Factors affecting chemical fixation, formaldehyde and glutaraldehyde (2023)

Factors affecting chemical fixation

There are a number of factors that affect the speed and effectiveness oftissue fixation.

Temperature:Increasing the fixation temperature increases the rate of diffusion of the fixative into the tissue and accelerates the rate of chemical reaction between the fixative and the tissue elements. It can also potentially increase the rate of tissue degeneration in unfixed areas of the specimen. For light microscopy, initial fixation is usually performed at room temperature and this may be followed by further fixation at temperatures up to 45°Ctissue processing. This is really a compromise that seems to be generally accepted in order to achieve high quality morphological conservation. Microwave fixation can involve the use of higher temperatures, up to 65°C, but for relatively short periods of time. Seepart 5for further discussion.

Time:The optimal time for fixation varies between fixatives. For fixation to occur, the fixative must penetrate to the center of the sample by diffusion, and then sufficient time must be allowed for the fixation reactions to take place. Both the diffusion time and the reaction time depend on the particular reagent used, and the optimal time varies from fixative to fixative. In busy diagnostic laboratories, there is significant pressure to reduce turnaround time and this can result in the processing of incompletely fixed tissues. This can result in poor quality sections showing tissue distortion and poor quality staining, as poorly fixed tissue cannot be processed well. Keep in mind that if incompletely fixed tissue is taken from formalin and placed in ethanol during processing, ethanol will continue to fix the tissue and the morphological image at the center of the specimen will be that of ethanol fixation.

Penetrationsrate:The penetration rate of a fixative depends on its diffusion behavior and varies from agent to agent. As developed by Medawar, it can be expressed as d = K√t, where d is the penetration depth, K is the diffusion coefficient (specific to each fixative), and t is time.1In practical terms, this means that the diffusion coefficient (K) is the distance in millimeters that the fixative has diffused into the tissue in one hour. For 10% formalin, K = 0.78. This means that your formalin fixative should not penetrate more than say 1mm in an hour and it will take around 25 hours to penetrate the center of a 10mm thick sample, i.e. 1mm thick. H. 5 mm (= 5² hours).


Factors affecting chemical fixation, formaldehyde and glutaraldehyde (1)

Figure 1: A composite photograph showing the rate at which 10% neutral buffered formalin penetrates 25 mm liver cubes. At the end of each time period, a cube was cut to reveal the advancing fixation front. A: one hour (about 0.8 mm penetration), B: two hours (about 1.2 mm penetration), C: four hours (about 1.6 mm penetration), and D: eight hours (about 2.2 mm penetration ). Note that after eight hours the center of the sample remains unfixed.

(Video) Formaldehyde vs. alcohol fixation for immunofluorescence (IF) | CST Tech Tips

Sample dimensions:The foregoing approximations emphasize the importance of sample dimensions in tissue fixation. A copy shouldnot be thicker than 4 mm. Ideally a 3mm thick disc should provide excellent fixation and finish. Remember that the sample well in a standard processing cassette is 5mm deep.

volume ratio:It is important to have an excess of fixative relative to the total volume of tissue, since with additive fixatives the effective concentration of reagent is depleted as fixation proceeds, and with a small total volume this could impact fixative quality. A 20:1 fixative-to-tissue ratio is considered the lowest acceptable ratio, but I would recommend a target50:1 ratio.

pH and buffers:At the light microscopic level, the pH of a fixative does not appear to affect the quality of the preservation much, as a number of formulations have a fairly low pH, such as e.g. B. those containing acetic or picric acid. However, pH can be important for other reasons, as in the case of formaldehyde solutions where the breakdown of formaldehyde to form formic acid produces an acidic solution which in turn reacts with hemoglobin to produce an artifact pigment (acidic formaldehyde hematin). For this reason, the formaldehyde solution most commonly used today is buffered to pH 6.8 – 7.2. For electron microscopy, the pH is more important and should correspond to the physiological pH.2

Osmolality:Again, the osmotic effects exerted by the fixative are more important at the ultrastructural level than at the light microscopic level, since the phospholipid membranes are easily damaged by overly hypotonic or hypertonic solutions, but osmolality has some relevance in routine histopathology. In general, the osmolality of the vehicle (buffer) is most important and in some formulations this is adjusted to resemble that of tissue fluid (e.g. formalin in isotonic saline). Prior to fixation, cells can certainly be damaged by non-isotonic fluids such as water, and if specimens cannot be fixed immediately, they can be kept moist with gauze soaked in isotonic saline for a short time. Soaking tissues in saline for long periods of time is not a good idea.

