Russell, Hugo and Ayliffe's Principles and Practice of Disinfection, Preservation and Sterilization

By Adam P. Fraise

The new edition of this established and highly respected text is THE definitive reference in its field. It details methods for the elimination or prevention/control of microbial growth, and features: 

• New chapters on bioterrorism and community healthcare
• New chapters on microbicide regulations in the EU, USA and Canada
• Latest material on microbial resistance to microbicides
• Updated material on new and emerging technologies, focusing on special problems in hospitals, dentistry and pharmaceutical practice
• Practical advice on problems of disinfection and antiseptics in healthcare
• A systematic review of sterilization methods, with uses and advantages outlined for each
• Evaluation of disinfectants and their mechanisms of action with respect to current regulations

The differences between European and North American regulations are highlighted throughout, making this a truly global work, ideal for worldwide healthcare professionals working in infectious diseases and infection control.

• Language: English
• Publisher: Wiley
• Release date: Nov 20, 2012
• ISBN: 9781118425862

Book preview

Preface to the Fifth Edition

It has been a particular privilege to be editors of this fifth edition, which has been substantially revised. Thirty-six of its 40 chapters are new and those from the previous edition have undergone major revisions/updates. Every attempt also has been made to cover the subject matter in all chapters from a global perspective.

Putting this edition together has been a daunting task in view of the rapidly expanding significance and scope of the subject matter covered while also considering the wide acceptance and utility of its previous editions. We thank the authors for their contributions and the publisher’s staff for coordinating all dealings between the contributors and the editors.

We are most grateful to our respective families for allowing us to devote the long hours needed to edit this book.

Adam P. Fraise

Jean-Yves Maillard

Syed A. Sattar

November 2012

Preface to the First Edition

Sterilization, disinfection and preservation, all designed to eliminate, prevent or frustrate the growth of microorganisms in a wide variety of products, were incepted empirically from the time of man’s emergence and remain a problem today. The fact that this is so is due to the incredible ability of the first inhabitants of the biosphere to survive and adapt to almost any challenge. This ability must in turn have been laid down in their genomes during their long and successful sojourn on this planet.

It is true to say that, of these three processes, sterilization is a surer process than disinfection, which in turn is a surer process than preservation. It is in the last field that we find the greatest interactive play between challenger and challenged. The microbial spoilage of wood, paper, textiles, paints, stonework, stored foodstuffs, to mention only a few categories at constant risk, costs the world many billions of pounds each year, and if it were not for considerable success in the preservative field, this figure would rapidly become astronomical. Disinfection processes do not suffer quite the same failure rate and one is left with the view that failure here is due more to uninformed use and naïve interpretation of biocidal data. Sterilization is an infinitely more secure process and, provided that the procedural protocol is followed, controlled and monitored, it remains the most successful of the three processes.

In the field of communicable bacterial diseases and some virus infections, there is no doubt that these have been considerably reduced, especially in the wealthier industrial societies, by improved hygiene, more extensive immunization and possibly by availability of antibiotics. However, hospital-acquired infection remains an important problem and is often associated with surgical operations or instrumentation of the patient. Although heat sterilization processes at high temperatures are preferred whenever possible, medical equipment is often difficult to clean adequately, and components are sometimes heat-labile. Disposable equipment is useful and is widely used if relatively cheap but is obviously not practicable for the more expensive items. Ethylene oxide is often used in industry for sterilizing heat-labile products but has a limited use for reprocessing medical equipment. Low-temperature steam, with or without formaldehyde, has been developed as a possible alternative to ethylene oxide in the hospital.

Although aseptic methods are still used for surgical techniques, skin disinfection is still necesssary and a wider range of non-toxic antiseptic agents suitable for application to tissues is required. Older antibacterial agents have been reintroduced, e.g. silver nitrate for burns, alcohol for hand disinfection in the general wards and less corrosive hypochlorites for disinfection of medical equipment.

Nevertheless, excessive use of disinfectants in the environment is undesirable and may change the hospital flora, selecting naturally antibiotic-resistant organisms, such as Pseudomonas aeru­ginosa, which are potentially dangerous to highly susceptible patients. Chemical disinfection of the hospital environment is therefore reduced to a minimum and is replaced where applicable by good cleaning methods or by physical methods of disinfection or sterilization.

A.D.R.

W.B.H.

G.A.J.A.

SECTION 1

Principles

1

Historical Introduction

Adam P. Fraise

Microbiology Department, Queen Elizabeth Medical Centre, Pathology – University Hospitals Birmingham NHS Foundation Trust, Birmingham, UK

Early concepts

Chemical disinfection

Sterilization

Future developments for microbicides

References

Further reading

Early Concepts

Disinfection and hygiene are concepts that have been applied by humans for thousands of years. Examples may be found in ancient literature such as the Bible where disinfection using heat was recorded in the Book of Numbers; the passing of metal objects, especially cooking vessels, through fire was declared to cleanse them. It was also noted from early times that water stored in pottery vessels soon acquired a foul odor and taste and Aristotle recommended to Alexander the Great the practice of boiling the water to be drunk by his armies. It may be inferred that there was awareness that something more than mechanical cleanness was required.

Chemical disinfection of a sort was practiced at the time of Persian imperial expansion, c. 450 BC, when water was stored in vessels of copper or silver to keep it potable. Wine, vinegar and honey were used on dressings and as cleansing agents for wounds and it is interesting to note that diluted acetic acid has been recommended comparatively recently for the topical treatment of wounds and surgical lesions infected by Pseudomonas aeruginosa.

The art of mummification, which so obsessed the Egyptian civilization (although it owed its success largely to desiccation in the dry atmosphere of the region), employed a variety of balsams containing natural preservatives. Natron, a crude native sodium carbonate, was also used to preserve the bodies of human and animal alike.

Practical procedures involving chemical agents were also applied in the field of food preservation. Thus tribes who had not progressed beyond the status of hunter-gatherers discovered that meat and fish could be preserved by drying, salting or mixing with natural spices. As the great civilizations of the Mediterranean and Near and Middle East receded, and European cultures arose, so the precepts of empirical hygiene were also developed. There was, of course, ongoing contact between Europe and the Middle and Near East through the Arab and Ottoman incursions into Europe, but it is difficult to find early European writers acknowledging the heritage of these empires.

An early account of procedures to try and combat the episodic scourge of the plague may be found in the writings of the 14th century, where Joseph of Burgundy recommended the burning of juniper branches in rooms where plague sufferers had lain. Sulfur, too, was burned in the hope of removing the cause of this disease. The association of malodor with disease and the belief that matter floating in the air might be responsible for diseases, a Greek concept, led to these procedures. If success was achieved it may have been due to the elimination of rats, later to be shown as the bearers of the causative organism.

In Renaissance Italy at the turn of the 15th century, a po­et, philosopher and physician, Girolamo Fracastoro, who was professor of logic at the University of Padua, recognized possible causes of disease, mentioning contagion and airborne infection; he thought there must exist seeds of disease. Robert Boyle, the skeptical chemist, writing in the mid-17th century, wrote of a possible relationship between fermentation and the disease process. In this he foreshadowed the views of Louis Pasteur. There is, however, no evidence in the literature that Pasteur even read the opinions of Robert Boyle or Fracastoro.

