The other option is to buy 10% neutral buffered formalin (4% formaldehyde) in a scientific supply house, use it for 3-6 months, and then dispose of it (as a dangerous commodity). There will be methanol in this solution (usually 1-2%), but if it is used shortly after purchase, it should not be important to most users. The buffered solution helps to slow down the acidification process. A one-liter bottle costs about $20 to $25. Store the fixative at room temperature. Local readers of this page should contact the MMT Histology Service Laboratory (626-4415) if they have difficulty finding a supplier. (Please note that different providers use different buffer solutions. For consistent immunohistochemistry and/or immunofluorescence, users should stick to a supplier.) Buffered formalin is the standard and preferred formalin fixer for tissue preservation. This solution is usually purchased as a prepared solution and avoids the additional handling risks required to mix the buffered formalin solution from a storage solution. In general, this buffered formalin solution is prepared by mixing part of the storage formalin with 9 parts of distilled water. To maintain buffering capacity, we can add reagents such as monobasic sodium hypophosphate and bibasic or anhydrous sodium hyperphosphate.
However, there is another method in which sodium chloride and dibasic sodium hyperphosphate are added to the mixture of formalin and water in a ratio of 1: 9. Please note the hazards associated with formaldehyde. The following statement is taken from an MSDS for 10% buffered formalin: Unbuffered formalin slowly oxidizes to formic acid, resulting in a decrease in pH. Under these conditions, formic acid reacts with hemoglobin and forms acid hematin formaldehyde, a brown-black granular artifact pigment that is deposited in blood-rich tissues. This pigment is a nuisance because it can be confused with microorganisms or other pathological pigments. 4 Although the pigment can be removed from saturated aqueous peckric acid sections before staining, it is best to avoid its formation in the first place. For this reason, and because formaldehyde reacts most effectively at about a neutral pH value, 10% formalin solutions are usually buffered at a pH of 6.8 to 7.2. The commonly used fixative is 10% formalin, or 3.7% formaldehyde in water with 1% methanol. It is an inexpensive and widely available fixative that does not cause excessive tissue shrinkage or distortion of cell structure. Commercial undiluted formaldehyde solutions contain 10% methanol as a preservative to prevent spontaneous condensation reactions. [1] For this reason, commercial formalin is a 2-phase fixative with an initial alcohol fixation phase followed by an aldehyde-mediated cross-linking phase.
Alcohol initially causes dehydration of the process and hardens the tissue and membrane. Formalin, when stored for a long time, is oxidized to form formic acid. Therefore, in the stored formaldehyde, the presence of unknown formic acid (also reacts with the blood to form a birefringent crystal called formalin pigments) is expected. [1] The following infographic shows more details on the differences between buffered formalin without buffer and neutralized formalin in tabular form for side-by-side comparison purposes. Time: The optimal time for fixation varies between fixatics. For a fixation to take place, the fixator must penetrate by diffusion in the center of the sample, and then sufficient time must be allowed for the reactions of the fixation to occur. The diffusion time and reaction time depend on the reagent used, and the optimal time varies from one fixator to another. In high-traffic diagnostic laboratories, there is considerable pressure to shorten the processing time, which can lead to the treatment of incompletely fixed tissues.
This can lead to poor quality sections that show tissue distortion and poor coloring quality, as poorly fixed tissues are not treated well. Remember that if incompletely fixed tissue is taken from formalin and placed in ethanol during processing, ethanol continues to fix the tissue and the morphological image in the middle of the sample is that of ethanol fixation. The initial fixation should be carried out at room temperature, since the penetration of formalin is related to the temperature of the solution. Formalin should be stirred thoroughly before use to avoid a concentration gradient in the vial. Formalin is the fixative widely used in pathology laboratories around the world due to its convenient handling, high precision and extreme adaptability. The basics of the chemical reactions involved in formalin fixation have been described in the literature. [4,5] Formalin penetration into the sample is a physical process in which the solution diffuses through the sample to reach the innermost cell layers. This movement of formalin is determined by several physical factors.
[3] The objective of this review is to examine the chemical basis of formalin fixation and to explain the effects of various factors on formalin fixation using known physical equations and basic chemical reactions. Formaldehyde reacts with protein side chains to form reactive hydroxymethyl groups. It can penetrate nuclear proteins and nucleic acids, stabilize the protein envelope of nucleic acids, and modify nucleotides by reacting with free amino groups. Formaldehyde can react with certain groups in unsaturated fats, especially when calcium ions are present, but tends not to react with carbohydrates. 5 Formaldehyde may react with groups of lysine, arginine, cysteine, tyrosine, threonine, serine and glutamine and form reactive complexes that can combine with each other to form methylene bridges (cross compounds) or with hydrogen groups. 5 It is generally accepted that washing tissues after formalin fixation can reverse some of these reactions, but important cross-connections remain. 6 It is formaldehyde`s ability to preserve peptides from cellular proteins that has made it so useful as a general fixative. 10% fixing buffered formalin is often used to preserve tissues for routine histology in many laboratories. Formaldehyde has a greater chance of oxidation in this concentration of tissue fixators and eventually the solution will decrease in pH despite the buffer. We recommend that 10% buffered formalin solutions should not be used for more than 3 months after the first mixture. The solution should be clear, colourless, without precipitation and the pH should not be less than 6.5.
Formalin without a buffer is a solution of formalin in water. This type of solution is formed when part of the formalin is mixed with 9 parts of water. This mixture of solution has a pH of about 3-4, which can vary depending on the concentration of the formalin stock that we use for this purpose. The literature has enough evidence to prove that the penetration of formalin into tissue is subject to Fick`s law of diffusion. [9] It has been studied several times and reported on the various physical factors that influence the penetration rate. Methylene glycol polymerized for a long time in polyoxymethylene glycol. In a neutral to alkaline buffered system such as tissues, it depolymerizes into methylene glycol, which dehydrates into carbonylformaldehyde (the one that contains C with a double bond with O, dehydrated form). Hydrated and unhydrated forms of formaldehyde fix tissues. [1,3,5] When tissues are immersed in formalin, they are rapidly penetrated by methylene glycol and the small amount of formaldehyde. The actual covalent chemical reaction of the fixing solution with the tissue depends on the fact that the existing formaldehyde is consumed after forming bonds with the tissue components. One by one, more formaldehyde is formed by dissociation of methylene glycol, which leads to a change in the equation so that more formaldehyde is formed. [1,5,6] The other problem with 10% buffered formalin is the slow increase in the concentration of methanol (an undesirable by-product of aging formaldehyde).
Methanol promotes protein clumping, rather than the cross-linking of proteins that formaldehyde performs. A methanol-free fixative offers the best preservation, especially if you want to use the fabric for antibody staining at a later date. 10% neutral buffered formalin is a general histological tissue fixative and a standard fixative for use in a diagnostic environment. .