(Video) Fixation and fixative | Easiest explanation with notes


There are a number ofreagentswhich can be used to fix tissue. Formaldehyde, by far the most popular agent for histopathology, and glutaraldehyde, which is commonly used for ultrastructural studies that require electron microscopy, are described here. Other reagents are discussed inpart 3.

Formaldehyde:Formaldehyde (CH2O) is the only gaseous aldehyde and is dissolved in water at 37% – 40% w/v to saturation. This solution is commonly referred to as "formalin" or "concentrated formaldehyde solution". For fixation, one part formalin is usually diluted with nine parts water or buffer. This produces a 10% formalin solution containing approximately 4% formaldehyde w/v, an optimal concentration for fixation. In concentrated solutions, formaldehyde exists as the monohydrate methylene glycol and as low molecular weight polymeric hydrates. In its diluted form, the monohydrate predominates. Paraformaldehyde, a highly polymerized form of formaldehyde, can separate out as a white precipitate in concentrated formaldehyde solutions. To prevent this, small amounts of methanol (up to 15%) are commonly added to proprietary solutions. Paraformaldehyde can be purchased as a dry powder and used to make high purity formaldehyde solutions needed for electron microscopy.2, 3

Unbuffered formalin slowly oxidizes to formic acid, causing the pH to drop. Under these conditions, formic acid reacts with hemoglobin to form acid formaldehyde hematin, a brown-black granular artifact pigment that is deposited in blood-rich tissues. This pigment is a nuisance because it can be confusedmicroorganismsor other pathological pigments.4Although pigment can be removed from sections prior to staining with saturated aqueous picric acid, it is preferable to avoid its formation in the first place. For this reason, and because formaldehyde reacts most effectively at around neutral pH, 10% formalin solutions are usually buffered to pH 6.8-7.2.


Factors affecting chemical fixation, formaldehyde and glutaraldehyde (2)

Figure 2: A formalin-fixed paraffin section of the kidney showing the typical deposition of acid formaldehyde hematin (formalin pigment) associated with red blood cells. The pigment is brown to black in color and is birefringent under polarized light. In this case, the sample remained in the fixative for a long time before processing.

(Video) Histotutor Chapter 1: Fixation

Formaldehyde reacts with the side chains of proteins to form reactive hydroxymethyl groups. It can penetrate core proteins and nucleic acids, stabilizes the nucleic acid-protein envelope and modifies the nucleotides by reacting with free amino groups. Formaldehyde can react with some groups in unsaturated lipids, particularly when calcium ions are present, but tends not to react with carbohydrates.5Formaldehyde can react with groups on lysine, arginine, cysteine, tyrosine, threonine, serine and glutamine to form reactive complexes that can combine to form methylene bridges (crosslinks) or with hydrogen groups.5It is generally accepted that washing tissues after formalin fixation can reverse some of these reactions, but important cross-links remain.6It is formaldehyde's ability to preserve the peptides of cell proteins that has made it so useful as an all-purpose fixative.

There are known hazards associated with the use of formaldehyde as a fixative through skin or eye contact, or via the respiratory tract. It is irritating, corrosive and can cause allergic sensitization. From 1981, formaldehyde was classified as "reasonably likely to cause human cancer" and in 2011 the listing was upgraded to "known to be a human carcinogen".7, 8Studies have shown that formaldehyde causes nasopharyngeal cancer, sinus cancer and myeloid leukemia. For these reasons, most countries have strict policies in place to limit worker exposure to formaldehyde in the workplace. For example, in the United States, the OSHA Permitted Exposure Limit (PEL) is 0.75 ppm (8-hour TWA) and a Short-Term Exposure Limit (STEL) is 2 ppm (15-minute exposure), and these recommendations are reinforced by a regular Monitoring program supported. In a well-equipped laboratory with modern extraction systems, these target values ​​should not be exceeded.9, 10