The next landmark in this history was the discovery by Antonie van Leeuwenhoek of small living creatures in a variety of habitats, such as tooth scrapings, pond water and vegetable infusions. His drawings, seen under his simple microscopes (×300), were published in the Philosophical Transaction of the Royal Society of London before and after this date. Some of his illustrations are thought to represent bacteria, although the greatest magnification he is said to have achieved was ×300. When considering Leeuwenhoek’s great technical achievement in microscopy and his painstaking application of it to original investigation, it should be borne in mind that bacteria in colony form must have been seen from the beginning of human existence. A very early report of this was given by the Greek historian Siculus, who, writing of the siege of Tire in 332 BC, states how bread, distributed to the Macedonians, had a bloody look. This was probably attributable to contamination by pigmented strains of Serratia marcescens and this phenomenon must have been seen, if not recorded, from time immemorial.

Turning back to Europe, it is also possible to find other examples of workers who believed, but could not prove scientifically, that some diseases were caused by invisible living agents, contagium animatum. Among these workers were Kircher (1658), Lange (1659), Lancisi (1718) and Marten (1720).

By observation and intuition, therefore, we see that the practice of heat and chemical disinfection, the inhibitory effect of desiccation and the implication of invisible objects with the cause of some diseases were known or inferred from early times.

Before moving on to a more formally scientific period in history it is necessary to report on a remarkable quantification of chemical preservation published in 1775 by Joseph Pringle. Pringle was seeking to evaluate preservation by salting and he added pieces of lean meat to glass jars containing solutions of different salts; these he incubated, and judged his end-point by the presence or absence of smell. He regarded his standard salt as sea salt and expressed the results in terms of the relative efficiency as compared with sea salt; niter, for example, had a value of 4 by this method. Rideal and Walker, 153 years later, were to use a similar method to measure the activity of phenolic disinfectants against Salmonella typhi; their standard was phenol.

Although the concept of bacterial diseases and spoilage was not widespread before the 19th century, procedures to preserve food and drink were used early in history. It is only more recently, that is in the 1960s, that the importance of microorganisms in pharmaceuticals was appreciated [1] and the principles of preservation of medicines introduced.

Chemical Disinfection

As the science of chemistry developed, newer and purer chemical disinfectants began to be used. Mercuric chloride, which had been used since the Middle Ages and was probably first used by Arab physicians, began to be used as a wound dressing. In 1798 bleaching powder was first made and a preparation of it was employed by Alcock in 1827 as a deodorant and disinfectant. Lefêvre introduced chlorine water in 1843, and in 1839 Davies had suggested iodine as a wound dressing. Semmelweis used chlorine water in his work on childbed fever occurring in the obstetrics division of the Vienna General Hospital, where he achieved a sensational reduction in the incidence of the infection by insisting that all attending the birth washed their hands initially in chlorine water and later (in 1847) in chlorinated lime.

Wood and coal tar were used as wound dressings in the early 19th century and, in a letter to the Lancet, Smith described the use of creosote (Gr. kreas flesh, soter savior) as a wound dressing [2]. In 1850 Le Beuf, a French pharmacist, prepared an extract of coal tar by using the natural saponin of quillaia bark as a dispersing agent. Le Beuf asked a well-known surgeon, Jules Lemair, to evaluate his product. It proved to be highly efficacious. Küchenmeister was to use pure phenol in solution as a wound dressing in 1860 and Joseph Lister also used phenol in his great studies on antiseptic surgery during the 1860s. It is also of interest to record that a number of chemicals were being used as wood preservatives. Wood tar had been used in the 1700s to preserve the timbers of ships, and mercuric chloride was used for the same purpose in 1705. Copper sulfate was introduced in 1767 and zinc chloride in 1815. Many of these products are still in use today.

Turning back to evaluation, Bucholtz in 1875 determined what is known today as the minimum inhibitory concentration of phenol, creosote and benzoic and salicylic acids against bacteria. Robert Koch made measurements of the inhibitory power of mercuric chloride against anthrax spores but overvalued the products as he failed to neutralize the substance carried over in his tests. This was pointed out by Geppert, who, in 1889, used ammonium sulfide as a neutralizing agent for mercuric chloride and obtained much more realistic values for the antimicrobial powers of mercuric chloride.

It will be apparent that, in parallel with these early studies, an important watershed had been passed; that is, the scientific identification of a microbial species with a specific disease. Credit for this should go to an Italian, Agostino Bassi, a lawyer from Lodi (a small town near Milan). Although not a scientist or physician, he performed exacting scientific experiments to equate a disease of silkworms with a fungus. Bassi identified plague and cholera as being of microbial origin and also experimented with heat and chemicals as antimicrobial agents. His work anticipated the great names of Pasteur and Koch in the implication of microbes with certain diseases, but because it was published locally in Lodi and in Italian it has not found the place it deserves in many textbooks.

Two other chemical disinfectants still in use today were early introductions. Hydrogen peroxide was first examined by Traugott in 1893, and Dakin reported on chlorine-releasing compounds in 1915. Quaternary ammonium compounds were introduced by Jacobs in 1916.

In 1897, Kronig and Paul, with the acknowledged help of the Japanese physical chemist Ikeda, introduced the science of disinfection dynamics; their pioneering publication [3] was to give rise to innumerable studies on the subject lasting through to the present day.

Since then other chemical microbicides, which are now widely used in hospital practice, have been introduced – such as chlorhexidine, an important cationic microbicide, whose activity was described in 1958 [4].

More recently, a better understanding of hygiene concepts has provided the basis for an explosion in the number of products containing chemicals. In particular, quaternary ammonium compounds are being developed with altered chemistry and improved activity. Peroxygen compounds are gaining popularity due to their good in vitro activity (including activity against spores), and mechanisms for preparing compounds that release hypochlorous acid are also being adopted widely in the healthcare, veterinary and food industries. This rise in microbicide-containing products has also sparked a major concern about the improper use of chemical disinfectants and a possible emergence of microbial resistance to these microbicides and possible cross-resistance to antibiotics. Among the most widely studied microbicides are chlorhexidine and triclosan. The bisphenol triclosan is unique, in the sense that it has been shown that at a low concentration it inhibits selectively an enoyl reductase carrier protein, which is also a target site for antibiotic chemotherapy in some microorganisms.

Sterilization

Heat has been known as a cleansing and purifying agent for centuries. In 1832, William Henry, a Manchester physician, studied the effect of heat on contaminated material, that is clothes worn by sufferers from typhus and scarlet fever. He placed the material in a pressure vessel and realized that he could achieve temperatures higher than 100°C by using a sealed vessel fitted with a safety valve. He found that garments so treated could be worn with impunity by others, who did not then contract the diseases. Louis Pasteur also used a pressure vessel with a safety valve for sterilization.

Sterilization by filtration has been observed from early times. Foul-tasting waters draining from ponds and percolating through soil or gravel were sometimes observed, on emerging at a lower part of the terrain, to be clear and potable (drinkable), and artificial filters of pebbles were constructed. Later, deliberately constructed tubes of unglazed porcelain or compressed kieselguhr, the so-called Chamberland or Berkefeld filters, made their appearance (in 1884 and 1891, respectively).

