Fixative brings about a crosslinking of protein which denature or coagulate proteins so that the semifluid state is converted into semisolid state so that it maintains everything in vivo in relation to each other.Therefore a semisolid state facilitates easy manipulation of tissues.
Choice of Fixative
The choice of fixative depends on the treatments a tissue is going to receive after fixation like
- what is the chemical structure that needs to be stained?
- If fat is to be demonstrated,formalin fixation for the tissue is recommended.
- For the demonstration of glycogen,formalin should never be used instead alcohol be the fixative of choice
Some Reagents Employed as Fixative (Simple Fixatives)
It is a gas but is soluble in water to the extent of 37 to 40 %w/v.The solution of formaldehyde in water is called formalin.It is one of the commonly used fixative since it is cheap,penetrate rapidly and does not harden the tissues
Properties of Formaldehyde
- It preserves the protein by forming cross linkage with them and the tissue component
- It denatures the protein
- Glycogen is partially preserved hence formalin is not a fixative of choice for carbohydrate
- Some enzymes can be demonstrated in formalin fixed tissues
- It neither preserves nor destroys fat.Complex lipids are fixed but has no effect on neutral fat.After the formalin fixation,fat may be demonstrated in frozen section.Also pure formalin is not as satisfactory fixatives as it over hardens the tissues.A 10% dilution in tap or distilled water is satisfactory.
Precaution when using formalin as fixatives
- Formalin oxidizes to formic acid if kept standing for a long period so it should be neutralized by phosphate or calcium carbonate otherwise it tends to form artifacts which is a brown pigment in tissue.To remove this pigment,It is treated with picric alcohol or saturated alcoholic sodium hydroxide.
- Concentrated formalin should never be neutralized as there is a great danger of explosion
- The commercial formalin becomes cloudy on standing especially when stored in a cool place due to formation of precipitate or paraformaldehyde which can be filtered
- Formalin on prolonged exposure can cause either dermatitis.It’s vapour may damage the nasal mucosa and cause sinusitis
Time required when fixing with formalin
|At room temperature (20 °C to 25 °C )||12 hours|
|For small biopsies||4-6 hours|
|At Temperature of 65°C||2 hours|
Alcohol (Ethyl alcohol)
Absolute alcohol alone has very little place in routine fixation for histopathology
Properties of Alcohol
- It acts as reducing agents,become oxidized to acetaldehyde and the to acetic acid
- It is slow to penetrate,hardens and shrinks the tissue
- Alcohol penetrates rapidly in the presence of other fixative hence in combination like Carnoy’s fixative is used to increase the speed of tissue processing
- Ethanol preserves some protein in relatively undenatured state so that it can be used for immunofluorescence or some histochemical methods to detect certain enzymes.
- It is a fat solvent hence it dissolve fats and lipids
- Methylene alcohol is used for fixing blood and bone marrow smears
Cold acetone is sometimes used as a fixative for the histochemical demonstration of some tissue enzymes like phosphatase and lipases
It’s mode of action as fixative is similar to that of alcohol
Mercuric chloride is a very good salt employed in fixing but is rarely used alone because it cases shrinkage of the tissue
Properties of Mercuric chloride
- It brings about precipitation of the proteins which are required to be removed before staining by using potassium iodide in which they are soluble
- The size that is thickness of the tissue to be fixed in mercuric chloride is important since if the tissue is more than 4 mm,then it hardends the tissue at the periphery whereas the centre remains soft and under fixed
- It penetrates rapidly without destroying lipids
- It neither fixes nor destroys carbohydrates,treatment of the tissue with mercuric chloride brings out more brilliant staining with most of the dyes
- Tissues fixed with mercuric chloride containing fixatives contain black precipitates of mercury which are removed by treating with 05% iodine solution in 70% ethanol for 5-10 minutes,sections are rinsed in water,decolourized for 5 minutes in 5 % sodium thiosulphate and washed in running water
- It produces marked cells shrinkage hence it is not used alone
- It has to be stored in a damp place because of its explosive nature and it is preferably stored under a layer of water
The advantage with picric acid is that it penetrate well and fixes rapidly.
It precipitates proteins and combines with to form picrates some of the picrates are water soluble so must be treated with alcohol before further processing where the tissue comes into contact with water
Note that all the tissues fixed in picric acid containing fixatives should be thoroughly washed to remove the yellow discolouration to ensure proper staining of tissue sections
If the fixative is not removed by washing thoroughly with time even the embedded tissue loses its staining quality
It fixes the cytoplasm without precipitation.Valuable in mixtures for the fixation of lipids especially phospholipids.
It is used for fixing phosphatides and mitochondria
Note thorough washing of the tissue fixed in dichromate is required to avoid forming an oxide in alcohol which cannot be removed later
It is a strong oxidizing agent and brings about fixation by forming cross links with proteins
- It gives excellent preservation of details of a cell,therefore exclusively used for electron microscopy
- It fixes fat like myelin
- It also demonstrates fat when 0.5-2% aqueous solution us used it gives a black colour to fat
It causes the cells to swell hence can never be used alone but should be used with fixatives causing cell shrinkage
It is used alone or in combination with osmium tetroxide for electron microscopy
Some fixatives are made by combining one or more fixative so that the disadvantage of one are reduced by use of another fixative as will be further describe below.
All these compound fixative have their own advantages and disadvantages and should be used judiciously