Despite the risks of using formaldehyde, most morphologists have built their knowledge of normal and diseased tissues by looking at formalin-fixed specimens. Now that we are aware of the toxic dangers of this reagent, many laboratories have sought, and continue to seek, a safer alternative. However, alternatives to formalin are generally judged on their ability to produce a similar, if not better, morphological image to that produced by formalin, allow for a full range of staining methods including molecular methods, and be just as cheap. Most labs continue to use formalin because they cannot find a completely satisfactory substitute. Therefore, it is important that employees are aware of the hazards involved. Some alternatives to formaldehyde are discussed inpart 3.

Glutaraldehyd:Glutaraldehyd oder Glutardialdehyd (CHO(CH2)3CHO) is considered a bifunctional aldehyde possessing aldehyde groups at both ends of the molecule that have the potential to react with the same chemical groups as formaldehyde. They will form addition junctions and methylene bridges, but even a single glutaraldehyde molecule can form direct crosslinks if the steric arrangement of neighboring peptides allows it. The amino groups of lysine are of particular importance. Tissue fixed in glutaraldehyde is more cross-linked than tissue fixed in formalin and also possesses some unreacted aldehyde groups which, unless chemically blocked, can be used in procedures such as e.g. B. can cause background stainingNOT. Extensive cross-linking is detrimental to immunohistochemical staining but provides excellent ultrastructural preservation, explaining its extensive use as a primary fixative for electron microscopy. Crosslinking reactions of glutaraldehyde are largely irreversible. Glutaraldehyde penetrates very slowly and it is recommended that the tissue be less than 1 mm thick in at least one dimension.5, 11

Glutaraldehyde slowly decomposes into glutaric acid and also polymerizes into cyclic and oligomeric compounds. Glutaraldehyde is therefore best obtained in sealed ampoules in a convenient form "stabilized for electron microscopy" and can be added to a suitable buffer at pH 7.2 - 7.4 (usually cacodylate, phosphate or maleate) to give a 3% strength glutaraldehyde concentration for use. For electron microscopy, primary fixation in glutaraldehyde is usually followed by secondary fixation in osmium tetroxide. Glutaraldehyde is not typically used for routine histopathology.11


(Video) 1.2 Fixation–Fixed cell imaging: 5 steps for publication-quality images

Factors affecting chemical fixation, formaldehyde and glutaraldehyde (3)

Figure 3: An electron micrograph showing a Schmidt-Lanterman section in a myelinated nerve fiber. This sample was fixed in buffered glutaraldehyde, washed in buffer, and then secondarily fixed in buffered osmium tetroxide, a standard procedure in preparing samples for transmission electron microscopy. The multiple layers of phospholipid membrane that make up the myelin sheath are well preserved by this procedure.


Factors affecting chemical fixation, formaldehyde and glutaraldehyde (4)

Figure 4: The Schmidt-Lanterman section in a myelinated nerve fiber shown at a higher magnification than in Figure 2. The multiple layers of phospholipid membrane that make up the myelin sheath are well preserved by glutaraldehyde/osmium tetroxide fixation.

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(Video) PhotoFix: Photo-Oxidation vs. Glutaraldehyde Fixation

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How is formaldehyde different from glutaraldehyde fixation? ›

Glutaraldehyde and formaldehyde have been used to fix chromatin core particles for electron microscopy. Glutaraldehyde crosslinks protein only, whereas formaldehyde crosslinks protein and DNA.

What is the chemical basis of fixation by glutaraldehyde? ›

Besides its application as disinfectant and medication, glutaraldehyde is used in biomedical research to fix cells. The principle behind the fixation is the binding of glutaraldehyde to nucleophiles of which the amino groups are the most abundant but binding to, e.g., sulfhydryl groups also occurs (Griffiths, 1993).

