INTERNATIONAL

BIODETERIORATION
&
BIODEGRADATION
International Biodeterioration & Biodegradation 41 (1998) 269-272

The testing of disinfectants

Gerald Reybrouck
Hospital Hygiene and Infection Conirol Department. Katholieke Uniclersiteit Leucen, Lewen (Lourain), Belgium

Accepted: 29 January 1998

Abstract

Although all disinfectant tests have the same final purpose, namely measuring the antimicrobial activity of a chemical substance
or preparation, a large number of testing methods has been described. They are subdivided into suspension tests, carrier and surface
disinfection tests and other practice-mimicking tests. The suspension tests comprise qualitative and quantitative suspension tests,
and, as derived tests, the determination of the phenol coefficient and capacity tests. There is an essential difference between a carrier
test and a surface disinfectant test: in the former case the carrier is submerged in the disinfectant solution during the whole exposure
time, whereas in the latter case the disinfectant is applied on the carrier for the application time and thereafter the carrier is drying
during the exposure.
The basic principle now widely accepted is that the antimicrobial efficiency of a disinfectant is examined at three stages of
testing.
The first stage concerns laboratory tests in which it is verified whether a chemical compound or a preparation possesses antimicrobial
activity. For these preliminary screening tests, suspension tests are considered.
In the second stage of tests, disinfection procedures and not disinfectants are examined. It is determined under which conditions
and at which use-dilution for a given application the preparation is active: the tests simulate real-life situations; such tests are carrier
tests for the disinfection of materials by submersion and surface disinfection tests.
The last stage takes place in the field, and comprises the in-loco or in-situ tests with as variants the in-use tests, which examine
whether, after a normal period of use, germs are still killed by the disinfectant solution.
It is the task of the European and international standardization organisations to develop new standards and to elaborate tests,
which predict the effectiveness of a preparation in practice under variable circumstances. c%, 1998 Elsevier Science Ltd. All rights
reserved.

Keywords; Testing; Efficacy assessment: Disinfectant.

1. Introduction tralization of the disinfectant at the end of the exposure

was studied by Geppert (1890).
It is scarcely surprising that, very early after the detection The greatest problem in disinfectant testing is that of
of bacteria as the cause of infections and deterioration, the repeatability (intralaboratory spread) and of repro-
Robert Koch tried to kill bacteria and to quantify this ducibility (interlaboratory spread) of the results. This is
antibacterial effect. He described a disinfectant test in the clearly demonstrated in the development of the early tests
article ober Desinfektion, on disinfection (Koch, 188 1). for the determination of the phenol coefficient (Rideal
It was the most simple carrier test: a silk thread was and Walker, 1903). The number of bacteria dried on a
contaminated by submersion in a liquid culture of the carrier is hardly to standardize. The survival of the germs
test organism, Bacillus anthracis; after drying, the con- on the carrier during drying is not constant. Therefore
taminated thread was immersed in several disinfectant suspension tests are preferred to the use of carriers: a
solutions for a given exposure time; thereafter the thread sample of the bacterial culture is suspended into the dis-
was cultured in a nutrient broth; no growth after incu- infectant solution and after exposure it is verified by
bation indicated activity of the product tested. Koch subculture whether this inoculum is killed or not. Samples
concluded that, from the disinfectant solutions were taken as it was usual in that time: by a loop; the
compared, mercuric chloride was the most active prod- inoculum was a loopful. The problem of the changing
uct. These erroneous results were due to the carry-over resistance of the test organisms was overcome by the
of bacteriostatic residues of the disinfectant into the sub- simultaneous testing of a phenol solution, which acted as
culture medium. The remedy of this problem by the neu- an internal standard. Nevertheless skips were frequent.

SO964-8305/98/$19.00 c: 1998 Elsevier Science Ltd. All rights reserved

PII: SO964-8305(98)00024-9
270 G. Revbrouck/International Biodeterioration & Biodegradation 41 (1998) 269-272

