The capacity of buffers occurring naturally in cells for maintaining a constant pH in the presence of fixatives is not effective to avoid damage or artifacts.
Buffer solutions contain a weak acid and its salt, or a weak base and its salt.
They resist changes in H+ concentration when small amounts of a strong acid or base are added to it.
Helps keep the fixative mixture near isotonic conditions with tissue.
Each buffer has a point at which the acid and base are of equal concentration (pKa) where buffering capacity is greatest. The pKa can be affected by temperature, concentration of buffer, the pH of the buffer
If there are physiologically active ions present in the buffer (e.g. Ca2+, Na+, K+ and Cl-) biochemical alterations can occur before immobilization by the fixative.
Compatibility – some buffers are not stable with OsO4 or aldehydes at high concentrations.
Buffering capacity decreases with increasing dilution of the buffer.
Buffer should be tissue compatible if used as a rinse or to store the tissue for any length of time. Otherwise toxic effects will be seen (dead or dying tissue or autolysing before fixation). Typical "vital buffers" include PBS, Tris, Hepes, Pipes.
Types of Buffers:
Cacodylate (Cacodylic Acid)
Cacodylate is a strong buffer and resistant to bacterial contamination. Its effective range is 6.4 - 7.4.
Avoids presence of phosphates which may interfere with cytochemical studies.
It is incompatible with uranyl salts and should be rinsed out thoroughly if planning to do "en-bloc" staining with UA. It is used extensively with animal tissues.
- NaCacodylate contains arsenic, which is toxic. Avoid contact with acids to avoid production of arsenic gas.
Phosphate Buffers (Millonigs, Sorensens, PBS)
More physiological than other buffers, as they are found in living systems in the form of inorganic phosphates and esters. Effective range 5.0-8.0
The buffers are non-toxic to cells grown in culture.
They are not as sensitive to temperature changes
They can cause artefacts such as electron dense particles or nuclear shrinkage.
Phosphate buffers should not be used when calcium is to be added to the fix solution.
Zwitterionic buffer with a pKa of 7.3 at 37 C
May interfere with amine-aldehyde rxns.
Appears to stabilize membranes.
Another zwitterionic buffer with pKa of 6.66 at 37 C.
Thought to be beeter buffer and reduce lipid loss
Has been reported to produce multivesicular myelin figures in rat cortex.
Pyridine derivative with buffering capacity is 6-8 but most efficient around 7.4.
Primarily used with Formaldehyde in tissue storage
Does not react with OsO4
May possibly extract proteins, but is good with high concentrations of aldehydes
Is not recommended for EM
Has a bad smell.
Effective between 4.2 and 5.2
Should not be used with aldehydes, since it reacts with these fixatives
Does not precipitate UA
Preserves membranes well when used with OsO4
Poor buffering capacity below 7.5 and is a biological inhibitor.
Reacts with GA
Causes excessive extraction of cellular components
Not recommended for EM