Although it was known that sunlight helped wound healing and in checking the spread of disease, it was Downes and Blunt in 1887 who first set up experiments to study the effect of light on bacteria and other organisms. Using Bacillus subtilis as the test organism, Ward, in 1892, attempted to investigate the connection between the wavelength of light and its antimicrobial activity; he found that blue light was more active than red.

In 1903, using a continuous arc current, Barnard and Morgan demonstrated that the maximum bactericidal effect resided in the range 226–328 nm, that is, light in the ultraviolet range. Ultraviolet light is now a well-established agent for water and air decontamination.

At the end of the 19th century, a wealth of pioneering work was being carried out in subatomic physics. In 1895, the German physicist Röntgen discovered X-rays, and 3 years later Rieder found these rays to be harmful to common pathogens. X-rays of a wavelength between 10−10 and 10−11 are emitted by ⁶⁰Co and are now used extensively in sterilization processes.

Another major field of research in the concluding years of the 19th century was that of natural radioactivity. In 1879, Becquerel found that, if left near a photographic plate, uranium compounds would cause the plate to fog. He suggested that rays, later named Becquerel rays, were being emitted. Rutherford, in 1899, showed that when the emission was exposed to a magnetic field three types of radiation (α, β and γ) were given off. The γ-rays were shown to have wavelengths of the same order as X-rays. Beta-rays were found to be electrons, and α-rays were helium nuclei. These emissions were demonstrated to be antimicrobial by Mink in 1896, and by Pancinotti and Porchelli 2 years later. High-speed electrons generated by electron accelerators are now used in sterilization processes.

Thus, within 3 years of the discovery of X-rays and natural radiation, their effect on the growth and viability of microorganisms had been investigated and published. Both were found to be lethal. Ultraviolet light was shown in 1993 to be the lethal component of sunlight.

For more information on this aspect of sterilization see Hugo [5].

Sterilization can also be achieved by chemicals, although their use for this purpose does not offer the same quality assurance as heat or radiation sterilization. The term chemosterilizer was first defined by Borick in 1968. This has now been replaced by the term chemical sterilants, which is used to refer to those chemicals used in hospital for sterilizing reusable medical devices. Among the earliest used chemical sterilants were formaldehyde and ethylene oxide. Another aldehyde, glutaraldehyde, has been used for this purpose for almost 40 years [6]. Compounds such as peracetic acid, chlorine dioxide and ortho-phthalaldehyde (OPA) have been introduced as substitutes for the dialdehyde and these compounds have been widely adopted for the decontamination of flexible fiberoptic endoscopes.

In the latter half of the 20th century the science of sterilization and disinfection followed a more ordered pattern of evolution, culminating in new technologies such as radiation sterilization and gas plasma sterilization. However, no method is foolproof and human error will always occur. Therefore, whatever technologies are used, all staff working in the field of sterilization must be vigilant and maintain a critical approach where evaluation of methodologies is an integral part of the process.

Future Developments for Microbicides

This is a very interesting time for those involved in the use of microbicides. For the last 50 years, our knowledge of microbicides has increased, but so have our concerns about their extensive use in hospital and domiciliary environments. One encouraging sign is the apparent willingness of the industry to understand the mechanisms of action of chemical microbicides and the mechanisms of microbial resistance to microbicides. Although new microbicidal molecules might not be produced in the future, novel products might concentrate on synergistic effects between microbicides and the combination of microbicide and permeabilizer or other non-microbicidal chemicals, so that an increase in antimicrobial activity is achieved. The ways microbicides are delivered is also the subject of extensive investigations. For example, the use of polymers for the slow release of microbicidal molecules, the use of light-activated microbicides and the use of alcoholic rubs for antisepsis are all signs of current concerted efforts to adapt laboratory concepts to practical situations.

Although, this might be a golden age for microbicidal science, many questions remain unanswered, such as the significance of microbicide resistance, the fine mechanism of action of microbicides, the possibility of primary action sites within target microorganisms, and the effect of microbicides on emerging pathogens and microbial biofilms. Some of these concepts will be discussed further in following chapters.

References

1 Kallings, L.O. et al. (1966) Microbial contamination of medical preparations. Acta Pharmaceutica Suecica, 3, 219–228.

2 Smith, F. Sir (1836–1837) External employment of creosote. Lancet, ii, 221–222.

3 Kronig, B. and Paul, T. (1897) Die chemischen Gundlagen der Lehr von der Giftwirkung und Desinfection. Zeitschrift fur Hygiene und Infectionskrankheiten, 25, 1–112.

4 Denton, W. (2001) Chlorhexidine, in Sterilisation and Preservation, 5th edn (ed. S.S. Block), Lippincott Williams & Wilkins, Philadelphia, pp. 321–336.

5 Hugo, W.B. (1996) A brief history of heat, chemical and radiation preservation and disinfection. International Biodeterioration and Biodegradation, 36, 197–221.

6 Bruch, C.W. (1991) Role of glutaraldehyde and other chemical sterilants in the processing of new medical devices, in Sterilization of Medical Products, vol. 5 (eds R.F. Morrissey and Y.I. Prokopenko), Polyscience Publications, Morin Heights, Canada, pp. 377–396.

Further Reading

Brock, T.D. (ed.) (1961) Milestones in Microbiology, Prentice Hall, London.

Bullock, W. (1938) The History of Bacteriology, Oxford University Press, Oxford.

Collard, P. (1976) The Development of Microbiology, Cambridge University Press, Cambridge.

Hugo, W.B. (1991) A brief history of heat and chemical preservation and disinfection. Journal of Applied Bacteriology, 71, 9–18.

Reid, R. (1974) Microbes and Men, British Broadcasting Corporation, London.

2

Types of Microbicidal and Microbistatic Agents

Ibrahim Al-Adham¹, Randa Haddadin² and Phillip Collier³

¹ Faculty of Pharmacy & Medical Sciences, University of Petra, Amman, Jordan

² Faculty of Pharmacy, University of Jordan, Amman, Jordan

³ School of Contemporary Sciences, University of Abertay, Dundee, UK

Introduction

Phenols

Organic and inorganic acids: esters and salts

Aromatic diamidines

Biguanides

Surface-active agents

Aldehydes

Microbicidal dyes

Halogens

Quinoline and isoquinoline derivatives

Alcohols

Peroxygens

Chelating agents

Permeabilizers

Heavy metal derivatives

Anilides

Miscellaneous preservative

Vapor-phase disinfectants

Aerial disinfectants

Inactivation of prions

Other uses of microbicidal and microbistatic agents

Which microbicidal or microbistatic agent?

Other concepts

References

Introduction

This chapter serves as a source of reference for those interested in developing an initial or general understanding of the chemistry and mode of action of a particular group of microbicidal agents. It is not intended to provide a definitive description of individual agents, but rather to introduce the reader to the general concepts of those agents and to provide key references as a starting point for more thorough investigations. With this in mind, the authors have undertaken a hard edit of the previous version of this chapter, including the removal of dated information and updates to the chemical groups discussed. Given this approach, the authors wish to acknowledge those who have nurtured and developed this chapter in previous editions of the book: Barry Hugo and Denver Russell (first, second and third editions) and Suzanne Moore and David Payne (fourth edition).

