Factors affecting the fixaton process

Buffers: maintain a stable environment for chemical reactions; pH is often around 7

Penetration rate:different fixative penetrate at different rates.

Volume:50-100: 1 fixative to tissue volume ratio

Temperature:increase in temperature results in increase of rate of chemical reactions

Concentration:use lowest effective level to reduce cost

Duration of fixation:shorter duration reduces autolysis, cut tissue into smaller (2-3 nm thick) sections before placing into fixative to reduce the duration

Alcohols, chromium containing fixative: denature proteins and transform protoplasm into an interconnected network, thus called coagulants.

Aldehydes:crosslink with proteins, fix in situ; the best fixative for overall retention of structural details

Osmium tetroxide:crosslink with lkipds, excellent for preserving lipids (cell membranes, lipid bodies) in situ, non-coagulant, can be used for both electron and light microscopy.

Acids:often used with other fixatives to preserve cytoplasm and counteract the shrinking effect of alcohols-they cause the sample to swell

(toxic and volatile, handle in a fume hood and wear gloves and eye protection)

the typical compound is formaldehyde that exists in a gas state in the usual environemnt. the formaldehyde gas is available in one of the three forms: 1)pure saturated solution (37-40%) in ampules, 2)formalin, 3) paraformaldehyde

Formalin=37-40% formaldehyde+10% methanol; methanol stablizes formaldehyde

Aldehyde-containing fixatives(except the ones from formalin) tend to polymerize and form a precipitate, so they should be prepared fresh, or frozen in small aliquots.

Paraformaldehyde is a solid form of polymerized formaldehyde that breaks down into formaldehyde upon dissolving in water--easy to handle and the resulting formaldehyde gas can be accurately controlled. use 60C amd above 8pH(KOH) to dissolve paraformaldehyde in water.

Glutaraldehyde is sometimes combines with formaldehyde in a fixative

Fixatives such as aldehydes and osmium tetroxide become part of the specimen are also called additive fixatives, others are nonadditive since they do not become part of the specimen.

Plant tissue organization is retained more or less equally between fixative classes.

Only intracellular detail varies with different fixatives.

Not necessary to use the finest (and most expensive) fixative when a gross morphological study is the immediate goal

Some compounds can be used individually. Ex:aldehydes

Several compounds can also be combined to counteract inherent weaknesses in individual compounds. Ex: FAA is composed of formalin, acetic acid, and ethanol

FAA and FPA

Carnoys fixative

Farmers fixative

Aldehydes

Karnovskys fixative

OsO4

Ethanol (95%)     50

Glacial acetic acid 5

formalin (37% formaldehyde) 10

DI                       35

Ethanol (95%)     50

Propionic acid       5

formalin (37% formaldehyde) 10

DI                       35

EtOH (100%)   60

Glacial acetic acid    10

Chloroform     30

EtOH(100%)    75

Glacial acetic acid   25

alone is an excellent fixative, but if combined with glutaraldehyde it can be even better

Paraformaldehyde  2g

Glutaraldehyde      5ml

DI                        25 ml

Buffer(0.2M)         25 ml

(extremely toxic and volatile, use gloves, eye protection, and fume hood all the time!)

After fixation using a standard fixative(e.g. Karnovskys) and washing with buffer, samples may be postfixed with 1% OsO4 to stabliz membrane and other structures mostly made of lipids

Turns tissue black so the tissue has to be bleached for light microscopy

Render tissue samples to a reasonably small size

Place the tissue in fixative

Let the fixative penetrate the tissue

Remove the fixative if necessary

Proceed with dehydration before infiltration by embedding medium.

Tissues may be stored in fixatives that do not overfix. Ex: FAA

Fixative have to be replaced with the corresponding buffer if they overfix. Examples: most aldehydes, chromium-containing fixatives