Principles and practice of disinfection, preservation and sterilization 4th ed a fraise (blackwell, 2004) 1

Russell, Hugo & Ayliffe's

Principles and
Practice of
Disinfection,
Preservation &
Sterilization


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Russell, Hugo & Ayliffe's

Principles and
Practice of
Disinfection,
Preservation &
Sterilization
EDITED BY

Adam P Fraise MB BS FRCPath
Consultant Medical Microbiologist and Director
Hospital Infection Research Laboratory
City Hospital
Birmingham, UK

Peter A Lambert BScPhD DSc
Reader in Microbiology
Pharmaceutical and Biological Sciences

Aston University
Birmingham, UK

Jean-Yves Maillard BSc PhD
Senior Lecturer in Pharmaceutical Microbiology
School of Pharmacy and Biomolecular Sciences
University of Brighton
Brighton, UK

FOURTH EDITION

Blackwell
Publishing


© 1982, 1992, 1999 by Blackwell Science Ltd
© 2004 by Blackwell Publishing Ltd
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All rights reserved. No part of this publication may be reproduced, stored in a retrieval
system, or transmitted, in any form or by any means, electronic, mechanical,
photocopying, recording or otherwise, except as permitted by the UK Copyright,
Designs and Patents Act 1988, without the prior permission of the publisher.
First published 1982
Second edition 1992
Reprinted 1994 (twice)

Third edition 1999
Fourth edition 2004
Library of Congress Cataloging-in-Publication Data
Russell, Hugo & Ayliffe's Principles and practice of disinfection,
preservation and sterilization / edited by Adam P. Fraise, Peter A.
Lambert, Jean-Yves Maillard. — 4th ed.
p.; cm.
Rev. ed. of: Principles and practice of disinfection, preservation, and
sterilization, 1999.
Includes bibliographical references and index.
ISBN 1-4051-0199-7
1. Disinfection and disinfectants. 2. Sterilization. 3. Preservation
of materials.
[DNLM: 1. Disinfection—methods. 2. Sterilization—methods.
3. Anti-Infective Agents. 4. Preservatives, Pharmaceutical. WA 240 R963
2004] I. Title: Principles and practice of disinfection, preservation
and sterilization. II. Russell, A. D. (Allan Denver), 1936-. III. Hugo,
W.B.(William Barry). IV. Ayliffe, G. A. J. V. Fraise, Adam P.
VI. Lambert, Peter A. VII. Maillard, J.-Y. VIII. Principles and practice of
disinfection, preservation, and sterilization. IX. Title.
RA761.P84 2004
614.4'8-dc22
2003017281
ISBN 1-4051-0199-7
A catalogue record for this title is available from the British Library
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Contents

List of contributors, vii
Preface to the fourth edition, ix
Preface to the first edition, x

Part1: Principles
1 Historical introduction, 3
Adam P Praise
2 Types of antimicrobial agents, 8
Suzanne L Moore and David N Payne
3 Factors influencing the efficacy of
antimicrobial agents, 98
A Denver Russell
4 Biofilms and antimicrobial
resistance, 128
Peter Gilbert, Alexander H Rickard and
Andrew J McBain
5 Mechanisms of action of biocides, 139
Peter A Lambert
6 Bacterial resistance, 154
6.1 Intrinsic resistance of Gram-negative
bacteria, 154
David J Stickler
6.2 Acquired resistance, 170

Keith Poole
6.3 Resistance of bacterial spores to chemical
agents, 184
Peter A Lambert
6.4 Mycobactericidal agents, 191
Peter M Hawkey
7 Antifungal activity of disinfectants, 205
7.1 Antifungal activity of biocides, 205
Jean-Yves Maillard

7.2 Evaluation of the antibacterial and
antifungal activity of disinfectants, 220
Gerald Reybrouck
8 Sensitivity of protozoa to disinfectants, 241
8.1 Acanthamoeba, contact lenses and
disinfection, 241
Neil A Turner
8.2 Intestinal protozoa and biocides, 258
Jean-Yves Maillard
9 Viricidal activity of biocides, 272
Jean-Yves Maillard
10 Transmissible degenerative encephalopathies:
inactivation of the unconventional causal
agents, 324
David M Taylor

Part 2: Practice
11 Evaluation of antimicrobial efficacy, 345
Ronald J W Lambert
12 Sterilization, 361

12.1 Heat sterilization, 361
Grahame W Gould
12.2 Radiation sterilization, 384
Peter A Lambert
12.3 Gaseous sterilization, 401
Jean-Yves Dusseau, Patrick Duroselle
and Jean Freney
12.4 Filtration sterilization, 436
Stephen P Denyer and Norman A
Hodges
13 New and emerging technologies, 473
Grahame W Gould
14 Preservation of medicines and cosmetics, 484
Sarah J Hiom

v


Contents

15 Reuse of single-use devices, 514
Geoffrey W Hanlon
16 Sterility assurance: concepts, methods and
problems, 526
Rosamund M Baird
17 Special problems in hospital antisepsis, 540
Manfred L Rotter
18 Decontamination of the environment and
medical equipment in hospitals, 563
Adam P Fraise

