Accepted Manuscript

Title: Antimicrobial activity of disinfectants commonly used

in the food industry in Mexico

Authors: Maricarmen Iñiguez-Moreno, Marı́a Guadalupe

Avila-Novoa, Elizabeth Iñiguez-Moreno, Pedro Javier
Guerrero-Medina, Melesio Gutiérrez-Lomelı́

PII: S2213-7165(17)30100-5
DOI: http://dx.doi.org/doi:10.1016/j.jgar.2017.05.013
Reference: JGAR 424

To appear in:

Received date: 25-2-2017

Revised date: 3-5-2017
Accepted date: 5-5-2017

Please cite this article as: Maricarmen Iñiguez-Moreno, Marı́a Guadalupe Avila-Novoa,
Elizabeth Iñiguez-Moreno, Pedro Javier Guerrero-Medina, Melesio Gutiérrez-Lomelı́,
Antimicrobial activity of disinfectants commonly used in the food industry in Mexico
(2010), http://dx.doi.org/10.1016/j.jgar.2017.05.013

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Antimicrobial activity of disinfectants commonly used in the food

industry in Mexico

Maricarmen Iñiguez-Moreno 1, María Guadalupe Avila-Novoa 1, Elizabeth Iñiguez-Moreno 1,

Pedro Javier Guerrero-Medina 1 and Melesio Gutiérrez-Lomelí 1*.

1
Departamento de Ciencias Médicas y de la Vida, Universidad de Guadalajara Centro

Universitario de la Ciénega. Ocotlán. Jalisco. México.

* Corresponding author: Av. Universidad 1115, Col. Linda Vista, 47820, Ocotlán, Jalisco,

México, Tel:+52-3929259400 (Ext. 48462). E-mail address: mele.gtz@gmail.com (M. Gutiérrez-

Lomelí)

Highlights

 All disinfectants showed an effectiveness of 99.9999% with Pseudomonas aeruginosa.

 The MIC of all disinfectants was two to four times bigger at 5 min than at 10 min.

 The MIC tended to be similar to 10 or 15 min of exposure time.

 QACs and chlorine-based disinfectants had a bigger MIC than peracetic acid-based.

 Gram-negative bacteria have a bigger MIC of third generation QACs than S. aureus.

1
Abstract

Objectives: Disinfectants are widely used in food processing environments for microorganism

control; their activity can vary according the microorganism and their used in the appropriated

concentrations is vital. Hence, the aim of this study was determined the effectiveness and

minimum inhibitory concentration (MIC) of 15 disinfectants commonly used in the food industry

in Mexico.

Methods: The antimicrobial activity and the MIC were determined according to AOAC and

CLSI, respectively, with approved strains.

Results: Most disinfectants reduced 99.999% of microorganisms in suspension after 30 s of

contact, so reduction rate corresponded at least 5 Log10. Only for Pseudomonas aeruginosa

ATCC 15442, did all disinfectants have 99.999% effectiveness. For the MIC, only the third

generation quaternary ammonium compounds (QACs) in acid medium did not have values within

the range in which is used in the food industry for Staphylococcus aureus ATCC 25923. In

addition, for all disinfectants the MIC at 5 min was two to four times greater than the

concentration with the same effect at 10 min; moreover, in most cases there was no difference in

the MIC at 10 and 15 min (p>0.05).

Conclusions: At recommended concentrations, disinfectants had bactericidal activity for at least

three of the six microorganisms evaluated. However, the MIC was affected by the exposure time:

it was bigger at 5 min than at 10 min; moreover, in the majority of the cases, it was equal at 10

and 15 min; with the results we could have a better understanding of disinfectants use in food

processing environments.

Keywords: Disinfectants; Minimum Inhibitory Concentration; Microbicidal efficacy test.

2
 1. Introduction

Regular cleaning is the most important action to control the microbial populations and prevent

the biofilm formation in the food industry. The cleanup program involves the removal of organic

waste from the food processing surfaces [1]. Cleaning can remove about 90% of bacteria found

on surfaces without killing them [1]. Therefore, it is essential that disinfection is performed after

cleaning, in order to prevent the binding of the bacterial cells to contact surfaces and reduce the

number of foodborne microorganisms to safe levels [2]. The disinfection process plays a vital

role in the food industry; there are different methods of disinfecting surfaces: chemical, physical

and biological. [2]. Chemical methods are commonly used for control of pathogens such as

Escherichia coli, Listeria monocytogenes, S. aureus and Salmonella, as well as spoilage

microorganisms such as P. aeruginosa [2,3].

