Journal of Hospital Infection (2003) 55, 137–140

www.elsevierhealth.com/journals/jhin

SHORT REPORT

Efficacy of some neutralizers in suspension tests

determining the activity of disinfectants
E. Espigares, A. Bueno, M. Fernández-Crehuet, M. Espigares*

Department of Preventive Medicine and Public Health, University of Granada, Avda Madrid, 11, 18012
Granada, Spain

Received 10 March 2003; accepted 5 June 2003

KEYWORDS Summary The ability of six mixtures to neutralize glutaraldehyde, o-phthalaldehyde

Neutralizer; and peracetic acid was tested using four reference strains: Pseudomonas aeruginosa
Disinfectant; CIP A22, Escherichia coli CIP 54127, Staphylococcus aureus CIP 53154, and
Glutaraldehyde; Enterococcus faecium CIP 5855. Glutaraldehyde was the hardest to neutralize, and
O-phthalaldehyde; peracetic acid the easiest. The most effective mixture was Tween 80 with sodium
Peracetic acid; bisulphate, sodium thioglycolate, lecithin and cysteine, and the least effective was
Suspension test; Tween 80, lecithin and histidine. The efficacy of the neutralizers may indicate a
Dilution– neutralization propensity loss of activity from interfering substances when disinfectants are used in
method practice.
Q 2003 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.

Introduction are problematic. Peracetic acid has a broad

High-level disinfectants are sporicidal if given
enough time; in , 30 min they destroy a wide
variety of vegetative bacteria, and a large pro-
portion of spores.1,2 They include glutaraldehyde,
o-phthalaldehyde, and peracetic acid, and are
widely used to disinfect medical equipment.3
Glutaraldehyde is one of the most commonly
used agents worldwide. The buffered 2% solutions
have a half-life of 14 –28 days because of polym-
erization.2 Glutaraldehyde reacts with proteins and
prevents dissociation of ribosomes,4 has a broad
spectrum and rapid action,5 but polymerization
and potential mutagenic and carcinogenic effects
*Corresponding author. Dpto. de Medicina Preventiva y Salud
Pública, Facultad de Farmacia, Campus Universitario de Cartuja,
18071 Granada, Spain.
E-mail address: mespigar@ugr.es
spectrum of activity and the advantage that its
decomposition products are innocuous.4 Although
highly unstable when diluted, stable powder for-
mulations containing peroxides, organic acids and
stabilizers have been developed,5 and 0.26% per-
acetic acid has an equivalent activity to 2%
glutaraldehyde. O-phthalaldehyde is effective in
12 min, and so faster than any other high-level
disinfectant. It is chemically related to glutaralde-
hyde and has a similar mode of action.
The bactericidal activity of these disinfectants
can be compared using suspension tests, such as the
dilution – neutralization method.7 The disinfectant
is allowed to react with a bacterial suspension
under standard conditions, then the reaction is
stopped by dilution with a neutralizing mixture.
Neutralizers require validation, usually by mixing
with disinfectant for 10 min at 208C, then testing

0195-6701/03/$ - see front matter Q 2003 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/S0195-6701(03)00238-X
138 E. Espigares et al.