What is the chemical basis of fixation by formaldehyde? ›

Formaldehyde fixes tissue by cross-linking the proteins, primarily the residues of the basic amino acid lysine. Its effects are reversible by excess water and it avoids formalin pigmentation. Paraformaldehyde is also commonly used and will depolymerize back to formalin when heated, also making it an effective fixative.

What is the role of glutaraldehyde in fixation? ›

Besides its application as disinfectant and medication, glutaraldehyde is used in biomedical research to fix cells. The principle behind the fixation is the binding of glutaraldehyde to nucleophiles of which the amino groups are the most abundant but binding to, e.g., sulfhydryl groups also occurs (Griffiths, 1993).

What are five 5 factors affecting fixation of the cells in a physical and chemical state? ›

The number of factors affecting the fixation process includes buffering, penetration, volume, temperature and concentration.

What is the main disadvantage of glutaraldehyde? ›

Exposure to glutaraldehyde may cause the following symptoms: throat and lung irritation, asthma and difficulty breathing, dermatitis, nasal irritation, sneezing, wheezing, burning eyes, and conjunctivitis. Workers may be harmed from exposure to glutaraldehyde.

What are the advantages and disadvantages of glutaraldehyde fixative? ›

Glutaraldehyde is a dialdehyde and has two reactive groups for fixation. It is more rapid at fixing than formaldehyde and is more effective at forming crosslinks. However, this can be a disadvantage as it may destroy antibody binding sites. Glutaraldehyde also produces high auto fluorescence.

Which temperature is most effective for formaldehyde fixation? ›

This subjective analysis indicated that the best results were obtained with 2 hours of 4°C formalin followed by 2 hours of 45°C formalin (see Figure S1).

What is chemical basis of fixation? ›

Chemical fixation is a technique to fix a specimen with chemicals to prevent autolysis by the action of enzymes and deformation of morphologies during specimen preparation. Biological tissues start autolysis caused by their enzymes immediately after stopping the activities of them.

What is the main purpose of chemical fixation? ›

Chemical fixation is a common technique used to preserve cellular and tissue structure for biological samples. Ideal fixation processes should happen quickly to minimize artifacts. The fixatives enter the cells by diffusion.

What conditions should be avoided with glutaraldehyde? ›

Mixed Exposures

emphysema, and other respiratory problems. It may worsen respiratory conditions caused by chemical exposure.

How does temperature affect fixation? ›

Temperature: Increasing the temperature of fixation will increase the rate of diffusion of the fixative into the tissue and speed up the rate of chemical reaction between the fixative and tissue elements. It can also potentially increase the rate of tissue degeneration in unfixed areas of the specimen.

What is the mode of action of chemical fixation? ›

The two main mechanisms of chemical fixation are cross-linking and coagulation. Cross-linking involves covalent bond formation both within proteins and between them, which causes tissue to stiffen and therefore resist degradation.

What chemical reactions does formaldehyde have? ›

Formaldehyde is a good electrophile, participating in electrophilic aromatic substitution reactions with aromatic compounds, and can undergo electrophilic addition reactions with alkenes. In the presence of basic catalysts, formaldehyde undergoes a Cannizaro reaction to produce formic acid and methanol.

How often should glutaraldehyde be changed? ›

High-Level Disinfectant: Glutaraldehyde solution is a high-level disinfectant when used or reused, according to Directions for Use, at full strength for a maximum of 28 days at 25°C with an immersion time of at least 90 minutes.

What are the advantages and disadvantages of using formaldehyde as a fixative? ›

Its advantages are low cost, simplicity of use and good fixation traits, which are fast tissue penetration, good preservation of morphological structures and compatibility with downstream histological applications. Formaldehyde disadvantages are negative effects on nucleic acids.

How do you make glutaraldehyde fixation? ›

To prepare 100 mL of glutaraldehyde/paraformaldehyde:
  1. Add 2 g paraformaldehyde to approx 35 mL distilled water + 0.5 mL of approx. ...
  2. Heat the parafomaldehyde solution in a fume cupboard to 60°C when the paraformaldehyde dissolves (it is unnecessary to use a thermometer).
  3. Cool and add 8 mL of EM grade 25% glutaraldehyde.

