Comparative mycobactericidal efficacy of chemical disinfectants in suspension and carrier tests

The efficacy of nine disinfectants on Mycobacterium smegmatis was tested in the presence of sputum, using quantitative suspension and carrier tests. Glutaraldehyde, povidone iodine, and chlorhexidine gluconate produced at least a 6-log10 reduction in CFU in all tests. Four disinfectants (sodium dichloroisocyanurate, phenol, ethanol, and sodium hypochlorite) were not as effective in the carrier tests as in the suspension tests; this difference ranged from a 1- to a 5-log10 reduction in CFU. The efficacy of ethanol and sodium hypochlorite was further reduced (3- and 1-log10 reductions in CFU, respectively) in the presence of sputum. The quaternary ammonium compound and iodophor were ineffective in all tests. The findings of this study demonstrate the need for a quantitative carrier test such as the one presented here.     

Inactivation of Bacillus anthracis spores by liquid biocides in the presence of food residue

Biocide inactivation of Bacillus anthracis spores in the presence of food residues after a 10-min treatment time was investigated. Spores of nonvirulent Bacillus anthracis strains 7702, ANR-1, and 9131 were mixed with water, flour paste, whole milk, or egg yolk emulsion and dried onto stainless-steel carriers. The carriers were exposed to various concentrations of peroxyacetic acid, sodium hypochlorite (NaOCl), or hydrogen peroxide (H(2)O(2)) for 10 min at 10, 20, or 30 degrees C, after which time the survivors were quantified. The relationship between peroxyacetic acid concentration, H(2)O(2) concentration, and spore inactivation followed a sigmoid curve that was accurately described using a four-parameter logistic model. At 20 degrees C, the minimum concentrations of peroxyacetic acid, H(2)O(2), and NaOCl (as total available chlorine) predicted to inactivate 6 log(10) CFU of B. anthracis spores with no food residue present were 1.05, 23.0, and 0.78%, respectively. At 10 degrees C, sodium hypochlorite at 5% total available chlorine did not inactivate more than 4 log(10) CFU. The presence of the food residues had only a minimal effect on peroxyacetic acid and H(2)O(2) sporicidal efficacy, but the efficacy of sodium hypochlorite was markedly inhibited by whole-milk and egg yolk residues. Sodium hypochlorite at 5% total available chlorine provided no greater than a 2-log(10) CFU reduction when spores were in the presence of egg yolk residue. This research provides new information regarding the usefulness of peroxygen biocides for B. anthracis spore inactivation when food residue is present. This work also provides guidance for adjusting decontamination procedures for food-soiled and cold surfaces.     

Efficacy of a variety of disinfectants against Listeria spp.

The efficacy of 14 disinfectants against Listeria innocua and two strains of Listeria monocytogenes in the presence of organic matter was studied. Quantitative efficacy tests were used. Many of the disinfectants tested were not as effective on Listeria spp. when the test organisms were dried onto the surface of steel disks (carrier tests) as they were when the organisms were placed in suspension (suspension test). The presence of whole serum and milk (2% fat) further reduced the disinfectant capacities of most of the formulations studied. Only three disinfectants (povidone-iodine, chlorhexidine gluconate, and glutaraldehyde) were effective in the carrier test in the presence of serum; however, all three were ineffective when challenged with milk (2% fat). Only one solution, sodium dichloroisocyanurate, was effective in the presence of milk. All but four formulations (chloramine-T, phosphoric acid, an iodophor, and formaldehyde) were effective in the suspension tests, regardless of the organic load. L. monocytogenes was observed to be slightly more resistant to disinfection than L. innocua was. There was no difference in disinfectant susceptibility between the two strains of L. monocytogenes. These findings emphasize the need for caution in selecting an appropriate disinfectant for use on contaminated surfaces, particularly in the presence of organic material.     