19 Treatment of laundry and clinical waste in
hospitals, 586
Christina R Bradley

vi

20 Other health-related issues, 595
20.1 Special issues in dentistry, 595
Jeremy Bagg and Andrew Smith
20.2 Veterinary practice, 604
Anders Engvall and Susanna Sternberg
20.3 Recreational and hydrotherapy pools,
614
John V Dadswell
21 Good manufacturing practice, 622
Elaine Underwood
Index, 641


List of contributors

Jeremy Bagg PhD FDS RCS (Ed)
FDS RCPS (Glasg) FRCPath
Professor of Clinical Microbiology
University of Glasgow Dental School
Glasgow, UK

Rosamund M Baird BPharm PhD
MRPharmS
School of Pharmacy

University of Bath
Bath, UK

Anders Engvall DVM

Sarah J Hiom PhD MRPharmS

Professor and Chief Epizootiologist
National Veterinary Institute SVA
Uppsala
Sweden

Senior Pharmacist R&D, NHS Wales
St Mary's Pharmaceutical Unit
Cardiff, UK

Adam P Fraise MBBS FRCPath
Consultant Medical Microbiologist and
Director
Hospital Infection Research Laboratory
City Hospital
Birmingham, UK

Christina R Bradley AIBMS
Laboratory Manager
Hospital Infection Research Laboratory
City Hospital
Birmingham, UK

John V Dadswell MBBS

FRCPath
Former Director
Reading Public Health Laboratory
Reading, UK

Stephen P Denyer BPharm PhD
FRPharmS
Head of School
Welsh School of Pharmacy
Cardiff University
Cardiff, UK

Jean-Yves Dusseau MD
Specialiste des Hopitaux des armees
Hopital d'instruction des armees Desgenettes
Departement de Biologic Medicale
Lyon
France

Principal Lecturer in Pharmaceutical
Microbiology
School of Pharmacy and Biomolecular
Sciences
University of Brighton
Brighton, UK

Jean Freney PhD
Professor of Microbiology
Department of Bacteriology and Virology
Faculty of Pharmacy

Lyon
France

Peter Gilbert BSc PhD

Peter A Lambert BSc PhD DSc
Reader in Microbiology
Pharmaceutical and Biological Sciences
Aston University
Birmingham, UK

Ronald J W Lambert BA BSc PhD
CChem MRSC

Professor of Microbial Physiology
School of Pharmacy and Pharmaceutical
Sciences
University of Manchester
Manchester, UK

Director
R2-Scientific
Sharnbrook
Beds, UK

Grahame W Gould BSc MSc PhD

Andrew JMcBain

Visiting Professor of Microbiology

University of Leeds
Leeds, UK

Research Fellow
School of Pharmacy and Pharmaceutical
Sciences
University of Manchester
Manchester, UK

Patrick Duroselle PhD
Department of Bacteriology and Virology
Faculty of Pharmacy
Lyon
France

Norman A Hodges BPharm
MRPharmS PhD

Geoffrey W Hanlon BSc PhD
MRPharmS
Reader in Pharmaceutical Microbiology
School of Pharmacy and Biomolecular
Sciences
University of Brighton
Brighton, UK

Peter M Hawkey BSc DSc MB BS
MD FRCPath
Professor of Clinical and Public Health
Bacteriology and Honorary Consultant

The Medical School, University of
Birmingham
Health Protection Agency, Birmingham
Heartlands and Solihull NHS Trust
Birmingham, UK

Jean-Yves Maillard BSc PhD
Senior Lecturer in Pharmaceutical
Microbiology
School of Pharmacy and Biomolecular
Sciences
University of Brighton
Brighton, UK

Suzanne L Moore BSc PhD
External Innovation, Health and Personal
Care R&D
Reckitt Benckiser Healthcare (UK)
Hull, UK

vii


List of contributors

David N Payne MIBiol CBiol

Manfred L Rotter MD Dip Bact

David J Stickler BSc MA DPhil

Microbiology Manager
Reckitt Benckiser Healthcare (UK)
Hull, UK

Director and Professor of Hygiene and
Medical Microbiology
Department of Hygiene and Medical
Microbiology of the University of Vienna
Vienna
Austria

Senior Lecturer in Medical Microbiology
Cardiff School of Biosciences
Cardiff University
Cardiff, UK

Keith Poole PhD
Professor of Microbiology and Immunology
Queen's University
Kingston, ON
Canada

Gerald Reybrouck MD AggrHO
Professor
Hospital Hygiene and Infection Control
Department
Katholiecke Universiteit Leuven
Leuven
Belgium

Alexander H Rickard BSc MSc
PhD
Research Fellow
School of Pharmacy and Pharmaceutical
Sciences
University of Manchester
Manchester, UK

viii

A Denver Russell BPharm PhD
DSc FRCPath FRPharmS
Professor of Pharmaceutical Microbiology
Welsh School of Pharmacy
Cardiff University
Cardiff, UK

Andrew Smith BDS FDS RCS PhD
MRCPath
Senior Lecturer and Honorary Consultant in
Microbiology
University of Glasgow Dental School
Glasgow, UK

Susanna Sternberg DVM PhD
Laboratory Veterinary Officer
National Veterinary Institute S VA
Uppsala
Sweden

