Allan Bennett

Thomas Pottage

Formaldehyde is a monoaldehyde that exists as a gas freely soluble in water. It can be used both as an aqueous solution (formalin) for disinfection and as a gas for fumigation of indoor spaces and equipment. Apart from its use as a disinfectant, it is used in the preparation of human and veterinary vaccines and the preservation of tissues. Formaldehyde is highly efficacious against a broad range of microorganisms, but its activity is relatively slow acting. Its antimicrobial efficacy is reduced in the presence of organic matter. Formaldehyde vapor has a pungent odor and may cause allergic reactions in some workers; is an irritant of mucous membranes; and, as of 2014, was classified as a carcinogen (category 1B), mutagen (category 2), and sensitizer. For these reasons, it is declining in use as a general liquid disinfectant and as part of formulated disinfectants. However, it is still widely used as a gaseous disinfectant in the microbiological laboratory sector and agricultural sectors, where it can be generated by depolymerizing paraformaldehyde by heating, boiling off formalin and water, or historically by adding formalin to potassium permanganate crystals. It is also used as a tissue preservative and in embalming.1


Formaldehyde is the simplest of the aldehydes and is also known as methanal. Its chemical formula is CH2O. Its chemical properties are summarized in Table 36.1. It is obtained either in a polymeric form, paraformaldehyde (HO[CH2O]nH), or in a liquid aqueous suspension at ˜40% vol/vol stabilized with methanol (10%-15%) to prevent oxidation or polymerization generally referred to as formalin.2


The basic mode of action of formaldehyde as an antimicrobial agent is through the irreversible cross-linking or denaturing of protein and through DNA damage potentially though protein-DNA cross-linking.3 This prevents the operation of essential cell functions causing loss of viability. Formaldehyde has a broad spectrum of action on microorganisms, and it denatures proteins by alkylation with amino and sulfhydryl groups of proteins and ring nitrogen atoms of purine bases such as guanine.4 In the inactivation of spores, alkylation of nucleic acids may be more important than changes in protein constituents.3

Due to this broad mode of action, formaldehyde is considered to have a very wide range of activity against all types of microorganisms, and the development of resistance is considered limited. Some microorganisms have been found to have increased resistance to formaldehyde due to the expression of formaldehyde dehydrogenase.5 Formaldehyde is oxidized by formaldehyde dehydrogenase. In these experiments it was found that a glutathione-dependent formaldehyde dehydrogenase that oxidizes formaldehyde6 was found to be encoded by a gene, which can be transferred between the microorganisms via plasmids, and the mechanisms of resistance can be transferred to unrelated strains of the same bacterial species, such as Escherichia coli.7,8 However, Azachi et al9 described that formaldehyde dehydrogenase was not the sole reason for increased resistance to formaldehyde in two gram-negative bacteria. It was found that formaldehyde-resistant strains had the addition of a high-molecular-mass protein in the outer membrane, and therefore, formaldehyde resistance is conferred by the structure and composition of the outer membrane.9 Revival in formaldehyde-treated Bacillus subtilis spores was increased by up to 1 log using heat shock for 30 minutes before plating out. It was postulated by Williams and Russell10 that the heat shock could enhance and/or trigger germination after contact with formaldehyde.

Formaldehyde has been used for the inactivation of viruses in vaccine production for a number of years.11 Complete inactivation depends on the physical state of
the particles. There have been reported problems with reactivation of the virus particles when clumped prior to treatment with formalin.11

TABLE 36.1 Chemical properties of formaldehyde

EC No.




Molecular Weight

30.03 g/mol

Melting Point


Boiling Point


Conversion Factor

1 ppm = 1.23 mg/m3 (20°C, 101.3 kPa)

Formaldehyde has reduced activity against agents in organic materials.12 This was demonstrated by the Cutter incident regarding the ineffective inactivation of live polio virus used in the polio vaccination.13 Approximately 400 000 people, primarily children, were inoculated with a formaldehyde-inactivated vaccine, of these over 250 either of vaccinees or their contacts were reported with polio infections.14

Formaldehyde is ineffective against prions and has been shown in some instances to stabilize them and make them more resistant to other modes of disinfection such as autoclaving and incineration.15


Tissue Fixative

Formalin is commonly used as a tissue fixative. Solutions used include phosphate-buffered formalin or formalinsaline at a formalin concentration of 10%.2

Soil Disinfection or Sterilization

Chemical decontamination of soil can be carried out in small batches collected from a contaminated site or in in situ by the application of formalin to the soil. Formaldehyde was used to decontaminate a Scottish island that had been contaminated with Bacillus anthracis spores through the testing of a biological weapon during the Second World War.16 A 5% suspension of formaldehyde was sprayed at 50 L/m2 over the contaminated area after burn off of vegetation. The highest areas of contamination were treated by injection of undiluted formalin (38% formaldehyde) to a depth of 50 mm. Soil sampling showed that the process was effective. The soil type facilitated the process allowing easy absorption of the formaldehyde. This approach may not be suitable for different soil types. It was also costly; approximately 2 million liters of 5% formaldehyde were applied over a 4-hectare (40 000 m2) area with further direct treatments with formaldehyde necessary for small pockets of spores.