the mixture by addition of a bacterial suspension. prepared at double strength and later diluted to
After another 5 min, a sample of the mixture is normal strength with diluent.
plated out, and the number of survivors is compared Neutralizer A ( £ 2): Tween 80, 129 g (120 mL);
with the initial inoculum. There should be no 40% sodium bisulphate, 25 mL; sodium thiosulphate
significant reduction in bacterial numbers. Suspen- pentahydrate, 15.69 g; diluent to 1000 mL.
sion tests can also be used to test the influence of Adjusted to pH 7.0 and sterilized by filtration.
interfering substances, such as organic matter, Neutralizer B ( £ 2): Tween 80, 129 g (120 mL);
soap or ions, on disinfectant activity.6 40% sodium bisulphate, 25 mL; sodium thiosulphate
The aim of this study was to determine the pentahydrate, 15.69 g; sodium thioglycolate, 10 g;
effectiveness of various neutralizers against glutar- L -cysteine, 3 g; diluent to 1000 mL. Adjusted to pH
aldehyde, o-phthalaldehyde, and peracetic acid 7.0 and sterilized by filtration.
using Gram-positive and Gram-negative bacterial Neutralizer C ( £ 2): Tween 80, 129 g (120 mL);
reference strains. 40% sodium bisulphite, 25 mL; sodium thiosulphate
pentahydrate, 15.69 g; diluent to 1000 mL.
Adjusted to pH 7.0 and sterilized by filtration.
Methods Four grams of lecithin was added aseptically.
Neutralizer D ( £ 2): Tween 80, 129 g (120 mL);
40% sodium bisulphite, 25 mL; sodium thiosulphate
Bacterial strains
pentahydrate, 15.69 g; sodium thioglycolate, 10 g;
L -cysteine, 3 g; diluent to 1000 mL. Adjusted to pH
The following reference strains were used: Pseudo-
7.0 and sterilized by filtration. Four grams of
monas aeruginosa CIP A22, Escherichia coli CIP
lecithin was added aseptically.
54127, Staphylococcus aureus CIP 53154, and
Neutralizer E ( £ 2): Tween 80, 64.5 g (60 mL); L -
Enterococcus faecium CIP 5855.
histidine, 2 g; diluent to 1000 mL. Adjusted to pH
7.0 and sterilized by filtration. Six grams of lecithin
Disinfectants was added aseptically.
Neutralizer F ( £ 2): Tween 80, 64.5 g (60 mL);
Three high-level commercial disinfectants were saponin, 60 g; L -histidine, 2 g; L -cysteine, 2 g;
used: 2% glutaraldehyde (Instrunetw), 0.26% per- diluent to 1000 mL. Adjusted to pH 7.0 and
acetic acid (Perasafew), and 0.55% o-phthalalde- sterilized by filtration.
hyde (Cidexw OPA). The disinfectants were Diluent (tryptone –salt): Tryptone, 1 g; sodium
prepared at a one in five dilution of the rec- chloride, 8.5 g; distilled water, 1000 mL. The
ommended regular use concentration in order to mixture was boiled to dissolve the ingredients
establish a disinfecting: neutralizing concentration completely. Adjusted to pH 7.2, and sterilized at
ratio comparable with the evaluative tests for the 1218C for 20 min.
bactericidal activity.
Test method
Neutralizers
The neutralizers were tested according to norms
The six neutralizers tested were designated A, B, C, UNE-EN 1040 and AFNOR NF T 72-150 for the
D, E and F (summarized in Table I). They were evaluation of bactericidal activity by means of the
dilution – neutralization method7 – 9 at 208C with
10 min contact time for the disinfectant – neutral-
Table I Summary of the degree of complexity of the
neutralizers tested with respect to their composition izer mixture, and 5 min contact time with the
bacterial suspension before sampling. All tests were
Component Neutralizer carried out in triplicate using homogeneous suspen-
A B C D E F sions of 1 – 3 £ 108 cfu/mL of test strain in diluent.
For this purpose, the density of the bacterial
Tween 80 þ þ þ þ þ þ suspensions was adjusted spectrophotometrically,
Sodium thiosulphate þ þ þ þ 2 2 at 620 nm in cuvettes with a path length of 1 cm, to
Sodium bisulphate þ þ þ þ 2 2 0.2 – 0.3 for Gram-negative bacilli, and to 0.3 – 0.4
Lecithin 2 2 þ þ þ 2
for Gram-positive cocci. Plate counts were per-
Sodium thioglycolate 2 þ 2 þ 2 2
Cysteine 2 þ 2 þ 2 þ formed from this suspension. For neutralizer test-
Histidine 2 2 2 2 þ þ ing, a control tube containing sterile distilled water
Saponin 2 2 2 2 2 þ was used in order to verify that the neutralizer had
no bactericidal action.
Neutralizers in suspension tests 139

Table II Mean counts of bacteria (cfu/mL) grown after neutralization of stated agent with mixtures A– F/mean count of control
tubes containing neutralizing mixtures A–F alone using the four indicator strains of bacteria