Phenols

Hugo [1, 2] and Marouchoc [3] showed that phenols and natural product distillates containing phenols shared, with chlorine and iodine, an early place in the armory of antiseptics. Today, they are widely used as general disinfectants and as preservatives for a variety of manufactured products [4], except where there is risk of contamination of foods. As a result of their long history, a vast literature has accumulated dealing with phenol and its analogs and a comprehensive review of these compounds can be found in Goddard and McCue [5]. While many different parameters have been used to express their microbicidal and microbistatic power, the phenol coefficient is perhaps the most widely employed.

A reasonable assessment of the relationship between structure and activity in the phenol series was compiled by Suter [6]. The main conclusions from this survey were:

1. Para-substitutions of an alkyl chain up to six carbon atoms in length increases the bactericidal action of phenols, presumably by increasing the surface activity and ability to orientate at an interface. Activity falls off after this due to decreased water solubility. Straight chain para-substituents confer greater activity than branched-chain substituents containing the same number of carbon atoms.

2. Halogenation increases the bactericidal activity of phenols. The combination of alkyl and halogen substitution, which confers the greatest bactericidal activity, is that where the alkyl group is ortho- to the phenolic group and the halogen para- to the phenolic group.

3. Nitration, while increasing the toxicity of phenols towards bacteria, also increases the systemic toxicity and confers specific biological properties on the molecule, enabling it to interfere with oxidative phosphorylation. This has now been shown to be due to the ability of nitrophenols to act as uncoupling agents. Studies [7] have shown that the nitro group is not a prerequisite for uncoupling, as ethylphenol is an uncoupler. Nitrophenols have now been largely superseded as plant protection chemicals, whereas at one time they were in vogue, although 4-nitrophenol is still used as a preservative in the leather industry.

4. In the bisphenol series, activity is found with a direct bond between the two C6H5 groups or if they are separated by –CH2–, –S– or –O–. If a –CO–, –SO– or –CH(OH)– group separates the phenyl groups, activity is low. In addition, maximum activity is found with the hydroxyl group at the 2,2′- position of the bisphenol. Halogenation of the bisphenols confers additional microbicidal activity.

Chemistry of Phenols

The phenol parent compound C6H5OH (Figure 2.1) is a white crystalline solid (melting point (m.p.) 39–40°C), which becomes pink and finally black on long standing. It is soluble in water 1 : 13 and is a weak acid, pKa 10. Its biological activity resides in the undissociated molecule. Phenol is effective against both Gram-positive and Gram-negative vegetative bacteria, but is only slowly effective against bacterial spores and acid-fast bacteria.

Figure 2.1 Phenol, cresols, xylenols, ethylphenols and high-boiling tar acids.

c02f001

Phenols are the reference standard for the Rideal–Walker (RW) and Chick–Martin tests for disinfectant evaluations. They find limited application in medicine today, but are used as preservatives in such products as animal glues. Although first obtained from coal tar, they are now obtained largely by synthetic processes, which include the hydrolysis of chlorobenzene of the high-temperature interaction of benzene sulfonic acid and alkali.

Mode of Action

At low concentrations, phenols interact with bacterial enzymes needed for cell wall synthesis, resulting in cell lysis. High concentrations of phenols cause general coagulation of the cytoplasm and act as general protoplasmic poisons. In addition, phenols can affect the cytoplasmic membrane [8, 9] resulting in leakage of potassium ions first, then the cytosol. Hexachlorophene was found to have additional activity as an inhibitor of the electron transport chain, thus inhibiting the metabolic activities in bacteria [10].

Sources of Phenols: The Coal-Tar Industry

Most of the phenols used to make disinfectants are a by-product of the destructive distillation of coal. Coal is heated in the absence of air and the volatile products, one of which is tar, are condensed. The tar is fractionated to yield a group of products that include phenols (called tar acids), organic bases and neutral products, such as alkyl naphthalenes, which are known in the industry as neutral oils.

The cresols consist of a mixture of 2-, 3- and 4-cresol. The xylenols consist of the six isomeric dimethylphenols plus ethylphenols. The combined fraction, cresols and xylenols, is also available as a commercial product known as cresylic acid. High-boiling tar acids consist of higher alkyl homologs of phenols: for example the diethylphenols, tetramethylphenols and methylethylphenols, together with methylindanols, naphthols and methylresorcinols, the latter being known as dihydrics. There may be traces of 2-phenylphenol. The chemical constituents of some of the phenolic components are shown in Figure 2.1.

Properties of Phenolic Fractions

The passage from phenol (boiling point (b.p.) 182°C) to the higher-boiling phenols (b.p. up to 310°C) is accompanied by a well-defined gradation in properties, as follows: water solubility decreases, tissue trauma decreases, bactericidal activity increases, inactivation by organic matter increases. However, the ratio of activity against Gram-negative to activity against Gram-positive organisms remains fairly constant, although in the case of pseudomonads, activity tends to decrease with decreasing water solubility (Table 2.1).

Table 2.1 Phenol coefficients of coal-tar products against Salmonella typhi and Staphylococcus aureus.

c02tbl0001ta

Formulation of Coal-Tar Disinfectants

It is seen from the above data that the progressive increase in desirable biological properties of the coal-tar phenols with in­­creasing boiling point is accompanied by a decrease in water solubility. This presents formulation problems and part of the story of the evolution of the present-day products is found in the evolution of formulation devices.

Modern Range of Solubilized and Emulsified Phenolic Disinfectants

Black fluids are essential coal-tar fractions solubilized with soaps; white fluids are prepared by emulsifying tar fractions. Their composition as regards phenol content is shown in Figure 2.1. The term clear soluble fluid is also used to describe the solubilized products Lysol and Sudol.

Cresol and Soap Solution British Pharmacopoeia (BP) 1963 (Lysol)

This consists of cresol (a mixture of 2-, 3- and 4-cresols) solubilized with a soap prepared from linseed oil and potassium hydroxide. It forms a clear solution on dilution and is a broad-spectrum disinfectant showing activity against vegetative bacteria, mycobacteria, fungi and viruses [11]. Most vegetative pathogens, including mycobacteria, are killed in 15 min by dilutions of Lysol ranging from 0.3% to 0.6%. Bacterial spores are much more resistant, and there are reports of the spores of Bacillus subtilis surviving in 2% Lysol for nearly 3 days. Even greater resistance has been encountered among clostridial spores. Lysol still retains the corrosive nature associated with the phenols and should be used with care. Both the method of manufacture and the nature of the soap used have been found to affect the microbicidal properties of the product [12]. RW coefficients (British Standard (BS) 541: 1985) are of the order of 2.

Black Fluids

These consist of a solubilized crude phenol fraction prepared from tar acids, with a boiling range of 250–310°C (Figure 2.1). The solubilizing agents used to prepare the black fluids of commerce include soaps prepared from the interaction of sodium hydroxide with resins (which contain resin acids) and with the sulfate and sulfonate mixture prepared by heating castor oil with sulfuric acid (called sulfonated castor oil or Turkey red oil).