What are the five 5 Factors affecting the rate of chemical reaction? ›

There are five general properties that can affect the rate of a reaction:
  • The concentration of the reactants. The more concentrated the faster the rate.
  • Temperature. ...
  • Physical state of reactants. ...
  • The presence (and concentration/physical form) of a catalyst (or inhibitor). ...
  • Light.

What are the four 4 main factors which affect the rate of chemical reactions? ›

The rate of a chemical reaction is influenced by many different factors, including reactant concentration, surface area, temperature, and catalysts.

What are 5 factors that affect the rate of a chemical reaction explain briefly the factors that influence the rate of reaction? ›

We can identify five factors that affect the rates of chemical reactions: the chemical nature of the reacting substances, the state of subdivision (one large lump versus many small particles) of the reactants, the temperature of the reactants, the concentration of the reactants, and the presence of a catalyst.

Is glutaraldehyde more toxic than formaldehyde? ›

The toxicity of glutaraldehyde was much less than that of formaldehyde when calculated as dilution of the stock solutions (i.e., in btl/ml). Expressed in terms maximum nontoxic concentration, 19% formaldehyde was approximately 40 times more toxic than 2.5% glu- taraldehyde at all times of exposure.

Which of the following is a disadvantage of formaldehyde? ›

When formaldehyde is present in the air at levels exceeding 0.1 ppm, some individuals may experience adverse effects such as watery eyes; burning sensations in the eyes, nose, and throat; coughing; wheezing; nausea; and skin irritation.

What is the effectiveness of glutaraldehyde? ›

The in vitro inactivation of microorganisms by glutaraldehydes has been extensively investigated and reviewed. Several investigators showed that 2% or greater aqueous solutions of glutaraldehyde, buffered to pH 7.5 to 8.5 with sodium bicarbonate, were effective in killing vegetative bacteria in less than 2 minutes; M.

What are the effects of fixatives? ›

It is important to realise that a fixative will initially produce a number of changes to the tissues in what is usually an aqueous environment. These will include shrinkage, swelling and hardening of various components.

What are two functions of glutaraldehyde? ›

Glutaraldehyde is used for several applications in healthcare facilities: Disinfectant and sterilization of surfaces and equipment. A tissue fixative in pathology labs. A hardening agent used to develop X-rays.

Are there long term effects of glutaraldehyde? ›


Respiratory irritation and skin sensitizing effects of glutaraldehyde have been confirmed. Reports in the literature have implicated glutaraldehyde as a possible causal factor in occupational asthma.

How can we reduce the effects of formaldehyde? ›

How can you reduce exposure to formaldehyde indoors?
  1. Chose low-formaldehyde products when building or remodeling. ...
  2. Ventilate indoor spaces. ...
  3. Air out new furniture and pressed-wood products. ...
  4. Don't allow smoking indoors. ...
  5. Wash permanent press clothing before wearing.
Nov 17, 2022

What effect does an elevated temperature have on formaldehyde? ›

Chamber experiments were carried out to gauge the effect of temperature and humidity on formaldehyde emission factors. The experiments established that 10 °C variation in temperature increased the formaldehyde emissions 1.9–3.5 times, and a 35% increase in RH can increase the emissions by a factor of 1.8–2.6.

What is the usual fixation time for formaldehyde? ›

Time of fixation: 6-18 hrs for biopsy specimens and 24-72 hrs for standard samples.

What are the two common methods of fixation What are the purposes of fixation? ›

The fixation methods are classified into chemical fixation and physical fixation. The former method chemically fixes proteins, lipids, etc., by using chemicals and the latter method physically fixes water in cells or tissues by freezing them.

What are the different types of fixation? ›

Depending on your specimen, you can choose one of the three general types of fixation processes – heat fixation, perfusion fixation, and immersion fixation.

What are the advantages of chemical fixation? ›

Chemical fixation is superior in the preservation of morphology to coagulating fixatives (e.g., solvents and acetic acid) that are typically used for light microscopy. However, chemical fixation can also lead to protein denaturation, which may be problematic in downstream antibody-labeling techniques.