Efficacy of a variety of disinfectants against Listeria spp

The efficacy of 14 disinfectants against Listeria innocua and two strains of Listeria monocytogenes in the presence of organic matter was studied. Quantitative efficacy tests were used. Many of the disinfectants tested were not as effective on Listeria spp. when the test organisms were dried onto the surface of steel disks (carrier tests) as they were when the organisms were placed in suspension (suspension test). The presence of whole serum and milk (2% fat) further reduced the disinfectant capacities of most of the formulations studied. Only three disinfectants (povidone-iodine, chlorhexidine gluconate, and glutaraldehyde) were effective in the carrier test in the presence of serum; however, all three were ineffective when challenged with milk (2% fat). Only one solution, sodium dichloroisocyanurate, was effective in the presence of milk. All but four formulations (chloramine-T, phosphoric acid, an iodophor, and formaldehyde) were effective in the suspension tests, regardless of the organic load. L. monocytogenes was observed to be slightly more resistant to disinfection than L. innocua was. There was no difference in disinfectant susceptibility between the two strains of L. monocytogenes. These findings emphasize the need for caution in selecting an appropriate disinfectant for use on contaminated surfaces, particularly in the presence of organic material.     

Effects of disinfectants against norovirus virus‐like particles predict norovirus inactivation

Human noroviruses (NoVs) are a major cause of epidemic and sporadic acute gastroenteritis worldwide. Public and personal hygiene is one of the most important countermeasures for preventing spread of NoV infection. However, no a practicable cell culture system for NoV had been developed, initial tests of the virucidal effectiveness of anti‐NoV disinfectants and sanitizers have been performed using surrogate viruses. In this study, NoV virus‐like particles (VLPs) were used as a new surrogate for NoVs and a method for evaluating NoV inactivation using them developed. This method is based on morphological changes in VLPs after treatment with sodium hypochlorite. VLP specimens were found to become deformed and degraded in a concentration‐dependent manner. Based on these results, the effects of sodium hypochlorite on VLPs were classified into four phases according to morphological changes and number of particles. Using the criteria thus established, the efficacy of ethanol, carbonates and alkali solutions against VLPs was evaluated. Deformation and aggregation of VLPs were observed after treatment with these disinfectants under specific conditions. To determine the degradation mechanism(s), VLPs were examined by SDS‐PAGE and immunoblotting after treatment with sodium hypochlorite and ethanol. The band corresponding to the major capsid protein, VP1, was not detected after treatment with sodium hypochlorite at concentrations greater than 500 ppm, but remained after treatment with ethanol. These results suggest that VLPs have excellent potential as a surrogate marker for NoVs and can be used in initial virucidal effectiveness tests to determine the mechanism(s) of chemical agents on NoVs.     

Development of a standard test to assess the resistance of Staphylococcus aureus biofilm cells to disinfectants

A standardized disinfectant test for Staphylococcus aureus cells in biofilms was developed. Two disinfectants, the membrane-active compound benzalkonium chloride (BAC) and the oxidizing agent sodium hypochlorite, were used to evaluate the biofilm test. S. aureus formed biofilms on glass, stainless steel, and polystyrene in a simple system with constant nutrient flow that mimicked as closely as possible the conditions used in the current standard European disinfectant test (EN 1040). The biofilm that was formed on glass contained cell clumps and extracellular polysaccharides. The average surface coverage was 60%, and most (92%) of the biofilm cells were viable. Biofilm formation and biofilm disinfection in different experiments were reproducible. For biofilms exposed to BAC and hypochlorite the concentrations needed to achieve 4-log killing were 50 and 600 times higher, respectively, than the concentrations needed to achieve this level of killing with the European phase 1 suspension test cells. Our results show that a standardized disinfectant test for biofilm cells is a useful addition to the current standard tests.     