There is a wide variety of disinfectants: QACs, halogen release agents, peracetic acid, hydrogen

peroxide (H2O2), organic extracts, etc. The evaluation of disinfectant effectiveness comprises

three steps: i) evaluation with planktonic cells, ii) evaluation with cells in carriers and iii)

evaluation in plants (in situ). The evaluation with planktonic cells is mainly based on three tests:

i) minimum inhibitory concentration (MIC) is considered the “gold standard” for determining the

susceptibility of microorganisms to antimicrobials [4,5]; ii) the phenol coefficient compares the

effectiveness of water-soluble disinfectants against the effectiveness of phenol, which is

considered a good disinfectant [6]; iii) disinfectant effectiveness is used to evaluate the

effectiveness in 30 s [6,7]. The aim of this study was to determinate the effectiveness and MIC of

15 disinfectants commonly used in the food industry in Mexico.

3
 2. Materials and methods

2.1 Bacterial strains, culture media and culture conditions

The microorganisms used in this study were Escherichia coli ATCC 11229, Salmonella enterica

serovar Choleraesuis ATCC 10708, Pseudomonas aeruginosa ATCC 15442 and Staphylococcus

aureus ATCC 6538, all used for testing antimicrobial agents [6]. Moreover, Staphylococcus

aureus ATCC 25923 was included as it is a biofilm-forming microorganism [3,8] and Listeria

monocytogenes ATCC 19111 was also used. The subcultures were prepared from stock cultures

stored at -20°C, by inoculation in trypticase soy broth (TSB; Bioxon, Le Pont de Claix, France)

and incubated at 37°C for 24 h in aerobic and static conditions.

2.2 Disinfectants tested

A total of 15 disinfectants were evaluated, 11 of which are registered in the National Sanitization

Foundation (NSF) International, one in the Organic Materials Review Institute, and the three

remaining disinfectants have been recently developed (Table 1). The disinfectants had different

active ingredients and were classified into the following groups: 3rd-generation QAC, 5 th-

generation QAC, halogen-releasing agents, hydrogen peroxide-based, mixtures of H2O2 and

organic acids, organic extract of grapefruit seeds and disinfectant-detergent acid anionic grease.

All the disinfectants were provided by CIP & GROUP S. de R. L. (Jalisco, Mexico) in

concentrated-dosage forms and were employed according to the technical information (Table 1);

the only excipient of the disinfectants tested was water. The disinfectant solutions were prepared

in sterile distilled water.

4
2.3 Microbicidal efficacy test

Bactericidal efficacy assays were performed according to the method of NMX-BB-040-SCFI-

1999 [7] and AOAC Official Method 960.09 [6]. Briefly, 100 µL of overnight cultures (~107

CFU ml-1) were mixed by vortexing for 15 s (Vortex Genie 2, Model G-560), with 9.9 ml

disinfectant solution. After 30 s, 100 µL of the assay mix was transferred to a new Eppendorf

tube with 900 µL of Dey/Engley (D/E; Becton, Dickinson and Company, Le Pont de Claix,

France) broth to neutralize the disinfectant activity. After 30 min of contact with D/E medium,

the number of surviving bacterial was estimated by standard plate counting on standard agar

(Becton Dickinson, Le Pont de Claix, France) and incubated at 37°C for 24 h, in aerobic

conditions. Each assay was realized in triplicated. The percentage of reduction was calculated

with the following formula:

(100)
= 100 −

Where S = surviving bacterial (CFU ml-1) and ACP = Aerobic counting plate initial (CFU ml-1).

The disinfectant was considered as effective when it demonstrated a 99.999% of bacterial

reduction. The mean of the temperature during the assays was 27 ± 1.0°C.

