercellular spaces, cell walls, glycocalyx/extracellular matrix, then the fix may not penetrate, or be uneven in penetration.

E.g. Plants with cuticles and gaseous intercellular spaces typically will float and the surface tension is high. One can either add small amount of detergent (tween 20) or put sample under vacuum.
Insects, can be immersed in fixative and then remove the cuticle, if the interface between tissue and cuticle is not required for the project. Sometimes, with soft specimens, detergent will allow fixative to enter through breathing pores.

Some tissues are subject to cyclic changes – diurnal/nocturnal variations in physiology or circadian cycles. Mitochondria have variable morphology depending on activity states, Dictyosome (Golgi) cisternae can appear swollen or with variable numbers of cisternae.

Reactivity of tissue to fixatives and/or buffers – The reagents may precipitate cellular components or allow migration. They may change the nature of the membrane or leach out components.
E.g. Some bacteria lose extracellular matrix or collapse when exposed to sequential GA and OsO4 for SEM examination. Parduc’s fixative ( OsO4/Mercuric Chloride mixture) may work better. KMgO4 is good for membranes, but extracts cytoplasm and organelle ground material.
Some fungi produce a granular precipitate upon exposure to GA within the cytoplasm.
Some buffers cause precipitation of intercellular enzymes or proteins (KPO4). Different buffer may be required not containing phosphates. Or may interfere with cytochemical labeling. Some buffers are used to fix specific components(PIPES/Mg/CaCl2 for microtubules), but be poor for other organelles.

Other Variables:
During fixation, proteins are denatured (change in their original secondary and tertiary conformation, change in charge, etc…) Changes occur to the sample with each variable below.

pH – isoelectric point: H+ concentration for which the mean charge of the protein is 0
Most cells/tissues have a natural buffering system. Careful to know what your sample is, some organelles show a change with different pH.
Depending on tissues, the membrane permeability can be modified before the fixative has time to stabilize, allowing swelling, extraction of some Components.
Most animal cells the pH of cytoplasm is ~7.0 whereas nuclei are ~ 7.6-7.8.

Duration of fixation – Optimal time for most tissues is not known and must be determined empirically. If fixed in too short of time, obviously the penetration is limited and crosslinking is inadequate. It may even be reversed in some cases.
Standard fixations are 1-4 hours at room temperature or 4 C (refrigerator) are commonly used. Typically, 1-2 hours in GA and 1 hr in OsO4, however, shorter times are preferable. Duration is controlled by the nature of the tissue, fixative, sample size, temperature, buffer type and the objective of the study.
Little is known regarding overfixation, other than extraction of tissue constituents. OsO4 fixation timing is more critical, since it does not crosslink many proteins and some extraction occurs from the tissue. Leaching of calcium and glycogen can occur

Temperature – Cold temperature is typically used because it decreases extraction and provides a slower rate of autolysis. But other factors must be taken into account depending on the nature of the study and the sample.
Effects of cold temperature are highly complex. Only some of the changes at 4 C are known.
Cytosolic pH and membrane permeability are affected unless the fixation is instantaneous (which does not happen with chemical fix). Enzymatic activity is slowed as is the rate of transfer of components across the membrane.
With some specimens, cold is a problem. e.g. bacterial spores fix better at 40 C. Cold labile structures, (MTs) are preserved better at RT or physiological temps. In mitochondria, the metabolic processes are affected and may not appear as expected.

Concentration of the Fixative - In general, low concentrations of fixatives require a longer duration of fixation. The resulting problems discussed earlier with duration occur. Extraction of cellular materials, diffusion of enzymes, shrinkage or swelling.
Higher concentrations are also bad. Concentrated solutions destroy enzyme activity and damage the cellular fine structure.
Different organelles respond differently to fix or buffer. Mitochondria appear to more sensitive to increased buffer concentration, whereas ER is sensitive to low concentrations of fixative. Many times, the fixative concentration is offset by the osmolarity of the solution. GA has a wide range of concentrations that can be used as long as the tonicity of the system is maintained.

Osmolarity – Definitions: Tonicity and Osmolarity
Osmolarity indicates the molarity (mole/liter of solution) or molality (mole/kg of solvent) that an ideal solution of nondissociating substance must possess to exert the same osmotic pressure as the test solution. In biological systems, it is often expressed as mOsm/liter of water
Osmolarity measurements are independent of temperature
Osmotic pressure is a measurement of the movement of the solvent (e.g. water)

Electrolyte and nonelectrolyte solutions:
Because non electrolyte solutions do not ionize, their molar and osmolar concentrations are equivalent. Electrolyte solutions exert a greater osmotic pressure because of ion concentration.
As a result, two solutions of same molarity can have different osmolarity.

Tonicity- refers to the pressure or concentration relationship of the solute.
- Isotonic solutions exerts an osmotic pressure equal to that exerted by the cell cytoplasm.
- Hypotonic – the solute concentration of the fixative is lower than the cell cytoplasm and will cause the water to move into the cell – bursting or lysis. With plant tissue, algae or fungal cells, increases turgor pressure against wall.
- Hypertonic – the solute concentration is higher than cell and cause water to move out of cell – crenulation (shrinkage) of cytoplasm.

SEM specimens should be fixed under near isotonic conditions
TEM specimens should be fixed under slightly hypertonic conditions

Buffer osmolarity plays a major role in osmotic pressure, but GA exerts a pressure also. Determination of the effective osmotic pressure is complex.

Blood/tissue osmolality in groups of organisms:
Mammals – 290 mOsm (plasma)
reptiles – 325 mOsm
Marine animals – 250-375
Freshwater invertebrates – as low as 30 mOsm
Plants – differs with tissue and species

The buffer used to rinse the specimen after fixation should be of the same osmolarity of the fixative/buffer. Consequently, a salt or sugar is added to the rinse buffer. Because the membrane still has a differential permeability after this wash, the OsO4/buffer solution should ideally be at the same osmolarity.