David M Taylor PhD MBE
Consultant
SEDECON 2000
Edinburgh, UK

Neil A Turner BSc PhD
Postdoctoral Research Fellow
Department of Medical and Molecular
Parasitology
New York University School of Medicine
New York
USA

Elaine Underwood BSc PhD
Wyeth Pharmaceuticals
SMA Nutrition Division
Maidenhead, UK


Preface to the fourth edition

It has been a privilege to take on the editing of this
textbook. The major change that has taken place is
that the organization of the chapters has been altered such that Chapters 1-10 deal with the principles of disinfection, preservation and sterilization, and Chapters 11-21 deal with the practice.
Although the book has always been aimed at microbiologists, physicians and pharmacists, the content
of this fourth edition has been modified to reflect
this clinical emphasis more. Consequently, chapters
on textile, leather, paint and wood preservation
have been removed, whereas sections on biofilms,

prions and specific clinical areas such as dentistry
have been updated and expanded. All other chapters have been revised, with new material added
where appropriate.

Inevitably much of the content of the previous
editions is still valid and we are grateful for the efforts of the previous editorial team and authors,
without whom it would have been impossible to
achieve this fourth edition within the allotted
timescale. We are especially grateful to authors of
chapters in previous editions, who have allowed
their text to be used by new authors in this edition.
We also thank all contributors (both old and new)
for their hard work in maintaining this text as one
of the foremost works on the subject.

A.P.R
P.A.L.
J.-Y.M.

ix


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 naive 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, hospitalacquired infection remains an important problem
and is often associated with surgical operations or

x

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. Lowtemperature 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.


Parti
Principles


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Chapter 1

Historical introduction
Adam P Fraise
1 Early concepts
2 Chemical disinfection
3 Sterilization

4 Future developments for chemical biocides
5 References

1 Early concepts

native sodium carbonate, was also used to preserve
the bodies of human and animal alike.
Not only in hygiene but in the field of food preservation were practical procedures discovered. Thus
tribes which 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,
so arose the European high cultures and, whether
through reading or independent discovery, concepts
of empirical hygiene were also developed. There
was, of course, a continuum of 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 fourteenth century, where one
Joseph of Burgundy recommended the burning of
jumper branches in rooms where the plague sufferers had lain. Sulphur, too, was burned in the hope
of removing the cause of this terrible disease.
The association of malodour 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 be due to
the elimination of rats, later to be shown as the
bearers of the causal organism. In Renaissance Italy
at the turn of the fifteenth 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

Throughout history it is remarkable how hygienic
concepts have been applied. Examples may be
found in ancient literature of the Near and Middle
East, which date from when written records first
became available. An interesting example of early
written codes of hygiene may be found in the Bible,
especially in the Book of Leviticus, chapters 11-15.
Disinfection using heat was recorded in the Book
of Numbers, in which 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 odour 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 an awareness that
something more than mechanical cleanness was

required.
Chemical disinfection of a sort could be seen in
the practice recorded at the time of Persian imperial
expansion, c. 450 BC, of storing water 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
dilute 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
country), also employed a variety of balsams which
contained natural preservatives. Natron, a crude

3


Chapter 7

exist 'seeds of disease', as indeed there did! Robert
Boyle, the sceptical chemist, writing in the midseventeenth century, wrote of a possible relationship between fermentation and the disease process.
In this he foreshadowed the views of Louis Pasteur.
There is 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
(x 300), were published in the Philosophical Transactions of the Royal Society in 1677 and also in a
series of letters to the Society 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 times. 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 Tyre in 332 BC, states how bread, distributed to the Macedonians, had a bloody look. This
was probably attributable to infestation by Serratia
marcescens; 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 passing to a more rationally supported
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
4

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; nitre, for example, had a value
of 4 by this method. One hundred and fifty-three
years later, Rideal and Walker were to use a similar
method with phenolic disinfectants and Salmonella
typhi; their standard was phenol.
Although the concept of bacterial diseases and
spoilage was not used before the nineteenth century, very early in history procedures to ensure
preservation of water and food were designed and
used. It is only more recently (i.e. in the 1960s), that
the importance of microorganisms in pharmaceuticals was appreciated (Kallings et al., 1966) and the
principles of preservation of medicine introduced.

2 Chemical disinfection
Newer and purer chemical disinfectants began to
be used. Mercuric chloride, corrosive sublimate,
found use as a wound dressing; it had been used
since the Middle Ages and was introduced by Arab
physicians. In 1798 bleaching powder was first
made and a preparation of it was employed by
Alcock in 1827 as a deodorant and disinfectant.
Lefevre introduced chlorine water in 1843. In 1839
Davies had suggested iodine as a wound dressing.
Semmelweis was to use chlorine water in his work
on childbed fever occurring in the obstetrics division of the Vienna General Hospital. He achieved a

sensational reduction in the incidence of the infection by insisting that all attending the birth washed
their hands in chlorine water; later (in 1847) he
substituted chlorinated lime.
Wood and coal tar were used as wound dressings
in the early nineteenth century and, in a letter to
the Lancet, Smith (1836-37) describes the use of
creosote (Gr. kreas flesh, soter saviour) as a wound
dressing. 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. Kiichenmeister was to use pure phenol in
solution as a wound dressing in 1860 and Joseph