2.4 Minimum inhibitory concentration

The MIC was achieved according to the protocols of Clinical and Laboratory Standards Institute

(CLSI) guidelines [9] with some modifications. Briefly, eight new and sterile Eppendorf tubes

were numbered; 1 ml of sterile distilled water was added to tubes 2-8, 1 ml of disinfectant

solution was added to tubes 1 and 2 and serial dilutions were realized from tubes 2 to 8. After

that, bacterial suspension was adjusted to 0.5 McFarland standard, 1 ml was added to each of the

eight tubes and were mixed for 10 s by vortexing. After 5, 10 and 15 min of contact with the

5
disinfectant solution, 20 µl of mix was transferred to a new 96-well microtiter plate with 180 µl

of D/E broth. The microtiter plate was incubated for 48 h at 37°C under aerobic and static

conditions. The MIC was determined by glucose fermentation, which produces a change of color

in D/E broth. For P. aeruginosa, the MIC was estimated by optical density (OD) at 600 nm using

a Multiskan FC (Thermo Fisher Scientific Inc., Madison, WI, USA); the MIC was the

concentration with an OD in the range of the OD of the negative control plus three standard

deviations. The assays were enhanced at 26.3 ± 0.79°C; each assay was done in triplicate.

2.5 Statistical analysis

Statistical analysis was carried out using ANOVA, and the variances were examined by at least

significant difference test (LSD) test using Statgraphics Centurion XV.II software (Statpoint

Technologies, Inc., Warrenton, VA, USA).

6
 3. Results and discussion

In Mexico, there is little information about the regulation and evaluation of disinfectants used in

the food industry; there only exists a norm for the evaluation of disinfectant efficacy which is not

obligatory [7]. For this reason, the Mexican manufacturers of disinfectants take regulations and

methods from international organizations such as the AOAC International and countries such as

the United States. In order to give a better idea about the efficacy of 15 disinfectants commonly

used in the food industry in Mexico, their efficacy and MIC against six bacteria were compared.

Disinfectants are used in food processing environments to reduce microbial loads to reduce the

risk of acquiring a foodborne disease and extend the shelf life of the food products. The results of

the effectiveness of 15 disinfectants evaluated at concentrations established by the manufacturer,

on six microorganisms after 30 s of exposure are shown in the Fig. 1. Using this technique it was

observed that most disinfectants managed to reduce 99.999% of microorganisms in the

suspension. Hence, the disinfectants exhibited high efficacy in in vitro conditions. For the test,

the bacterial suspensions had a density ~7.72 ± 0.56 Log10 CFU ml-1, so the reduction rate

obtained corresponded to at least 5 Log10, which is acceptable bactericidal activity for

disinfectants at the evaluated concentration [6,10]. Disinfectants that showed differences in the

level of efficacy were C for E. coli ATCC 11229, S. aureus ATCC 25923 and L. monocytogenes

ATCC 19111, M against both strains of S. aureus and N against S. Choleraesuis 10708 and S.

aureus ATCC 25923. It is important to mention that the biofilm-forming microorganism S.

aureus ATCC 25923 showed resistance to two disinfectants (C and N) to which S. aureus ATCC

6538 was susceptible. Only for P. aeruginosa ATCC 15442, did all disinfectants have 99.999%

effectiveness (Fig. 1). In this research, the disinfectants seemed to have a broad spectrum of

7
activity against Gram-positive and Gram-negative bacteria. It has been recommended that

disinfectants for general use should be able to kill a wide range of common or pathogenic

microorganisms [11].

In the present research, the MIC was estimated after 5, 10 and 15 min of contact with the

disinfectant because the disinfection process in the food industry does not usually exceed 15 min.

Therefore, microorganisms in the food industry are not in contact with the disinfectant for 24 h,

as occurs in the original test [9,12]. In addition, the use of culture media in the test can affect the

disinfectant activity; as organic matter has long been identified as a factor that affects disinfectant

effectiveness [13]. QACs are extensively utilized due to their non-aggressive action and residual

activity, and hence could act to replace phenolic compounds; additionally, they can contribute to

control the microbial population on surfaces [14]. In this study, the fifth generation QACs

required a lower concentration to inhibit the growth of the Gram-negative bacteria evaluated,

compared with the disinfectant A (third generation QACs) (p<0.05) after 5 min of exposure (Fig.