Formaldehyde is the major chemical used to preserve cadavers. Its antimicrobial properties delay the decay processes and thus can preserve tissues without destroying their structural detail, which is essential for anatomical study.17 The embalming fluid fixes the proteins within the body or parts thereof limiting their ability to become a food source for microorganisms. The embalming fluid is either injected arterially or directly into the body’s cavities to displace the bodily fluids. The embalming fluid itself is made up of a mixture containing not only predominantly formalin but also glutaraldehyde and methanol, the concentration of these agents can be varied depending on the area to be injected.

Liquid Disinfection

Forty-percent formaldehyde gas dissolved in water constitutes a 100% solution of formalin; 8% formaldehyde in water is 20% formalin. Depending on its concentration, formaldehyde can be classified as a high-level (8% formaldehyde plus 70% alcohol) or intermediate- to high-level (4%-8% formaldehyde in water) disinfectant.18 Aqueous formaldehyde in concentrations less than 4% may have limited activity against Mycobacterium species, particularly the nontuberculous mycobacteria indigenous to certain potable water supplies.18 The action of formaldehyde on the protein coat of poliovirus progressively slows down the killing rate by obstructing penetration to the nucleic acid core.19 As mentioned, 8% formaldehyde in water is considered an intermediate- to high-level disinfectant; combining 8% formaldehyde in 65% to 70% isopropanol yields a compound that is rapidly bactericidal, tuberculocidal, and sporicidal, but the time required for achieving sterility using high numbers of spores as a challenge may be up to 18 hours or longer, depending on the test conditions.18

Experiments conducted by Sagripanti and Bonifacino20 in a comparison of different liquid chemical agents found that an 8% formaldehyde solution at different pH, ranging from 3 to 10, produced less than 90% inactivation of B subtilis subspecies Bacillus globigii (now Bacillus atrophaeus) spores after contact for 30 minutes at 20°C. The sporicidal properties of 8% formaldehyde was increased when the temperature was increased from 20°C to 40°C (at pH 3.2) to >99.9% spore inactivation in 20 minutes. A later set of experiments used 8% formaldehyde, this time at pH 3.4, against a range of vegetative bacteria in liquid suspensions. It was found that with a contact time
of 30 minutes, formaldehyde was able to reduce the recoverable numbers of bacteria to below the detection limit in all cases except Staphylococcus epidermidis21 (Table 36.2).

TABLE 36.2 Bacterial inactivation using 8% formaldehyde for 30 minutes at 20°Ca


Log10 Reduction (Replicates)

Bacillus cereus

>5.0 (2)

Clostridium perfringens

>6.3 (2)

Escherichia coli

>6.9 (2)

Listeria monocytogenes

>6.1 (1)

Pseudomonas aeruginosa

>6.1 (3)

Salmonella typhimurium

>6.2 (3)

Shigella sonnei

>6.3 (2)

Staphylococcus aureus

>6.5 (3)

Staphylococcus epidermidis

5.9 ± 1.1 (3)

Vibrio cholerae

>6.4 (2)

Vibrio parahaemolyticus

>6.2 (2)

Vibrio vulnificus

>6.3 (2)

Yersinia enterocolitica

>6.8 (2)

a Data from Sagripanti et al.21

Alasri et al22 investigated the minimum sporicidal concentration of formaldehyde to produce a 5 log reduction of Bacillus spores (three species of B subtilis and one species of Bacillus amyloliquefaciens) over a range of exposure periods. Formaldehyde concentrations of 7500 ppm (0.75 %) were required to inactivate spores (5 log reduction) of one wild type B subtilis species and B amyloliquefaciens within 5 minutes, with a lower formaldehyde concentration (3750 ppm) required for inactivation within a 7-hour period. The concentration of 3750 ppm was also found to inactivate the other B subtilis wild-type spores over the range of times investigated from 5 minutes up to 7 hours. Spores of a B subtilis-type strain (ATCC 6633) were found to be the least resistant to formaldehyde, being inactivated by 3750 ppm within 5 minutes decreasing to 1875 ppm for 7 hours.22

A far lower concentration of formaldehyde, 0.02%, was found to be able to inactivate E coli. Conversely, sporicidal activity of 4% (pH 8.0) formaldehyde against B anthracis spores was slower, >2 hours for a 104 reduction, than 1% and 2% glutaraldehyde treatment for 30 and 15 minutes, respectively, under the same conditions.23

Formaldehyde solutions have been demonstrated to be tuberculocidal by Rubbo et al.23 A concentration of 4% was shown to inactivate 104 colony-forming units of Mycobacterium tuberculosis within 5 minutes in liquid suspension.23

Möller et al24 have shown that a 2% solution of formaldehyde was effective against vaccinia, adenovirus, and murine norovirus at 25°C. However, long exposure times of 6 hours were required for the enveloped vaccinia virus, and inactivation took days at a lower temperature of 4°C.

Because it is not corrosive to equipment associated with hemodialysis systems, formaldehyde has been used in a concentration of 4% to disinfect dialysis systems and disposable hemodialyzers that are reused in the same patient. In both instances, however, the problem of residual formaldehyde constitutes a potential health hazard to dialysis patients, and hemodialysis systems and hemodialyzers must be thoroughly rinsed free of residual formaldehyde prior to use.25

Liquid formaldehyde has also been used to control outbreaks of parasites, such as the ectoparasite Ichthyophthirius multifiliis, in aquaculture systems. It was found in these studies not to harm the fish at the concentrations used, but the safety issues to workers using it and environmental discharge have led to alternative agents to be identified and investigated.26,27

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May 9, 2021 | Posted by in MICROBIOLOGY | Comments Off on Formaldehyde

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