Additional stability is conferred by the presence of coal-tar hydrocarbon-neutral oils. The actual mechanism whereby they stabilize the black fluids has not been adequately elucidated; however, they do prevent crystallization of naphthalene present in the tar acid fraction. Mixtures of cresol and soap solution (Lysol type) of the United States Pharmacopeia have varying concentrations of neutral oil. Using a phenol coefficient-type test and Salmonella typhi as the test organism, a product containing 30% cresols and 20% neutral oil was found to be twice as active as a similar product containing 50% cresols alone. However, the replacement of cresol by neutral oil caused a progressive decrease in phenol coefficient when a hemolytic Streptococcus and Mycobacterium tuberculosis were used as test organisms. The results were further checked using a pure 2-methylnaphthalene in place of neutral oil and similar findings were obtained.

Black fluids give either clear solutions or emulsions on dilution with water, those containing greater proportions of higher phenol homologs giving emulsions. They are partially inactivated by the presence of electrolytes.

White Fluids

These differ from the foregoing formulations in being emulsified, as distinct from solubilized, phenolic compounds. The emulsifying agents used include animal glue, casein and the carbohydrate extractable from seaweed called Irish moss. Products with a range of RW coefficients may be manufactured by the use of varying tar acid constituents.

As white fluids are already in the form of an oil-in-water emulsion, they are less liable to have their activity reduced on further dilution, as might happen with black fluids if dilution is carried out carelessly. They are much more stable in the presence of electrolytes. As might be expected from a metastable system, the emulsion, they are less stable on storage than black fluids, which are solubilized systems. As with the black fluids, products of varying RW coefficients may be obtained by varying the composition of the phenol. Neutral oils from coal tar may be included in the formulation.

Non-Coal-Tar Phenols

The coal-tar (and to a lesser extent the petrochemical) industry yields a large array of phenolic products. However, phenol itself is now made in large quantities by a synthetic process, as are some of its derivatives. Three such phenols, which are used in a variety of roles, are 4-tertiary octylphenol, 2-phenylphenol and 4-hexylresorcinol (Figure 2.2).

Figure 2.2 Examples of phenolic compounds.

c02f002

4-Tertiary Octylphenol

This phenol (often referred to as octylphenol) is a white crys­talline substance, with a melting point of 83°C. The cardinal property in considering its application as a preservative is its insolubility in water, 1 in 60,000 (1.6 × 10−3%). The sodium and potassium derivatives are more soluble. It is soluble in 1 in 1 of 95% ethanol and proportionally less soluble in ethanol containing varying proportions of water. It has been shown by animal feeding experiments to be less toxic than phenol or cresol.

Alcoholic solutions of the phenol are 400–500 times as effective as phenol against Gram-positive organisms, but against Gram-negative bacteria the factor is only one-fiftieth. Octylphenol is also fungistatic, and has been used as a preservative for proteinaceous products such as glues and non-food gelatins. Its activity is reduced in the presence of some emulgents, a property that might render it unsuitable for the preservation of soaps and cutting oils.

2-Phenylphenol (o-phenylphenol; 2-phenylphenoxide)

This occurs as a white crystalline powder, melting at 57°C. It is much more soluble than octylphenol, 1 part dissolving in 1000 parts of water, while the sodium salt is readily soluble in water. It is active against both bacteria and fungi and is used as a preservative, especially against fungi, in a wide variety of applications. Typical minimal inhibitory concentrations (MICs, µg/ml) for the sodium salt are: Escherichia coli, 32; Staphylococcus aureus, 32; B. subtilis, 16; Pseudomonas fluorescens, 16; Aspergillus niger (brasiliensis), 4; Epidermophyton spp., 4; Myrothecium verrucaria, 2; and Trichophyton interdigitale, 8. Many strains of Pseudomonas aeruginosa are more resistant, requiring higher concentrations than those listed above for their inhibition.

Its main applications have been as ingredients in disinfectants of the pine type, as preservatives for cutting oils and as a general agricultural disinfectant. It has been particularly useful as a slimicide and fungicide in the paper and cardboard industry, and as an additive to paraffin wax in the preparation of waxed paper and liners for bottle and jar caps. In combination with para-tertiary amylphenol, phenylphenol is effective against M. tuberculosis and HIV [13].

Studies on S. aureus have revealed that o-phenylphenol inhibits anabolism of many amino acids and highly downregulates the genes that encode the enzymes involved in the diaminopimelate (DAP) pathway. Lysine and DAP are essential for building up the peptidoglycan in cell walls. It was suggested that the mode of action of o-phenylphenol is similar to the mechanism of action of some antibiotics [14].

4-Hexylresorcinol

This occurs as white crystalline needles (m.p. 67°C). It is soluble 0.5% in water, but freely soluble in organic solvents, glycerol and glycerides (fixed oils). It is of low oral toxicity, having been used for the treatment of roundworm and whipworm infections in humans. It is used as a 0.1% solution in 30% glycerol as a skin antiseptic and in lozenges and medicated sweets for the treatment of throat infections, where it has local anesthetic effect [15].

Halo and Nitrophenols

The general effect of halogenation (see Figure 2.2) upon the microbicidal activity of phenols is to increase their activity – with the para- position being more effective than the ortho- position – but reduce their water solubility. There is also a tendency for them to be inactivated by organic matter.

In order to illustrate the effect of chlorination on the micro­bicidal activity of phenols, RW coefficients are as follows: 2-chlorophenol, 3.6; 4-chlorophenol, 4; 3-chlorophenol, 7.4; 2,4-dichlorophenol, 13; 2,4,6-trichlorophenol, 22; 4-chloro-3-methylphenol, 13; 4-chloro-3,5-dimethylphenol, 30.

Chlorophenols are made by the direct chlorination of the corresponding phenol or phenol mixture, using either chlorine or sulfuryl chloride.

2,4,6-Trichlorophenol

This is a white or off-white powder, which melts at 69.5°C and boils at 246°C. It is a stronger acid than phenol with a pKa of 8.5 at 25°C. It is almost insoluble in water, but is soluble in alkali and organic solvents. This phenol has been used as a bactericidal, fungicidal and insecticidal agent. It has found application in textile and wood preservation, as a preservative for cutting oils and as an ingredient in some antiseptic formulations. Its phenol coefficient against S. typhi is 22 and against S. aureus 25.

Pentachlorophenol (2-phenylphenoxide, PCP)

A white to cream-colored powder (m.p. 174°C), it can crystallize with a proportion of water, and is almost insoluble in water, but is soluble in organic solvents. PCP and its sodium or potassium derivatives have micromicrobicidal, fungicidal and algicidal effects. Pentachlorophenol or its sodium derivative is used as a preservative for adhesives, textiles, wood, leather, paper and cardboard. It has been used for the in-can preservation of paints, but it tends to discolor in sunlight. However, over the counter sales of PCP has been banned in the USA due to potential contamination to drinking water [16]. As with other phenols, the presence of iron in the products, which it is meant to preserve, can also cause discoloration.