A further step in the standardization of disinfectant of the phenol coefficient (Rideal and Walker, 1903) and
tests was the introduction of quantitative tests after the the quantitative suspension tests. They all have the fol-
second World War. From the study of the kinetics of lowing features in common; an appropriate volume of
disinfection, it was clear that the end-point of no-sur- bacterial suspension, the inoculum, is added to the dis-
viving-organisms is determined by the size of the inocu- infectant in the concentration to be tested. After a given
lum, i.e. the number of organisms present in the bacterial exposure time, a sample is examined to determine
suspension that is brought into contact with the disin- whether the inoculum has been killed or not. Initially this
fectant. If the inoculum consists of only a few bacteria, was done in a qualitative way. A loopful of bacterial
then the end-point “no growth” will be reached after a suspension was brought into contact with the disinfectant
shorter exposure than when more bacteria were present and again a loopful of this mixture was cultured for
in the beginning. To overcome this difficulty, it is better surviving organisms. Results were expressed as ‘growth’
to calculate the real reduction in a quantitative way. (+ ) or ‘no growth’ ( - ). At present the number of sur-
A good test must be able to predict the value of the viving organisms is counted and compared to the original
disinfectant in practice, and this covers a broad field. inoculum size. By subtracting the logarithm of the former
Disinfectants are applied to treat surfaces, instruments, from the logarithm of the latter, the decimal log reduction
water, linen, waste, etc., but also living tissues as hands or microbicidal effect (ME) is obtained. An ME of 1
and skin, mucous membranes and wounds. They are equals to a killing of 90% of the initial number of
applied in medicine, in veterinary medicine, in agriculture bacteria, an ME of 2 means 99% killed. A generally
and food industry, in the domestic area. It is impossible accepted requirement is an ME that equals or is greater
that one test can cover all these fields and domains. More than 5: at least 99.999% of the germs is killed. Several
specific tests were developed to give a picture of the value concentrations and exposure periods can easily be exam-
of a disinfectant procedure in a defined application. A ined. Potentially inhibitory substances such as organic
large number of testing methods has been the result. matter or soap can be added, or the influence of water
Therefore a subdivision in function of their structure hardness can be determined.
is helpful (Cremieux and Fleurette, 1991; Reybrouck, A quantitative suspension test is the basic test in all
1992a). contemporary testing schemes. Examples are the sus-
pension tests of the Association franguise de Nor-
malisation (AFNOR, 1989) of the German Society for
2. The disinfectant tests Hygiene and Microbiology (Borneff et al., 1981), of the
German veterinary Society (DVG, 1988), the European
2.1. Carrier tests suspension test (Council of Europe, 1987) and the pro-
posal of the new basic bactericidal test of the European
Carrier tests are the oldest tests. The structure is as Committee for Standardization (CEN, 1996).
follows: the carrier (a silk or catgut thread, a penicylinder, It is examined in these suspension tests whether micro-
a little stick) is contaminated by submersion in a liquid organisms are killed by a disinfectant in terms of a range
culture of the test organism; after drying it is brought of concentrations and several times of exposure. There is
into the use-dilution of the disinfectant for a given an intense contact between the disinfectant solution and
exposure time, after which it is cultured in a nutrient the bacterial cells. As a result these tests are generally well
broth; no growth indicates activity of the product tested; standardized. This approach, however, is less practical. It
growth indicates a failing. By multiplying the number of was realized that not disinfectants themselves, but their
test concentrations and the contact times an overview of use in a given field of application, this is a disinfection
potentially active concentration-time relationships of the procedure, should be tested. Therefore capacity tests and
disinfectant is obtained. Examples of a carrier test are practical tests were developed in order to simulate real-
the former use-dilution test of the American Association life situations, and to obtain results that offer more pre-
of Official Analytical Chemists (AOAC, 1990) and the cise information on the effective use-dilution for a given
Keimtriigerversuch of the German Society of Hygiene and fields of application. Indeed suspension tests and carrier
Microbiology (Borneff et al., 1981). The great short- tests are less severe than capacity tests and practical tests
coming of the carrier tests is that the survival of the (Reybrouck, 1992b).
inoculum on the carrier is not constant and hard to stan-
dardize.
2.3. Capacity tests
2.2. Suspension tests
The structure of a capacity test can be understand as
The most simple disinfectant tests are the suspension follows: each time a soiled instrument is placed into a
tests. There are different kinds of suspension tests: the container with disinfectant, or a mop is soaked in a
qualitative suspension tests, the test for the determination bucket containing a disinfectant solution, a certain quan-
 G. Re~brouck/international Biodeterioration & Biodegradation 41 (1998) 269-272 271