Fluorescent assay based on resazurin for detection of activity of disinfectants against bacterial biofilm

A new, quick method, using the resazurin dye test as a bacterial respiration indicator, has been developed to assay the antibacterial activity of various substances used as disinfectants against bacterial biofilm growth on clinical devices. Resazurin was used to measure the presence of active biofilm bacteria, after adding disinfectant, in relation to a standard curve generated from inocula in suspension of the same organism used to grow the biofilm. The biofilm was quantified indirectly by measuring the fluorescent, water-soluble resorufin product produced when resazurin is reduced by reactions associated with respiration. Four products used as disinfectants and the biofilm growth of five bacterial species on carriers made of materials commonly found in clinical devices were studied. Under test conditions, chlorhexidine, NaOCl, ethanol, and Perasafe at concentrations of 0.2, 0.01, 350, and 0.16 mg/ml, respectively, all produced 5-log reductions in biofilm cell numbers on the three different carriers. The redox-driven test depends on bacterial catabolism, for which reason resazurin reduction produces an analytic signal of the bacterial activity in whole cells, and therefore could be used for determining disinfectant efficacy in an assay based on the metabolic activity of microorganisms grown as biofilm or in suspension.     

Assessment of the efficacy of disinfectants on surfaces

The Literature on testing the efficacy of disinfectants covers a century. Most predominant and standardized are the so called suspension tests that allow for the quantitative estimation of the microbicidal activity (log reduction factors) of disinfectants on test organisms suspended in solutions of these products.Since the outcome of suspension tests might be a poor predictor for the efficacy of a disinfectant under practical circumstances, especially with regard to bacteria attached to surfaces, a variety of test procedures have been designed to mimic those conditions. Within the framework of CEN/TC 216 a quantitative surface test has been developed to assess the activity of disinfectants on bacteria or fungi attached to steenless steel surfaces. Preliminary data suggest that covering a dried inoculum with disinfectant without any further mechanical action to improve contact between organisms and disinfectant, will usually result in lower reduction factors than those obtained with suspension tests. Comparative testing further suggests that by applying mechanical action, with the effect of resuspending cells in the liquid on the surface,—similar to mopping, brushing etc.— will result in higher reduction rates. Although not unexpected these findings emphasize the importance of designing test methods based on practical applications of disinfectants.     

Development of a Standard Test To Assess the Resistance of Staphylococcus aureus Biofilm Cells to Disinfectants

A standardized disinfectant test for Staphylococcus aureus cells in biofilms was developed. Two disinfectants, the membrane-active compound benzalkonium chloride (BAC) and the oxidizing agent sodium hypochlorite, were used to evaluate the biofilm test. S. aureus formed biofilms on glass, stainless steel, and polystyrene in a simple system with constant nutrient flow that mimicked as closely as possible the conditions used in the current standard European disinfectant test (EN 1040). The biofilm that was formed on glass contained cell clumps and extracellular polysaccharides. The average surface coverage was 60%, and most (92%) of the biofilm cells were viable. Biofilm formation and biofilm disinfection in different experiments were reproducible. For biofilms exposed to BAC and hypochlorite the concentrations needed to achieve 4-log killing were 50 and 600 times higher, respectively, than the concentrations needed to achieve this level of killing with the European phase 1 suspension test cells. Our results show that a standardized disinfectant test for biofilm cells is a useful addition to the current standard tests.     

Sporicidal effect of disinfectants on Bacillus cereus isolated from the milk processing environment

The sporicidal efficacy of sodium hypochlorite and a combination of peracetic acid and hydrogen peroxide on Bacillus cereus spores isolated from the milk processing environment was examined using the European Suspension Test and by a surface disinfection test on stainless steel and rubber. The results of the laboratory tests were compared with field trials in a milking installation. In general, it was difficult to obtain consistent results, as the repeatability and reproducibility of the tests were found to vary according to the test strain, spore suspension preparation, disinfectant test solution, organic load, contact time and temperature. The sporulation medium used to obtain spores influenced the sporicidal effect considerably. To overcome this problem a standard method for preparation of spore suspensions should be prescribed. The various disinfectants were more effective in suspensions than on surfaces and in field trials. For the suspension tests SE values ranging from 1.0 to 3.0 were reached within 10 min at 50°C, depending on the disinfectant used. Sodium hypochlorite-based products were most effective. The activity on spores on surfaces and in field trials was limited. In surface tests reductions of 0.4–0.8 were observed within 10 min at 50°C depending on the type of surface. The SE values obtained for rubber were lower compared with stainless steel. The decrease in spore levels found in the milking installation was comparable with the surface experiments, i.e. 0.4–1.0. It is important to develop standard test procedures to assess the sporicidal efficacy of disinfectants used in food hygiene. Surface tests should be included to reflect the in-use conditions more closely and minimum standards should be determined for both suspension tests and surface tests.     