T

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 sulphate was introduced in
1767 and zinc chloride in 1815. Many of these
products are still in use today.
Turning back to evaluation, Bucholtz (1875)
determined what is called today the minimum
inhibitory concentration of phenol, creosote and
benzoic and salicylic acids to inhibit the growth of
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 sulphide 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, parallel with these early
studies, an important watershed already alluded to
in the opening paragraphs of this brief history 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 medical man, 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 was to give rise to
innumerable studies on the subject lasting through
to the present day.
Since then other chemical biocides, which are
now widely used in hospital practice, have been
introduced, such as chlorhexidine, an important
cationic biocide which activity was described in
1958 (Hugo, 1975).
More recently, a better understanding of hygiene
concepts has provided the basis for an explosion in
the number of products containing chemical biocides. Of those, quaternary ammonium compounds
and phenolics are the most important. This rise in
biocide-containing products has also sparked a
major concern about the improper use of chemical
disinfectants and a possible emergence of microbial resistance to these biocides and possible
cross-resistance to antibiotics. Among the most
widely studied biocides are chlorhexidine and triclosan. The bisphenol triclosan is unique, in the
sense that it has recently 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.
These important aspects in biocide usage will be
discussed later.

3 Sterilization
As has been stated above, heat sterilization has been
known since early historical times as a cleansing
and purifying agent. In 1832 William Henry, a
Manchester physician, studied the effect of heat on
contagion by placing contaminated material, i.e.
clothes worn by sufferers from typhus and scarlet

fever, in air heated by water sealed in a pressure vessel. He realized that he could achieve temperatures
higher than 100 °C by using a closed vessel fitted
with a proper 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 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, spring-like, at a
5


Chapter 1

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
test organism, Ward in 1892 attempted to investigate the connection between the wavelength of light
and its toxicity; he found that blue light was more
toxic 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,

i.e. in the ultraviolet light, and this is now a wellestablished agent for water and air sterilization
(see Chapter 12.2).
At the end of the nineteenth century, a wealth of
pioneering work was being carried out in subatomic physics. In 1895, the German physicist, Roentgen, discovered X-rays, and 3 years later Rieder
found these rays to be toxic to common pathogens.
X-rays of a wavelength between 10-10 and 10-11 nm
are one of the radiations emitted by 60Co, now used
extensively in sterilization processes (Chapter
12.2).
Another major field of research in the concluding
years of the nineteenth century was that of natural
radioactivity. In 1879, Becquerel found that, if left
near a photographic plate, uranium compounds
would cause it 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
(a, B and y) were given off. The y-rays were shown to
have the same order of wavelength as X-rays.
(B-Rays were found to be highspeed electrons, and
(X-rays were helium nuclei. These emissions were
demonstrated to be antimicrobial by Mink in 1896
and by Pancinotti and Porchelli 2 years later. Highspeed electrons generated by electron accelerators
are now used in sterilization processes (Chapter
12.2).
Thus, within 3 years of the discovery of X-rays
6

and natural radiation, their effect on the growth of
microorganisms had been investigated and published. Both were found to be lethal. Ultraviolet
light was shown in 1893 to be the lethal component

of sunlight.
These and other aspects have been discussed by
Hugo (1996).
Sterilization can also be achieved by chemicals,
although their use for this purpose do not offer the
same quality assurance as heat- or radiation-sterilization. The term 'chemosterilizer' was first defined
by Borick in 1968. This term has now been replaced
by 'liquid chemical sterilants', which defined those
chemicals used in hospital for sterilizing reusable
medical devices. Among the earliest used 'liquid
chemical sterilants' were formaldehyde and ethylene oxide. Another aldehyde, glutaraldehyde has
been used for this purpose for almost 40 years
(Bruch, 1991). More recently compounds such as
peracetic acid and ortho-phthalaldehyde (OPA)
have been introduced as alternative substitutes for
the di-aldehyde.
After this time, the science of sterilization and
disinfection followed a more ordered pattern of
evolution, culminating in the new technology of
radiation sterilization. However, mistakes—often
fatal—still occur and the discipline must at all times
be accompanied by vigilance and critical monitoring and evaluation.

4 Future developments for chemical
biocides
This is a very interesting time for biocides. For the
last 50 years, our knowledge of biocides has increased, but also 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 biocides and the mechanisms of microbial
resistance to biocides. Although, 'new' biocidal
molecules might not be produced in the future,
novel 'disinfection/antisepsis' products might concentrate on synergistic effects between biocides
or/and the combination of biocide and permeabilizer, or other non-biocide chemicals, so that an increase in antimicrobial activity is achieved. The


Historical introduction

ways in which biocides are delivered is also the subject of extensive investigations. For example, the
use of polymers for the slow release of biocidal molecules, the use of light-activated biocides and the
use of alcoholic gels for antisepsis are all signs of
current concerted efforts to adapt laboratory concepts to real life situations.
Although, this might be a 'golden age' for
biocidal science, many questions remain unanswered, such as the significance of biocide
resistance in bacteria, the fine mechanism of action
of biocides and the possibility of primary action
sites within target microorganisms, and the effect of
biocides on new emerging pathogens and microbial
biofilms. Some of these concepts will be discussed
further in several chapters.