2A). Hence, for Gram-positive bacteria, a lower concentration of disinfectant A was required to

inhibit their development in comparison with Gram-negative bacteria for the three exposure times

evaluated (p<0.05) (Fig. 2). The effectiveness of QACs increases according to the generation;

third generation QACs are mixtures of equal quantities of first and second generation QACs, and

fifth generation QACs are mixtures of second and fourth generation QACs [15,16]. In addition,

the C-chain length is important in the antimicrobial activity of QACs: the bacterial resistance

increases with their length [17]. The results obtained with third generation QACs differed from

those reported by Deshaies et al. [15]; however, the results for fifth generation QACs were

similar to their results obtained using fourth generation QACs. Surprisingly, E. coli ATCC

11229, S. Choleraesuis ATCC 10708 and P. aeruginosa ATCC 15442 showed more resistance to
8
third generation QACs in comparison with S. aureus. However, it is known that the resistance to

QACs is not exclusive to S. aureus; in other microorganisms such as E. coli and P. aeruginosa

the cross-resistance seems to be mediated by non-specific multidrug efflux pumps encoded by

various qac genes located frequently on plasmids [18–21]. Moreover, microorganism resistance

to disinfectants is mediated by multiple resistance mechanisms at the same time, such as cell wall

changes, gene acquisition, efflux pumps and others [22]. It is noteworthy that for disinfectant C

we do not found an MIC within the range in which is used in the food industry against S. aureus

ATCC 25923. Therefore, solutions at different concentrations of disinfectant C were prepared

and we found that five times more than the recommended concentration was required to inhibit to

S. aureus ATCC 25923 in a period of 5 min (Fig. 2A) and 2.5 times more after 10 and 15 min of

exposure (Fig. 2B-C). This is related to the fact that QACs have greater effectiveness at basic pH,

than in an acid medium [16]. Disinfectants N and O, in the most cases required the same

concentration to inhibit Gram-positive and Gram-negative microorganisms in most cases,

affected by the exposure time; nevertheless, the concentration was bigger than the MIC of A and

B (Fig. 2).

Halogen-releasing agents represent an important group of disinfectants used in the food industry;

chlorination is an effective process for food processing environments which has been used since

the 19th century [23]. Halogen-releasing agents act by oxidizing the proteins and hence destroying

the cellular activity [11]. The MIC for chlorine from sodium hypochlorite for the microorganisms

tested was between 50 and 125 µg ml-1, while for chlorine dioxide the concentrations fluctuated

between 19 and 125 µg ml-1. In a similar study, the concentration of chlorine reported to inhibit S.

aureus ATCC 25923, E. coli ATCC 25922 and P. aeruginosa ATCC 27853 after 5 min of

contact was 82 µg ml-1 [15]. However, the most effective halogen-releasing agent was
9
disinfectant F (iodine-based) (p<0.05), which inhibited the microorganisms at concentrations

between 1.56 and 6.25 µg ml-1 at different exposure times (Fig. 2). In line with this, Joseph et al.

[24] found that 2.4 x 106 cells ml-1 of S. Weltevreden were completely killed by exposure to 10

µg ml-1 Cl2 for 10 min; on the other hand, 1.9 x 106 cells ml-1 were killed after 5 min exposure to

10 µg ml-1 I2.

H2O2 is a powerful oxidizing agent, easily handled and non-toxic, applied on non-critical items. It

is a mainstay in metal surface treatment. It is also used for treating huge volumes of wastewater,

and in water and food disinfection applications [11]. Disinfectant K (H2O2), failed to achieve an

effectiveness of 99.999% against S. aureus ATCC 25923 and S. aureus ATCC 6538 in the

microbicidal efficacy test. This could be because the MIC for both strains was higher than the

concentration recommended by the manufacturer. The MIC of K for S. aureus ATCC 25923 was

10000 and 7500 µg ml-1 at 5 and 10 min of exposure, respectively, while for S. aureus ATCC

6538 16800 and 13800 µg ml-1 were required to obtain the same results for the same periods of

exposure (p<0.05) (Fig. 2A-B). Based on these results it can be emphasized that each strain of the

same species in the planktonic stage has a different behavior. Besides the fact that S. aureus

ATCC 25923 is a biofilm-former, it does not provide an advantage over S. aureus ATCC 6538 in

the planktonic stage. However, in the evaluations with the disinfectants C and D, higher

concentrations were required (p<0.05) to inhibit S. aureus ATCC 25923 in comparison with the

results for S. aureus ATCC 6538 (Fig. 2).