Chemical disinfection of high-consequence transboundary animal disease viruses on nonporous surfaces

Disinfection is a critical part of the response to transboundary animal disease virus (TADV) outbreaks by inactivating viruses on fomites to help control infection. To model the inactivation of TADV on fomites, we tested selected chemicals to inactivate Foot and Mouth Disease virus (FMDV), African Swine Fever virus (ASFV), and Classical Swine Fever virus (CSFV) dried on steel and plastic surfaces. For each of these viruses, we observed a 2 to 3 log reduction of infectivity due to drying alone. We applied a modified surface disinfection method to determine the efficacy of selected disinfectants to inactivate surface-dried high-titer stocks of these three structurally different TADV. ASFV and FMDV were susceptible to sodium hypochlorite (500 and 1000 ppm, respectively) and citric acid (1%) resulting in complete disinfection. Sodium carbonate (4%), while able to reduce FMDV infectivity by greater than 4-log units, only reduced ASFV by 3 logs. Citric acid (2%) did not totally inactivate dried CSFV, suggesting it may not be completely effective for disinfection in the field. Based on these data we recommend disinfectants be formulated with a minimum of 1000 ppm sodium hypochlorite for ASFV and CSFV disinfection, and a minimum of 1% citric acid for FMDV disinfection.     

Antimicrobial activity and effectiveness of a combination of sodium hypochlorite and hydrogen peroxide in killing and removing Pseudomonas aeruginosa biofilms from surfaces

AIMS: To evaluate both the antimicrobial activity and the effectiveness of a combination of sodium hypochlorite and hydrogen peroxide (Ox-B) for killing Pseudomonas aeruginosa ATCC 19142 cells and removing P. aeruginosa biofilms on aluminum or stainless steel surfaces. METHODS AND RESULTS: Pseudomonas aeruginosa biofilms were developed in tryptic soy broth containing vertically suspended aluminium or stainless steel plates. Biofilms were exposed to a mixed sodium hypochlorite and hydrogen peroxide solution as a sanitizer for 1, 5 and 20 min. The sanitizer was then neutralized, the cells dislodged from the test surfaces, and viable cells enumerated. Cell morphologies were determined using scanning (SEM) and transmission electron microscopy (TEM). Cell viability was determined by confocal scanning laser microscopy (CSLM). Biofilm removal was monitored by Fourier transform infrared (FTIR) spectrophotometry. Cell numbers were reduced by 5-log to 6-log after 1 min exposure and by 7-log after 5 min exposure to Ox-B. No viable cells were detected after a 20 min exposure. Treatment with equivalent concentrations of sodium hypochlorite reduced viable numbers by 3-log to 4-log after 1 min exposure and by 4-log to 6-log after 5 min, respectively. A 20 min exposure achieved a 7-log reduction. Hydrogen peroxide at test concentration treatments showed no effect. FTIR analysis of treated pseudomonad biofilms on aluminium or stainless steel plates showed either a significant reduction or complete removal of biofilm material after a 5 min exposure to the mixed sodium hypochlorite and hydrogen peroxide solution. SEM and TEM images revealed damage to cell wall and cell membranes. CONCLUSIONS: A combination of sodium hypochlorite and hydrogen peroxide effectively killed P. aeruginosa cells and removed biofilms from both stainless steel and aluminium surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of sodium hypochlorite and hydrogen peroxide can be used as an alternative disinfectant and/or biofilm remover of contaminated food processing equipment.     