Test strain Disinfectant A B C D E F

Pseudomonas aeruginosa O-phthalaldehyde 39/156 34/145 43/174 154/164 8/80 168/148

Peracetic acid 57/140 35/133 57/122 87/126 0/149 5/138
Glutaraldehyde 0/155 41/156 0/169 11/170 0/149 0/164
Escherichia coli O-phthalaldehyde 9/132 21/155 12/152 137/133 0/199 0/226
Peracetic acid 62/111 69/157 73/102 103/99 0/178 61/159
Glutaraldehyde 0/140 18/140 0/121 26/130 6/167 0/180
Staphylococcus aureus O-phthalaldehyde 81/300 107/326 135/321 181/114 22/222 67/208
Peracetic acid 348/304 259/266 196/348 189/97 0/287 81/329
Glutaraldehyde 0/202 294/231 0/231 1/148 0/182 0/172
Enterococcus faecium O-phthalaldehyde 67/245 67/245 123/249 228/233 220/239 250/239
Peracetic acid 255/249 245/267 270/247 247/278 0/158 242/263
Glutaraldehyde 0/188 65/140 0/189 14/198 0/178 0/170

Results Discussion

The results are listed in Table II as the mean counts
(cfu/mL) of the bacterial suspensions in tubes
containing neutralizer, and their control tubes. A
neutralizer was considered valid when it produced a
reduction of # 50% (Table III).
The control tubes showed that the neutralizers
had no antibacterial activity (results not shown).
Neutralizer A was effective against peracetic
acid except with P. aeruginosa, which gave a
reduction of 59.3%. Similar results were obtained
with neutralizer C, which also had some activity
against o-phthalaldehyde with E. faecium as
the test organism. Neutralizer B was effective
against peracetic acid and glutaraldehyde using
S. aureus; and against peracetic acid with
E. faecium. Neutralizer D was effective against o-
phthalaldehyde and peracetic acid, for all strains
tested, but failed to inactivate glutaraldehyde.
Neutralizer E only worked with o-phthalaldehyde
and E. faecium. Finally, neutralizer F was effective
against o-phthalaldehyde with E. faecium and
P. aeruginosa, and against peracetic acid with
E. faecium.
Disinfectant tests require the use of an appropriate
neutralizer, but many references have been made to
the lack of a ‘universal’ neutralizer.10 Our results
showed that glutaraldehyde was the most difficult
disinfectant to neutralize and was only neutralized
with neutralizer B using S. aureus as an indicator
strain. In a similar study using vancomycin-resistant
enterococci, a mixture containing lecithin and poly-
sorbate 80 effectively neutralized 2% glutaralde-
hyde,11 underlining the need to test each
neutralizer against each test bacterium. Neutralizer
D, the most complex, was the most effective overall
(glutaraldehyde excepted), while E was the least
effective. Neutralizer F was also poorly affective,
although its effectiveness against a hand gel contain-
ing 85% ethanol has been demonstrated.12
Interfering substances such as organic matter,
surface-active agents or metal ions are of great
importance in the use and evaluation of disinfec-
tants, and a number of standardized tests have
been devised to take these into account.7,13 A
neutralizer is a mixture of interfering substances,
so if a disinfectant, such as glutaraldehyde, is hard

Table III Efficacy of the neutralizers

O-phthalaldehyde Peracetic acid Glutaraldehyde

Pseudomonas aeruginosa D, F (A), (C), D 2

Escherichia coli D A, (B), C, D 2
Staphylococcus aureus D A, B, C, D B
Enterococcus faecium (C), D, E, F A, B, C, D, F (B)

(): Reduction between 50% and 60% of the mean N values with respect to C.
140
to neutralize, it implies a lesser sensitivity to
interfering substances; this is supported by other
studies.4,13 Conversely peracetic acid was readily
neutralized, and is presumptively susceptible to
interfering substances, thus stressing the import-
ance of cleaning when using this agent.
Acknowledgements
The authors thank Mr César Criado Sánchez for his
assistance in the laboratory, and Jean Sanders for
her helpful suggestions while translating and editing
the manuscript.
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