5 References
General references
Brock, T.D. (ed.) (1961) Milestones in Microbiology.
London: Prentice Hall.
Bullock, W. (1938) The History of Bacteriology. Oxford:
Oxford University Press.
Collard, P. (1976) The Development of Microbiology.
Cambridge: Cambridge University Press.

Crellin, J.K. (1966) The problem of heat resistance of
micro-organisms in the British spontaneous generation
controversies of 1860-1880. Medical History, 10,50-59.

Gaughran, E.R. & Goudie, A.J. (1975). Heat sterilisation
methods. Acta Pharmaceutica Suecica, 12 (Suppl.), 15-25.
Hugo, W.B. (1978) Early studies in the evaluation of disinfectants. Journal of Antimicrobial Chemotherapy,
4,489–494.
Hugo, W.B. (1978) Phenols: a review of their history and development as antimicrobial agents. Microbios, 23, 83–85.
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. London: British Broadcasting Corporation.
Selwyn, S. (1979) Early experimental models of disinfection
and sterilization. Journal of Antimicrobial Chemotherapy,
5,229-238.

Specific references
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. Morrissey,
R.F. and Prokopenko, Y.I.), pp. 377-396. Morin Heights
Canada: Polyscience Publications Inc.
Hugo, W.B. (1975) Disinfection. In Sterilization and Disinfection, pp. 187–276. London: Heinemann.
Hugo, W.B. (1996) A brief history of heat, chemical and
radiation preservation and disinfection. International
Biodeterioration and Biodegra dation, 36, 197–221.
Kallings, L.O., Ringertz, O., Silverstone, L. & Ernerfeldt, F.
(1966) Microbial contamination of medical preparations.
Acta Pharmaceutica Suecica, 3, 219–228.
Smith, Sir F. (1836-7) External employment of creosote.
Lancet, ii, 221-222.

Chapter 2
Types of antimicrobial agents
Suzanne L Moore and David N Payne

1 Introduction
2 Phenols
2.1
Sources of phenols—the coal-tar industry
2.2
Properties of phenolic fractions
2.3
Formulation of coal-tar disinfectants
2.4
The modern range of solubilized and emulsified
phenolic disinfectants
2.4.1 Cresol and soap solution British
Pharmacopoeia (BP) 1963 (Lysol)
2.4.2 Black fluids
2.4.3 White fluids
2.5
Non-coal-tar phenols
2.5.1 4-Tertiary octylphenol
2.5.2 2-Phenylphenol (2-phenylphenoxide)
2.5.3 4-Hexylresorcinol
2.6
Halo and nitrophenols
2.6.1
2,4,6-Trichlorophenol

2.6.2 Pentachlorophenol (2-phenylphenoxide)
2.6.3 4-Chloro-3-methylphenol
(chlorocresol)
2.6.4
4-Chloro-3,5-dimethylphenol
(chloroxylenol; para-chloro-metaxylenol; PCMX)
2.6.5
2,4-Dichloro-3,5-dimethylphenol
(dichloroxylenol;dichloro-meta-xylenol;
DCMX)
2.6.6 4-Chloro-3-methylphenol (para-chlorometa-cresol; PCMC)
2.6.7
Monochloro-2-phenylphenol
2.6.8 2-Benzyl-4-chlorophenol (chlorphen;
ortho-benzyl-para-chlorophenol;
OBPCP)
2.6.9 Mixed chlorinated xylenols
2.6.10 Other halophenols
2.6.11 Nitrophenols
2.6.12 Formulated disinfectants containing
chlorophenols
2.6.13 Phenol
2.7
Pine disinfectants
2.8
Theory of solubilized systems
2.9
The bisphenols
2.9.1 Derivatives of
dihydroxydiphenylmethane

2.9.2 Derivatives of hydroxydiphenylether
2.9.3 Derivatives of diphenylsulphide
3 Organic and inorganic acids: esters and salts
3.1
Introduction

8

3.2

4

5

6

7

8

Physical factors governing the antimicrobial
activity of acids
3.3
Mode of action
3.4
Individual compounds
3.4.1 Acetic acid (ethanoic acid)
3.4.2 Propionic acid
3.4.3 Undecanoic acid (undecylenic acid)
3.4.4 2,4-Hexadienoic acid (sorbic acid)

3.4.5 Lactic acid
3.4.6 Benzoicacid
3.4.7 Salicylic acid
3.4.8 Dehydroacetic acid (DHA)
3.4.9 Sulphur dioxide, sulphites, bisulphites
3.4.10 Esters of p-hydroxybenzoic acid
(parabens)
3.4.11 Vanillic acid esters
Aromatic diamidines
4.1
Propamidine
4.2
Dibromopropamidine
Biguanides
5.1
Chlorhexidine
5.2
Alexidine
5.3
Polymeric biguanides
Surface-active agents
6.1
Cationic agents
6.1.1 Chemical aspects
6.1.2 Antimicrobial activity
6.1.3 Uses
6.2
Anionic agents
6.3
Non-ionic agents