4-Chloro-3-methylphenol (Chlorocresol)

Chlorocresol is a colorless crystalline compound, which melts at 65°C and is volatile in steam. It is soluble in water at 3.8 g/l and readily soluble in ethanol, ether and terpenes. It is also soluble in alkaline solutions. Its pKa at 25°C is 9.5. Chlorocresol is used as a preservative in pharmaceutical products and as an adjunct in a former UK pharmacopoeial sterilization process called heating with a bactericide, in which a combination of heat (98–100°C) and a chemical microbicide enabled a sterilization process to be conducted at a lower temperature than the more usual 121°C (see Chapter 3). Its RW coefficient in aqueous solution is 13 and nearly double this value when solubilized with castor oil soap. It has been used as a preservative for industrial products, such as glues, paints, sizes, cutting oils and drilling muds and for pharmaceutical products such as topical preparations, injections and cosmetics.

4-Chloro-3,5-dimethylphenol (Chloroxylenol, para-chloro-meta-xylenol, PCMX)

PCMX is a white crystalline substance. It has microbicidal activity against bacteria, fungi and algae [17]. It is used chiefly as a topical antiseptic and a disinfectant. In order to improve solubility, PCMX is often solubilized in a suitable soap solution and often in conjunction with terpineol or pine oil. The British Pharmacopoeia [18] contains a model antiseptic formulation for a chloroxylenol solution containing soap, terpineol and ethanol.

Phenol coefficients for the pure compound are: S. typhi, 30; S. aureus, 26; Streptococcus pyogenes, 28; Trichophyton rosaceum, 25; P. aeruginosa, 11. It is not sporicidal and has little activity against the tubercle bacillus. It is also inactivated in the presence of organic matter. Its properties have been re-evaluated [19].

2,4-Dichloro-3,5-dimethylphenol (Dichloroxylenol, Dichloro-Meta-Xylenol, DCMX)

This is a white powder with a melting point of 94°C. Although it is slightly less soluble than PCMX, it has similar properties and microbicidal spectrum. It is used as an ingredient in pine-type disinfectants and in medicated soaps and hand scrubs.

4-Chloro-3-methylphenol (Para-Chloro-Meta-Cresol, PCMC)

PCMC is more water soluble than other phenols with a solubility of 4 g/l at 20°C. It retains a reasonably broad spectrum of activity of microbicidal activity over a wide pH range due to its solubility. This makes it suitable as an industrial preservative for products such as thickeners, adhesives and pigments [5].

Monochloro-2-phenylphenol

This is obtained by the chlorination of 2-phenylphenol and the commercial product contains 80% of 4-chloro-2-phenylphenol and 20% of 6-chloro-2-phenylphenol. The mixture is a pale straw-colored liquid, which boils over the range 250–300°C. It is almost insoluble in water, but may be used in the formulation of pine disinfectants, where solubilization is effected by means of a suitable soap.

2-Benzyl-4-chlorophenol (Chlorphen; Ortho-Benzyl-Para-Chlorophenol, OBPCP)

This occurs as a white to pink powder, which melts at 49°C. It has a slight phenolic odor and is almost insoluble in water (0.007 g/l at 20°C), but like PCMX is more soluble in alkaline solution and organic solvents. Suitably formulated by solubilization with vegetable-oil soaps or selected anionic detergents, it has a wide microbicidal spectrum, being active against Gram-positive and Gram-negative bacteria, Mycobacterium tuberculosis, viruses, protozoa and fungi. It is used as a sanitizer and disinfectant for cooling towers, poultry houses, food-processing plants and surfaces surrounding swimming pools [20]. However, OBPCP is more commonly used in combination with other phenolics in disinfectant formulations [5].

Mixed Chlorinated Xylenols

A mixed chlorinated xylenol preparation can be obtained for the manufacture of household disinfectants by chlorinating a mixed xylenol fraction from coal tar.

Formulated Disinfectants Containing Chlorophenols

A formulation device, such as solubilization, can be used to prepare liquid antiseptics and disinfectants. This is based on the good activity and low level of systemic toxicity, and of the likelihood of tissue damage shown by chlorinated cresols and xylenols.

In 1933, Rapps [21] compared the RW coefficients of an aqueous solution and a castor oil soap-solubilized system of chlorocresol and chloroxylenol and found the solubilized system to be superior by a factor of almost 2. This particular disinfectant recipe received a major advance (also in 1933) when two gynecologists, seeking a safe and effective product for midwifery and having felt that Lysol, one of the few disinfectants available to medicine at the time, was too caustic, made an extensive evaluation of the chloroxylenol–castor oil product. Their recipe also contained terpineol [22]. It was fortunate that this preparation was active against β-hemolytic streptococci, which are a hazard in childbirth, giving rise to puerperal fever. A chloroxylenol–terpineol soap preparation is the subject of a monograph in the British Pharmacopoeia [18].

The bacteriology of this formulation has turned out to be controversial. The original appraisal indicated good activity against β-hemolytic streptococci and E. coli, with retained activity in the presence of pus, but subsequent bacteriological examinations by experienced workers gave divergent results. Thus, Colebrook in 1941 cast doubt upon the ability of solubilized chloroxylenolterpineol to destroy staphylococci on the skin, a finding which was refuted by Beath [23]. Ayliffe et al. [24] indicated that the product was more active against P. aeruginosa than S. aureus. As so often happens, however, P. aeruginosa was subsequently shown to be resistant and Lowbury [25] found that this organism would actually multiply in dilutions of chloroxylenol soap.

Although still an opportunistic organism, P. aeruginosa has become a dangerous pathogen, especially as more and more pa­­tients receive radiotherapy or radiomimetic drugs. Attempts have been made to potentiate chlorophenol disinfection and to widen its spectrum so as to embrace the pseudomonads. It is well known that ethylenediamine tetraacetic acid (EDTA) affected the permeability of pseudomonads and some enterobacteria to drugs to which they were normally resistant [26] and both Dankert and Schut [27] and Russell and Furr [28] were able to demonstrate that chloroxylenol solutions with EDTA were most active against pseudomonads. Hatch and Cooper [29] exhibited a similar po­­tentiating effect with sodium hexametaphosphate. However, it is worth noting that the German industry trade association have undertaken to eliminate EDTA in products released to the aquatic environment, which would include disinfectants.

Pine Disinfectants

As long ago as 1876, Kingzett took out a patent in Germany for a disinfectant deodorant made from oil of turpentine and camphor that had been allowed to undergo oxidation in the atmosphere. This was marketed under the trade name Sanitas. Later, Stevenson [30] described a fluid made from pine oil solubilized by a soap solution.

The chief constituent of turpentine is the cyclic hydrocarbon pinene (Figure 2.3), which has little or no microbicidal activity. The terpene alcohol terpineol (Figure 2.3) is another ingredient of pine disinfectants and had already been exploited as an ingredient of the Colebrook and Maxted [22] chloroxylenol formu­lation. Unlike pinene, it possesses microbicidal activity in its own right, and it shares with pinene the property of modifying the action of phenols in solubilized disinfectant formulations, although not in the same way for all microbial species.

Figure 2.3 Pinene and terpineol.