What are the guidelines for glutaraldehyde? ›

Use glutaraldehyde only in designated areas where traffic and ventilation can be controlled. Ensure that the ventilation system is operating prior to handling glutaraldehyde solutions. (Consult your facilities department for help on how to check the operation of your ventilation system.)

Which items are critical factors influencing disinfection? ›

Several physical and chemical factors also influence disinfectant procedures: temperature, pH, relative humidity, and water hardness. For example, the activity of most disinfectants increases as the temperature increases, but some exceptions exist.

Is glutaraldehyde related to formaldehyde? ›

Glutaraldehyde is very similar to formaldehyde in its structure and uses in the healthcare industry. Chemically, Glutaraldehyde exists at room temperature as a liquid, whereas formaldehyde exists under those conditions as a gas, which can easily be dissolved in water.

What are the effects of fixation? ›

Fixation, then, is a compromise between time-progressive formaldehyde damage of protein antigens and autolytic degradation. Therefore, any method that accelerates fixation would limit autolysis and time spent immersed in formaldehyde and thereby better preserve antigen and DNA.

What is the procedure of tissue fixation? ›


The specimen is placed in a liquid fixing agent (fixative) such as formaldehyde solution (formalin). This will slowly penetrate the tissue causing chemical and physical changes that will harden and preserve the tissue and protect it against subsequent processing steps.

What are the aims and effects of fixation? ›

The aim of fixation is to preserve cells or tissues in as near a life-like condition as possible, prevent autolysis and putrefaction, and protect the tissue from damage during subsequent processing. Fixatives have different actions: for example, crosslinking, precipitative, coagulative.

What are fixation issues? ›

Fixation is the human tendency to approach a given problem in a set way that limits one's ability to shift to a new approach to that problem. As such, fixation impairs ideation for designers and results in impasses. It can also cause the Einstellung effect, the phenomenon of overlooking better ways of solving problems.

What are the types of fixation? ›

Depending on your specimen, you can choose one of the three general types of fixation processes – heat fixation, perfusion fixation, and immersion fixation.

What are the properties of fixation? ›

Fixation results in denaturation and coagulation of protein in the tissues. The fixatives have a property of forming cross links between proteins, thereby forming a gel, keeping everything in their in vivo relation to each other.

How do you make glutaraldehyde solution? ›

Dilute 1 mL of 250 g/L aqueous glutaraldehyde to 25 mL with distilled, deionized water to make the glutaraldehyde stock solution. Put 10.0 mL of 1.13 mol/L sodium sulfite solution in a beaker and stir magnetically. Adjust pH to between 8.5 and 10 with base or acid.

How is chemical fixation done? ›

Chemical fixation is usually achieved by immersing the specimen in the fixative (immersion fixation) or, in the case of small animals or some whole organs such as a lung, by perfusing the vascular system with fixative (perfusion fixation).

What are the two types of fixation? ›

Mechanism of Fixation

The two main mechanisms of chemical fixation are cross-linking and coagulation. Cross-linking involves covalent bond formation both within proteins and between them, which causes tissue to stiffen and therefore resist degradation.

What is used for chemical fixation of tissues? ›

Aldehydes such as glutaraldehyde and formaldehyde are the most commonly used fixatives. Glutaraldehyde is great for crosslinking proteins while formaldehyde, due to its small molecular size, can penetrate into cells quickly; however, aldehydes lack the ability to fix lipids present in the sample.

What are the 2 reasons of fixation? ›

Fixation consists of two steps: cessation of normal life functions in the tissue (killing) and stabilization of the structure of the tissue (preservation). The goal of fixation is to preserve structure as faithfully as possible compared to the living state.

What is the effect of poor fixation? ›

Poor fixation, or delay in fixation, causes loss of antigenicity or diffusion of antigens into the surrounding tissues. Poorly fixed blocks do not process to paraffin adequately. Alcohol used in the tissue processing dehydrating steps is an excellent fixative and an excellent dehydrating agent.

Why fixation is the most important step? ›

Without question, the most important step to viewing biological tissue is fixation. The purpose of fixation preserves the structure of the tissue permanently in as life-like a state as possible.


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