6.4
Amphoteric (ampholytic) agents
Aldehydes
7.1
Glutaraldehyde (pentanedial)
7.1.1 Chemical aspects
7.1.2 Interactions of glutaraldehyde
7.1.3 Microbicidal activity
7.1.4 Uses of glutaraldehyde
7.2
Formaldehyde (methanal)
7.2.1 Chemical aspects
7.2.2 Interactions of formaldehyde
7.2.3 Microbicidal activity
7.2.4 Formaldehyde-releasing agents
7.2.5 Uses of formaldehyde
7.3
Ortho-phthalaldehyde
7.4
Other aldehydes
Antimicrobial dyes
8.1
Acridines


Types of antimicrobial agents

9

10

11

12

13

14

15

16

8.1.1 Chemistry
8.1.2 Antimicrobial activity
8.1.3 Uses
8.2
Triphenylmethane dyes
8.3
Quinones
Halogens
9.1
Iodine compounds
9.1.1 Free iodine
9.1.2 lodophors
9.1.3 lodoform
9.2
Chlorine compounds
9.2.1 Chlorine-releasing compounds
9.2.2 Uses of chlorine-releasing compounds

9.2.3 Chloroform
9.3
Bromine
Quinoline and isoquinoline derivatives
10.1 8 -Hy droxyquinoline derivatives
10.2 4-Aminoquinaldinium derivatives
10.3 Isoquinoline derivatives
Alcohols
11.1 Ethyl alcohol (ethanol)
11.2 Methyl alcohol (methanol)
11.3 Isopropyl alcohol (isopropanol)
11.4 Benzyl alcohol
11.5 Phenylethanol (phenylethyl alcohol)
11.6 Bronopol
11.7 Phenoxyethanol (phenoxetol)
11.8 Chlorbutanol (chlorbutol)
11.9 2,4-Dichlorobenzyl alcohol
Peroxygens
12.1 Hydrogen peroxide
12.2 Peracetic acid
Chelating agents
13.1 Ethylendiamine tetraacetic acid
13.2 Other chelating agents
Permeabilizers
14.1 Polycations
14.2 Lactoferrin
14.3 Transferrin
14.4 Citric and other acids
Heavy-metal derivatives
15.1 Copper compounds

15.2 Silver compounds
15.3 Mercury compounds
15.3.1 Mercurochrome (disodium-2,7dibromo-4-hydroxymercurifluorescein)
15.3.2 Nitromersol (anhydro-2hydroxymercuri-6-methyl-3nitrophenol)
15.3.3 Thiomersal (merthiolate; sodium-o(ethylmercurithio)-benzoate)
15.3.4 Phenylmercuric nitrate (PMN)
15.3.5 Phenylmercuric acetate (PMA)
15.4 Tin and its compounds (organotins)
15.5 Titanium
Anilides
16.1 Salicylanilide

17

18

19
20

21

22
23

16.2 Diphenylureas (carbanilides)
16.3 Mode of action
Miscellaneous preservatives
17.1 Derivatives of 1,3-dioxane
17.1.1 2,6-dimethyl-l,3-dioxan-4-ol acetate
(dimethoxane)

17.1.2 5-Bromo-5-nitro-l,3-dioxane (Bronidox:
Care Chemicals)
17.2 Derivatives of imidazole
17.2.1 l,3-Di(hydroxymethyl)-5,5-dimethyl2,4-dioxoimidazole; 1, 3-dihydroxymethyl)-5,5-dimethylhydantoin
(Dantoin)
17.2.2 N, N"-methylene bis [5'[1hydroxymethyl]-2,5-dioxo-4imidazolidinyl urea] (Germall 115: ISP,
Wayne, New Jersey, USA)
17.2.3 Diazolidinyl urea
17.3 Isothiazolones
17.3.1 5-Chloro-2-methyl-4-isothiazolin-3-one
(CMIT) and 2-methyl-4-isothiazolin-3one (MIT)
17.3.2 2-n-Octyl-4-isothiazolin-3-one (Skane:
Rohm & Haas)
17.3.3 l,2-Benzisothiazolin-3-one (BIT)
17.3.4 Mechanism of action
17.4 Derivatives of hexamine
17.5 Triazines
17.6 Oxazolo-oxazoles
17.7 Sodium hydroxymethylglycinate
17.8 Methylene bisthiocyanate
17.9 Captan
17.10 1,2-dibromo-2,4-dicyanobutane (Tektamer 38)
17.11 Essential oils
17.12 General statement
Vapour-phase disinfectants
18.1 Ethylene oxide
18.2 Formaldehyde-releasing agents
18.3 Propylene oxide
18.4 Methyl bromide
18.5 Ozone

18.6 Carbon dioxide
18.7 Mechanism of action
Aerial disinfectants
Other uses of antimicrobial agents
20.1 Disinfectants in the food, dairy, pharmaceutical
and cosmetic industries
20.2 Disinfectants in recreational waters
Which antimicrobial agent?
21.1 Regulatory requirements
21.2 Which preservative ?
21.3 New concepts
The future
References