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Terpineol is a colorless oil, which tends to darken on storing. It has a pleasant hyacinth odor and is used in perfumery, especially for soap products, as well as in disinfectant manufacture. Many solubilized products has been marketed, with active ingredients ranging from pine oil, pinene through terpineol to a mixture of pine oil and/or terpineol and a suitable phenol or chlorinated phenol. This gave rise to a range of products, extending from those which are really no more than deodorants to effective disinfectants.

Bisphenols

Hydroxy halogenated derivatives (Figure 2.4) of diphenyl meth­ane, diphenyl ether and diphenyl sulfide have provided a number of useful microbicides active against bacteria, fungi and algae. In common with other phenolics they all seem to have low activity against P. aeruginosa; they also have low water solubility and share the property of the monophenols in that they are inactivated by non-ionic surfactants.

Figure 2.4 Bisphenols.

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Ehrlich and co-workers were the first to investigate the microbicidal activity of the bisphenols and published their work in 1906. Klarmann and Dunning and colleagues described the preparation and properties of a number of these compounds [31, 32]. A useful summary of this early work has been made by Suter [6]. Later, Gump and Walter [33–35] and Walter and Gump [36] made an exhaustive study of the microbicidal properties of many of these compounds, especially with a view to their use in cosmetic formulations.

Derivatives of Dihydroxydiphenylmethane

Dichlorophen, G-4,5,5′-dichloro-2,2′-dihydroxydiphenylmeth­ane (Panacide, Rotafix, Swansea, UK) is active to varying degrees against bacteria, fungi, helminths and algae. It is soluble in water at 30 µg/ml but more soluble (45–80 g/100 ml) in organic solvents. The pKa values at 25°C for the two hydroxyl groups are 7.6 and 11.6, and it forms a very alkaline solution when diluted. It is typically used as an algicide, fungicide and at a dilution of 1 in 20 as a surface microbicide. It has found application as a preservative for toiletries, textiles and cutting oils and to prevent the growth of bacteria in water-cooling systems and humidifying plants. It is used as a slimicide in paper manufacture. It is used as antihelminthic in poultry, cats and dogs [37, 38]. It may be added to papers and other packing materials to prevent microbial growth and has been used to prevent algal growth in greenhouses.

Hexachlorophene, 2,2′-dihydroxy-3,5,6,3′,5′,6′-hexachlorodi­phenylmethane, G11 is almost insoluble in water but soluble in ethanol, ether and acetone and in alkaline solutions. The pKa values are 5.4 and 10.9. Its mode of action has been studied in detail by Gerhardt, Corner and colleagues [39–43]. It is used mainly for its bactericidal activity but it is much more active against Gram-positive than Gram-negative organisms. Typical MICs (bacteriostatic) in µg/ml are: S. aureus, 0.9; B. subtilis, 0.2; Proteus vulgaris, 4; E. coli, 28; and P. aeruginosa, 25. It has found chief application as an active ingredient in surgical scrubs and medicated soaps and has also been used to a limited extent as a preservative for cosmetics. Its use is limited by its insolubility in water, its somewhat narrow bactericidal spectrum, and by the fact that in the UK it is restricted by a control order made in 1973. In general, this order restricted the use of this product to 0.1% in human medicines and 0.75% in animal medicines. Its toxicity has restricted its use in cosmetic products, and the maximum concentration allowed is 0.1%, with the stipulation that it is not to be used in products for children or personal hygiene products.

Bromochlorophane, 3,3′-dibromo-5,5′-dichlor-2,2′-dihydro­xy­diphenylmethane is soluble in water at 100 µg/ml and is markedly more active against Gram-positive organisms than bacteria. Strains of S. aureus are inhibited at concentrations from 8 to 11 µg/ml, whereas 100 times these concentrations are required for E. coli and P. aeruginosa. It has been used as the active ingredient in deodorant preparations and toothpastes. It also has antiplaque activity [44].

Derivatives of Hydroxydiphenylether

Triclosan, 2,4,4′-trichlor-2′-hydroxydiphenylether (Irgasan, re­­gistered Ciba Speciality Chemicals, Basle, Switzerland) is only sparingly soluble in water (10 mg/l) but is soluble in solutions of dilute alkalis and organic solvents. Its activity is not compromised by soaps, most surfactants, organic solvents, acids or alkalis. However ethoxylated surfactants such as polysorbate 80 (Tween-80) entrap triclosan within micelles, thus preventing its action [45]. Triclosan is generally bacteriostatic against a broad range of Gram-positive and Gram-negative bacteria and also demonstrates some fungistatic activity. Triclosan has a wide range of activity. In general it is more active against Gram-positive bacteria than Gram-negative bacteria, particularly Pseudomonas spp. It is active against some fungi, Plasmodium falciparum and Toxoplasma gondii although bacterial spores are unaffected [46]. It inhibits staphylococci at concentrations ranging from 0.1 to 0.3 µg/ml. Paradoxically, a number of E. coli strains are inhibited over a similar concentration range. Most strains of P. aeruginosa require concentrations varying from 100 to 1000 µg/ml for inhibition. It inhibits the growth of several species of mold at concentrations from 1 to 30 µg/ml.

Triclosan is commonly found in a wide range of personal care products such as handwashes, shower foams, medicated soaps, deodorants and hand scrubs. It is also used in toothpastes and it has been suggested that it has antiplaque activity [44]. It is ideally suited to these applications as it has a low toxicity and irritancy and is substantive to the skin [45]. More recently it has been used in a range of other applications such as incorporation in plastics and fabrics to confer microbicidal activity. This, and the link made between triclosan-resistant bacteria and antibiotic resistance, has led to concerns about its use [47–49]. However, with the correct usage of this microbicidal, there is no direct evidence to suggest that a proliferation of antibiotic resistant bacteria will occur [50]. Some reports have shown that the inhibitory activity of triclosan results from blocking lipid synthesis by specifically inhibiting an NADH-dependent enoyl-acyl carrier protein (ACP) reductase, or FabI [46, 51]. Based on this mode of action, triclosan-based molecules are considered to be potential candidates for novel antituberculosis and antimalarial drugs [46, 52, 53].

Derivatives of Diphenylsulfide

Fenticlor, 2,2′-dihydroxy-5,5′-dichlorodiphenylsulfide, is a white powder soluble in water at 30 µg/ml, but is much more soluble in organic solvents and oils. It shows more activity against Gram-positive organisms and a "Pseudomonas gap". Typical inhibitory concentrations (µg/ml) are: S. aureus, 2; E. coli, 100; and P. aeruginosa, 1000. Typical inhibitory concentrations (µg/ml) for some fungi are: Candida spp., 12; Epidermophyton interdigitale, 0.4; and Trichophyton granulosum, 0.4. Fenticlor has found chief application in the treatment of dermatophytic conditions. However, it can cause photosensitization and as such its use as a preservative is limited [5]. Its low water solubility and narrow spectrum are further disadvantages, but it has potential as a fungicide. Its mode of action has been described by Hugo and Bloomfield [54–56] and Bloomfield [57].

The chlorinated analog of fenticlor, 2,2′-dihydroxy-3,4,6,3′,4′,6′-hexachlorodiphenylsulfide or 2,2′-thiobis (3,4,6-trichlorophenol) is almost insoluble in water. In a field test, it proved to be an effective inhibitor of microbial growth in cutting-oil emulsions. An exhaustive study of the antifungal properties of hydroxydiphenylsulfides was made by Pflege et al. [58].