tity of dirt and bacteria is added to the solution. The 3. Testing schemes
ability to retain activity in the presence of an increasing
load is the capacity of the disinfectant. Capacity tests The antimicrobial efficiency of a disinfectant is exam-
simulate the practical situations of housekeeping and ined at three stages of testing (Reybrouck, 1992a). The
instrument disinfection. The best known capacity test is first phase concerns laboratory tests in which it is verified
the Kelsey-Sykes test (Kelsey and Sykes, 1969). whether a chemical compound or a preparation possesses
antimicrobial activity: for these preliminary screening
tests essentially quantitative suspension tests are
2.4. Practical tests considered. The second stage is still carried out in the
laboratory but in conditions simulating real-life
The practical tests under real-life conditions are per- conditions. Not disinfectants, but disinfection procedures
formed after measuring the time-concentration relation- are examined. It is determined in the practical tests in
ship of the disinfectant in a quantitative suspension test. which conditions and at which use-dilution after a given
The objective is to verify whether the proposed use- contact time the preparation is active. The third phase
dilution is still adequate in the conditions under which it comprises the field tests or pilot studies, and the variant
would be used. Practical tests have the advantage that of in-use tests. In these tests it is verified whether, after a
the experiments still take place in the laboratory and can normal period of use, germs in the disinfectant solution
be better standardized than in field tests. are still killed.
The best known practical tests are the surface dis- All these tests and testing schedules apply for as well
infection tests. There exist many variants of these tests: bacteria as other micro-organisms. Most studied are the
the Fltichendesinfektionversuche unter praxisnahen Bedin- bactericidal tests in which the activity towards vegetative
gungen of the German Society for Hygiene and Micro- bacteria is examined. But most testing schedules are
biology (DGHM, 1991), the MPthodes de portes-germes applicable for fungi and yeasts too (fungicidal tests), for
of the Association frcqaise de Normalisation (AFNOR, mycobacteria (tuberculocidal tests), for viruses (virucidal
1989) and the new proposal of the European Committee tests) and for spores of bacteria (sporicidal tests).
for Standardization (Bloomfield et al., 1994). All these
tests do not show the same severity (Reybrouck, 1990,
Reybrouck, 1992b). The test schedule is as follows: the 4. The present situation and further developments
test surface (a small tile, a microscopic slide, a piece of
PVC, a stainless steel disc, etc.) is contaminated with a As the unification of disinfection tests is a priority for
standardized inoculum of the test bacteria and dried: then the European industry, the English, French, German and
a definite volume of the disinfectant solution is dis- Swiss standard institutes asked the CEN (ComitP Euro-
tributed over the carrier; after the given exposure time p&en de Normalisation, European Committee for Stan-
the number of survivors is determined by impression on dardization) to develop European standards. The CEN
a contact plate or by a rinsing technique, in which the is under patronage of the 12 EC (European Community)
carrier is rinsed in a diluent, and the number of bacteria member states as well as the 6 member states of the EFTA
is determined in the rinsing fluid. In order to determine (European Free Trade Association). In April 1990 the
the spontaneous dying rate of the organisms caused by CEN started a new Technical Committee TC 216
drying on the carrier, a control series is included in which “Chemical disinfectants and antiseptics” with as scope:
the disinfectant is substituted by distilled water; from the “the standardization of the terminology, requirements,
comparison of the survivors in this control series with the test methods including potential efficacy under in use
test series, the reduction is determined quantitatively. conditions, recommendations for use and labelling in the
There is an essential difference between a carrier test whole field of chemical disinfection and antisepsis; areas
and a surface disinfectant test: in the former case the of activity include agriculture (but not crop protection
carrier is submerged in the disinfectant solution during chemicals), domestic service, food hygiene and other
the whole exposure time, whereas in the latter case the industrial fields, institutional, medical, and veterinary
disinfectant is applied on the carrier for the application applications”.
time and thereafter the carrier is drying during the The testing scheme accepted by the CEN TC 216 is
exposure. fully in concordance with the above-mentioned testing
Other practical tests are described for other appli- schemes. The phase 1 tests comprise the basic bacteri-
cations, e.g. instrument disinfection, swimming-pool cidal, respectively fungicidal, tuberculocidal, virucidal,
disinfection, water treatment. sporicidal tests; they are essentially quantitative sus-
For some applications, e.g. hand decontamination or pension tests in which the biocidal activity is determined
antisepsis, practical tests are indispensable since sus- towards a limited number of test organisms: for the bac-
pension tests can not present the total picture of activity tericidal tests they are Staph~~lococcus aweus and Pseudo-
to possible inefficacy. monas aeruginosa. From the results of the phase 2 tests it
212 G. Rqvbrouckllnternational Biodeterioration & Biodegradation 41 (1998) 269-272

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