9


Chapter 2

1 Introduction
Many different types of antimicrobial agents are
now available and serve a variety of purposes in the
medical, veterinary, dental and other fields (Russell
et al., 1984; Gorman & Scott, 1985; Gardner &
Peel, 1986, 1991; Russell & Hugo, 1987; Russell,
1990a,b, 1991a,b; Russell & Gould, 1991a,b;
Fleurette et al., 1995; Merianos, 1995; Rossmore,
1995; Russell & Russell, 1995; Rutala, 1995a,b;
Ascenzi, 1996a; Russell & Chopra, 1996). Subsequent chapters will discuss the factors influencing
their activity and their role as disinfectants and

antiseptics and as preservatives in a wide range
of products or materials (Akers, 1984; Pels et al.,
1987; Eklund, 1989; Gould & Jones, 1989; Wilkins
& Board, 1989; Russell & Gould, 1991a,b; Kabara
& Eklund, 1991; Seiler & Russell, 1991). Lists of
preservatives are provided by Denyer and
Wallhausser (1990) and by Hill (1995). Additional
information is provided on their mechanism of action and on the ways in which microorganisms
show resistance.
The present chapter will concentrate on the
antimicrobial properties and uses of the various
types of antimicrobial agents. Cross-references to
other chapters are made where appropriate. A comprehensive summary of inhibitory concentrations,
toxicity and uses is provided by Wallhausser (1984).

2 Phenols
The historical introduction (Chapter 1) and the
papers by Hugo (1979, 1991) and Marouchoc
(1979) showed that phenol and natural-product
distillates containing phenols shared, with chlorine
and iodine, an early place in the armoury of antiseptics. Today they enjoy a wide use as general disinfectants and as preservatives for a variety of
manufactured products (Freney, 1995). The main
general restriction is that they should not be used
where they can contaminate foods. As a result of
their long history, a vast literature has accumulated
dealing with phenol and its analogues and comprehensive review of these compounds can be found in
Goddard and McCue (2001). Unfortunately, many

10

different parameters have been used to express their
biocidal and biostatic power but the phenol coefficient (Chapters 7.2 and 11) has probably been the
most widely employed and serves as a reasonable
cross-referencing cipher for the many hundreds of
papers and reports written.
A reasonable assessment of the relationship between structure and activity in the phenol series was
compiled by Suter (1941). The main conclusions
from this survey were:
1 para-Substitutions of an alkyl chain up to six
carbon atoms in length increases the antibacterial
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. Again, due to the conferment of
polar properties, straight chain para-substituents
confer greater activity than branched-chain substituents containing the same number of carbon
atoms.
2 Halogenation increases the antibacterial activity
of phenol. The combination of alkyl and halogen
substitution which confers the greatest antibacterial 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 phenol
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 (Hugo & Bowen, 1973)
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, where at one

time they enjoyed a large vogue, although 4nitrophenol 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


Types of antimicrobial agents

of the bisphenols confers additional biocidal
activity.
2.1 Sources of phenols—the coal-tar industry
Most of the phenols that are used to manufacture
disinfectants are obtained from the tar obtained as a
by-product in the destructive distillation of coal.
Coal is heated in the absence of air and the volatile
products, one of which is tar, condensed. The
tar is fractionated to yield a group of products,
which 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 4cresol. The 'xylenols' consist of the six isomeric dimethylphenols plus ethylphenols. The combined
fraction, cresols and xylenols, is also available as a
commercial product, which is known as cresylic
acid. High-boiling tar acids consist of higher alkyl
homologues of phenol: e.g. the diethylphenols,

tetramethylphenols, methylethylphenols, together
with methylindanols, naphthols and methylresorcinols, the latter being known as dihydries. There
may be traces of 2-phenylphenol. The chemical
constituents of some of the phenolic components
are shown in Fig. 2.1.
Extended information on coal tars and their constituents is given in the Coal Tar Data Book (1965).
As tar distillation is a commercial process, it should
be realized that there will be some overlap between
fractions. Phenol is obtained at 99% purity. Cresol
of the British Pharmacopoeia (2002) (2-, 3- and 4cresols) must contain less than 2% of phenol. A
commercially mixed xylenol fraction contains no
phenols or cresols but may contain 22 of the higherboiling phenols. High-boiling tar acids may contain
some of the higher-boiling xylenols, for example
3,4-xylenol (boiling-point (b.p.) 227 °C).
Mention must be made of the neutral oil fraction,
which has an adjuvant action in some of the formulated disinfectants to be considered below. It is
devoid of biocidal activity and consists mainly
of hydrocarbons, such as methyl- and dimethylnaphthalenes, w-dodecane, naphthalene,
tetramethylbenzene, dimethylindenes and tetrahy-

dronaphthalene. Some tar distillers offer a neutral
oil, boiling range 205-296°C, for blending with
phenolics destined for disinfectant manufacture
(see also section 2.4.2).
2.2 Properties of phenolic fractions
The passage from phenol (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. The ratio of activity against Gram-negative to activity against
Gram-positive organisms, however, remains fairly

constant, although in the case of pseudomonads,
activity tends to decrease with decreasing watersolubility; see also Table 2.1.
2.3 Formulation of coal-tar disinfectants
It will be seen from the above data that the progressive increase in desirable biological properties
of the coal-tar phenols with increasing boilingpoint 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.
The antiseptic and disinfectant properties of coal
tar had been noted as early as 1815, and in 1844 a
Frenchman called Bayard made an antiseptic powder of coal tar, plaster, ferrous sulphate and clay, an
early carbolic powder. Other variations on this
theme appeared during the first half of the nineteenth century. In 1850, a French pharmacist, Ferdinand Le Beuf, prepared an emulsion of coal
tar using the bark of a South American tree, the
quillaia. This bark contained a triterpenoid glycoside with soap-like properties belonging to the class
of natural products called saponins. By emulsifying
coal tar, Le Beuf made a usable liquid disinfectant,
which proved a very valuable aid to surgery. A 'solution' of coal tar prepared with quillaia bark was
described in the Pharmaceutical Codex (1979).
Quillaia is replaced by polysorbate 80 in formulae
for coal-tar 'solutions' in the British Pharma-