Organic and Inorganic Acids: Esters and Salts

A large family of organic acids (Figure 2.5), both aromatic and aliphatic, and one or two inorganic acids have found application as preservatives, especially in the food industry. Some, for example benzoic acid, are also used in the preservation of pharmaceutical products; others (salicylic, undecylenic and benzoic acids) have been used, suitably formulated, for the topical treatment of fungal infections of the skin.

Figure 2.5 Organic acids and esters.

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Vinegar, containing acetic acid (ethanoic acid), has been found to act as a preservative. It was also used as a wound dressing. This application has been revived in the use of dilute solutions of acetic acid as a wound dressing where pseudomonal infections have occurred.

Hydrochloric and sulfuric acids are two mineral acids sometimes employed in veterinary disinfection. Hydrochloric acid at high concentrations is sporicidal and has been used for disinfecting hides and skin contaminated with anthrax spores. Sulfuric acid, even at high concentrations, is not sporicidal, but in some countries it is used, usually in combination with phenol, for the decontamination of floors, feed boxes and troughs.

Citric acid is an approved disinfectant against foot-and-mouth disease virus. It also appears, by virtue of its chelating properties, to increase the permeability of the outer membrane of Gram-negative bacteria [59] when employed at alkaline pH. Malic acid and gluconic acid, but not tartaric acid, can also act as permeabilizers at alkaline pH [59].

Chemistry of Organic and Inorganic Acids

If an acid is represented by the symbol AH, then its ionization will be represented by A− H+. Complete ionization, as seen in aqueous solutions of mineral acids, such as hydrogen chloride (where AH = ClH), is not found in the weaker organic acids and their solutions will contain three components: A−, H+ and AH. The ratio of the concentration of these three components is called the ionization constant of that acid, Ka, and Ka = A− × H+/AH. By analogy with the mathematical device used to define the pH scale, if the negative logarithm of Ka is taken, a number is obtained, running from about 0 to about 14, called pKa. Some typical pKa values are shown in Table 2.2.

Table 2.2 pKa values of acids and esters used as microbicidal agents.

An inspection of the equation defining Ka shows that the ratio A−/AH must depend on the pH of the solution in which it is dissolved, and Henderson and Hasselbalch derived a relationship between this ratio and pH as follows:

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An inspection of the formula will also show that at the pH value equal to the pKa value the product is 50% ionized. These data enable an evaluation to be made of the effect of pH on the toxicity of organic acids. Typically it has been found that a marked toxic effect is seen only when the conditions of pH ensure the presence of the un-ionized molecular species AH. As the pH increases, the concentration of AH falls and the toxicity of the system falls; this may be indicated by a higher MIC, longer death time or higher mean single-survivor time, depending on the criterion of toxicity (i.e. microbicidal activity) chosen.

An inspection of Figure 2.6 would suggest that HA is more toxic than A−. However, an altering pH can alter the intrinsic toxicity of the environment. This is due to H+ alone, the ionization of the cell surface, the activity of transport and metabolizing enzymes, and the degree of ionization of the cell surface and hence sorption of the ionic species on the cell.

Figure 2.6 A generalized diagram of the effect of pH on the ionization and microbicidal activity of an acid (HA) of pKa 4.1. A− is the acid anion.

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Predictions for the preservative ability of acids validated at one pH are rendered meaningless when such a preservative is added without further consideration to a formulation at a higher pH. The pKa of the acid preservative should always be ascertained, and any pH shift of 1.5 units or more on the alkaline side of this can be expected to cause progressive loss of activity quite sufficient to invalidate the originally determined performance. That pH modifies the microbicidal effect of benzoic acid has been known for a long time [60]. For more detailed accounts of the effect of pH on the intensity of action of a large number of ionizable micro­bicides, the papers of Simon and Blackman [61] and Simon and Beevers [62, 63] should be consulted.

Mode of Action

The mode of action of acids used as food preservatives has been reviewed by Eklund [64], Booth and Kroll [65] Cherrington et al. [66] and Russell [67]. Convincing evidence has been produced that many acid preservatives act by preventing the uptake of substrates, which depend on a proton-motive force for their entry into the cell; in other words, they act as uncoupling agents. In addition to acids such as benzoic, acetic and propionic, the esters of p-hydroxybenzoic acid (parabens) were also included in some of the above studies; they too acted as uncoupling agents, but also inhibited electron transport.

Equally interesting were experiments on the pH dependence of the substrate uptake effect. The intensity of uptake inhibition by propionate, sorbate and benzoate declined between pH 5 and 7, while that induced by propyl-p-hydroxybenzoic acid (pKa 8.5) remained constant over the same pH range. The growth-inhibitory effect of ionizable microbicides shows pH dependence and this, as might be expected, is applicable to a biochemical effect upon which growth in turn depends.

Organic acids, such as benzoic acid and sorbic acid, are deliberately used as preservatives. Acids such as acetic, citric and lactic are often employed as acidulants, that is to lower artificially the pH of foods. However, a low pKa value is not the only significant feature of acidulants, since: (i) sorbate and acetate have similar pKa values but the latter is a less potent preservative; (ii) organic acids used as preservatives are more potent inhibitors than other weak acids of similar pH; and (iii) weak organic acid preservatives are more effective inhibitors of pH homeostasis than other acids of similar structure.

Individual Compounds

Acetic Acid (Ethanoic Acid)

This acid, as a diluted petrochemically produced compound or as the natural product vinegar, is used primarily as a preservative for vegetables. The toxicity of vinegars and diluted acetic acid must rely to an extent on the inhibitory activity of the molecule itself, as solutions of comparable pH made from mineral acid do not exhibit the same preservative activity. A 5% solution of acetic acid contains 4.997% CH3COOH and 0.003% H+. As might be expected from the pKa value, 4.7, the activity is rapidly lost at pH levels above this. This suggests that the acetate ion is less toxic than the undissociated molecule, although, as has been said, the concomitant reduction in hydrogen ion concentration must play some part in the reduction of toxicity. As has been stated, diluted 1–5% acetic acid has been used as a wound dressing where infection with Pseudomonas has occurred [68]. Acetic acid is also used as topical otological preparation [69].

Propionic Acid

This acid is employed almost exclusively as the sodium, and to a lesser extent the calcium, salt in the baking industry, where it is used to inhibit mold and bacterial growth in breads and cakes. It is particularly useful in inhibiting the growth of the spore-forming aerobe Bacillus macerans, which gives rise to ropy bread. Manufacturers give explicit directions as to the amount to be used in different products, but in general 0.15–0.4% is added to the flour before processing. Other products that have been successfully preserved with propionates include cheeses and malt extract. In addition to foods, wrapping materials for foods have also been protected from microbial damage with propionates. It is added to poultry feed to protect from the growth of many bacteria like E. coli, Salmonella spp. and molds [70, 71].

Butyric Acid

The sodium salt of butyric acid is commonly used as an additive to poultry feed to protect from Salmonella spp. infection and shedding [72, 73].

Caprylic Acid

Caprylic acid is a natural eight-carbon fatty acid present