Mixtures of H2O2 and organic acids, allow stabilization and accelerate the biological activity of

H2O2, which has weak and slow microbicidal activity [23]. The MIC was lower for H compared

with G (p<0.05) at the three exposure times (Fig. 2). In addition, the disinfectant I had an MIC
10
higher than J (p<0.05), after 5 min of exposure (Fig. 2A). However, at 10 and 15 min the MIC for

both disinfectants tended to be similar (p>0.05) (Fig. 2B-C). H2O2 showed the higher MIC; it was

reported that 5000 µg ml-1 of that disinfectant only reduces 2 Log CFU ml-1 of Aeromonas

hydrophila after 5 min [25] and the MIC for E. coli and S. aureus was 2505 and 938 µg ml-1,

respectively [13]. The disinfectants with peracetic acid were most effective and had a lower MIC

than H2O2. The mean MIC for S. aureus isolated from food and food environments was 78.13 µg

ml-1 [22], which is higher than our results, and could be related to the increment of resistance in

wild strains, due to the exposure to sublethal concentrations in food processing environments.

In conclusion, 15 disinfectants were evaluated and 14 of these showed an MIC was between 1.14

and 128 times lower than the recommended concentration and within that concentration

established in CFR 21 178.1010 [9]. In addition, for all disinfectants the MIC at 5 min was two to

four times greater than the concentration with the same effect at 10 min; moreover, in most cases

there was no difference in the MIC at 10 and 15 min. However, in the test conditions, organic or

inorganic residues of food processing environments, water harness, the effect of the surface, and

other factors that affect the disinfectant efficacy were not considered [14]. This study

demonstrated the efficacy of different disinfectants; their effectiveness and MIC depend on the

microorganism tested, and MIC only represents the bacteriostatic, not the bactericidal

concentration. The information obtained can be used for risk assessment in the food industry and

in the validation of sanitization procedures. Furthermore, disinfectants must be used at the

established concentrations as inappropriate use can contribute to microbial resistance.

11
Declarations

Funding: The authors thank CIP & GROUP S. de R. L. for financing for this project, particularly

Ing. Jesús Casillas (General Director) and Ing. Sergio Miramontes (Technical Assessor). We also

thank the National Council on Science and Technology of Mexico for the scholarship granted to

Maricarmen Iñiguez Moreno [No. 287793].

Ethical Approval: Not required

Competing interests: The authors declare that they have no conflict of interest.

12
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15
Fig. 1. Disinfectants effectiveness. The graph represents the reduction percentage of each

microorganism after 30 s of exposure to the 15 disinfectants. E. coli ATCC 11229 ■; S.

Choleraesuis ATCC 10708 ■; P. aeruginosa ATCC 15442 ■; S. aureus ATCC 25923 ■; S.

aureus ATCC 6538 ■ and L. monocytogenes ATCC 19111 ■. L1 and L8 are the disinfectant L at

1000 and 8000 µg ml-1, respectively.

16
Fig. 2. MIC of disinfectants at three time exposure. Each graphic represents the MIC of 15

disinfectants evaluated at three times exposure A) MIC at 5 min, B) MIC at 10 min and C) MIC

15 min of time exposure. The microorganism used in this test were E. coli ATCC 11229 ■; S.

Choleraesuis ATCC 10708 ■; P. aeruginosa ATCC 15442 ■; S. aureus ATCC 25923 ■; S.

aureus ATCC 6538 ■ and L. monocytogenes ATCC 19111 ■.

17
Table 1. Classification of disinfectants
Manufacturer recommended Registration
Disinfectant Active compounds*
concentrations (µg ml-1) number
A Quaternary ammonium-based 3rd generation 200 144383
B Quaternary ammonium-based 5th generation 400 146540
Quaternary ammonium-based 3rd generation
C 200 146539
plus phosphoric acid
D Sodium hypochlorite 200 146538
E Chlorine dioxide 4000 †
F Iodine 25 144382
G 80 ent-2949
Hydrogen peroxide and peracetic acid
H 375 144380
I Hydrogen peroxide, peracetic acid and acetic 53 146655
J acid 200 144381
K Hydrogen peroxide 20000 †
L Hydrogen peroxide and enzymes 1000 and 8000 †
M Organic extract of grapefruit seeds 2000 144378
N Mixed of anionic surfactant and phosphoric, 2000 146654
O succinic and octanoic acids 2000 144379
* The only excipient used in the formulations is water.
† Recently developed.

18