11


Chapter 2

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

copoeia (2002). In 1887 the use of soap and coal tar
was first promulgated, and in 1889 a German experimenter, T. Damman, patented a product which
was prepared from coal tar, creosote and soap and
which involved the principle of solubilization.
12

Thus, between 1850 and 1887, the basis for the formulation of coal-tar disinfectants had been laid and
subsequent discoveries were either rediscoveries or
modifications of these two basic themes of emulsification and solubilization. Better-quality tar acid


Types of antimicrobial agents
Table 2.1 Phenol coefficients of coal-tar products against Salmonella typbi and Staphylococcus aureus.
Phenol coefficient
Product and m.p., m. range (°C)
Phenol
182
Cresols
190–203
4-Ethylphenol
195
Xylenols
210-230
High-boiling tar acids
230-270
High-boiling tar acids
250-275

S. typhi

Staph. aureus

Water solubility (g/100 mL)

1

1

6.6

2.5

2.0

2.0

6

6

Slightly

5

4.5

Slightly

40

25

Insoluble

60

40

Insoluble

fractions and products with clearer-cut properties
aided the production of improved products. At the
same time, John Jeyes of Northampton patented a
coal-tar product, the well-known Jeyes fluid, by
solubilizing coal-tar acids with a soap made from
the resin of pine trees and alkali. In 1897, Engler
and Pieckhoff in Germany prepared the first Lysol
by solubilizing cresol with soap.
2.4 The 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 Fig. 2.1. The term 'clear
soluble fluid' is also used to describe the solubilized
products Lysol and Sudol.
2.4.1 Cresol and soap solution British
Pharmacopoeia (BP) 1963 (Lysol)
This consists of cresol (a mixture of 2-, 3- and 4cresols) 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 (British Association of Chemical Specialities, 1998). Most vegeta-

tive 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 biocidal properties of the product (Tilley
& Schaffer, 1925; Berry & Stenlake, 1942).
Rideal-Walker (RW) coefficients [British Standard
(BS) 541:1985] are of the order of 2.
2.4.2 Black fluids
These are defined in a British Standard (BS 2462:
1986) which has now been superceeded by specific
European standard methods for products in
medical, veterinary, industrial, domestic and institutional usage. They consist of a solubilized crude
phenol fraction prepared from tar acids, of the boiling range 250–310 °C (Fig. 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 sulphate and
sulphonate mixture prepared by heating castor oil
13


Chapter 2

with sulphuric acid (called sulphonated castor oil or
Turkey red oil).
Additional stability is conferred by the presence
of coal-tar hydrocarbon neutral oils. These have
already been referred to in section 2.1 and comprise
such products as the methyl naphthalenes, indenes
and naphthalenes. The actual mechanism whereby
they stabilize the black fluids has not been adequately explained; however, they do prevent crystallization of naphthalene present in the tar acid
fraction. Klarmann and Shternov (1936) made a
systematic study of the effect of the neutral oil fraction and also purified methyl- and dimethylnaphthalenes on the bactericidal efficiency of a coal-tar
disinfectant. They prepared mixtures of cresol and
soap solution (Lysol type) of the United States Pharmacopeia with varying concentrations of neutral
oil. They found, using a phenol coefficient-type test
and Salmonella typhi as test organism, that a product containing 30% cresols and 20% neutral oil
was 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 haemolytic 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.
Depending on the phenol fraction used and its
proportion of cresylic acids to high-boiling tar acid,
black fluids of varying RW coefficients reaching as
high as 30 can be produced; however, as shown in
section 2.2, increasing biocidal activity is accompanied by an increasing sensitivity to inactivation by
organic debris. To obtain satisfactory products, the
method of manufacture is critical and a considerable expertise is required to produce active and
reproducible batches.
Black fluids give either clear solutions or emulsions on dilution with water, those containing

greater proportions of higher phenol homologues
giving emulsions. They are partially inactivated by
the presence of electrolytes.
2.4.3 White fluids
White fluids are also defined in BS 2462: 1986,
which has since been superceeded by specific
14

European standard methods. They 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 the seaweed
called Irish moss. Products with a range of RW coefficients may be manufactured by the use of varying
tar-acid constituents.
As they 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
the 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.
An interesting account of the methods and pitfalls of manufacture of black and white fluids is
given by Finch (1958).
2.5 Non-coal-tar phenols
The coal-tar (and to a lesser extent the petrochemical) industry yields a large array of phenolic products; phenol itself, however, 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, 2phenylphenol and 4-hexylresorcinol (Fig. 2.2).
2.5.1 4-Tertiary octylphenol
This phenol (often referred to as octylphenol) is a
white crystalline substance, melting-point (m.p.)
83 °C. The cardinal property in considering its application as a preservative is its insolubility in water,
1 in 60 000 (1.6 x 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