Wide variation in effectiveness of laboratory disinfectants against bacteriophages

Aims: The purpose of this study was to identify an effective disinfectant for the inactivation of the bacteriophages (phages) being used in our laboratory, as published studies on phage inactivation are far from unanimous in their conclusions. Methods and Results: The phages studied were three closely related strains of Myoviridae and three strains of Siphoviridae. Three disinfectants which are used commonly in microbiology laboratories were evaluated: Virkon (1%), ethanol (75%) and sodium hypochlorite (2500 ppm available chlorine). The most effective of these was Virkon, which inactivated all six phages rapidly. Ethanol was effective against the Myoviridae but had little effect on the Siphoviridae. Sodium hypochlorite was the least effective of the disinfectants evaluated. Conclusions: The findings of this study demonstrate a wide diversity in the effectiveness of disinfectants tested for inactivation of phages. Significance and Impact of the Study: Of the disinfectants tested Virkon is the most suitable choice for those unable to carry out disinfection validation studies, or where a broad spectrum disinfectant against phages is required. All of the phages in this study showed resilience to inactivation by sodium hypochlorite, and therefore this disinfectant is an unwise choice for use against phage without first assessing its effectiveness.     

The effect of small amounts of sodium hypochlorite on the growth of Earl's "L" cells in suspension growth

Of several candidate disinfectants for use in tissue culture work, especially suspension cultures, sodium hypochlorite solution was selected to test its effect on growing cells. Metabolizing cells reduce, sodium hypochlorite oxidizes ; therefore NaOCl leakage into such systems must be neutralized with no untoward effects on the cells. Dilutions of routine disinfectant-grade sodium hypochlorite were tested against cell cultures. Those exposed to 15.62 to 31.25 ppm of NaOCl grew with no apparent cell damage.     

Effectiveness of disinfectants in killing Enterobacter sakazakii in suspension, dried on the surface of stainless steel, and in a biofilm

The effectiveness of 13 disinfectants used in hospitals, day-care centers, and food service kitchens in killing Enterobacter sakazakii in suspension, dried on the surface of stainless steel, and in biofilm was determined. E. sakazakii exhibited various levels of resistance to the disinfectants, depending on the composition of the disinfectants, amount and type of organic matrix surrounding cells, and exposure time. Populations of planktonic cells suspended in water (7.22 to 7.40 log CFU/ml) decreased to undetectable levels (<0.30 log CFU/ml) within 1 to 5 min upon treatment with disinfectants, while numbers of cells in reconstituted infant formula were reduced by only 0.02 to 3.69 log CFU/ml after the treatment for 10 min. The presence of infant formula also enhanced the resistance to the disinfectants of cells dried on the surface of stainless steel. The resistance of cells to disinfectants in 6-day-old and 12-day-old biofilms on the surface of stainless steel was not significantly different. The overall order of efficacy of disinfectants in killing E. sakazakii was planktonic cells > cells inoculated and dried on stainless steel > cells in biofilms on stainless steel. Findings show that disinfectants routinely used in hospital, day-care, and food service kitchen settings are ineffective in killing some cells of E. sakazakii embedded in organic matrices.     

Fungicidal effects of chemical disinfectants, UV light, desiccation and heat on the amphibian chytrid Batrachochytrium dendrobatidis

The efficacy of a number of disinfection treatments was tested on in vitro cultures of the fungus Batrachochytrium dendrobatidis, the causative agent of chytridiomycosis in amphibians. The aim was to evaluate the fungicidal effects of chemical disinfectants, sterilising ultraviolet (UV) light, heat and desiccation, using methods that were feasible for either disinfection in the field, in amphibian husbandry or in the laboratory. The chemical disinfectants tested were: sodium chloride, household bleach (active ingredient: sodium hypochlorite), potassium permanganate, formaldehyde solution, Path-X agricultural disinfectant (active ingredient: didecyl dimethyl ammonium chloride, DDAC), quaternary ammonium compound 128 (DDAC), Dithane, Virkon, ethanol and benzalkonium chloride. In 2 series of experiments using separate isolates of B. dendrobatidis, the fungicidal effect was evaluated for various time periods and at a range of chemical concentrations. The end point measured was death of 100% of zoospores and zoosporangia. Nearly all chemical disinfectants resulted in 100%, mortality for at least one of the concentrations tested. However, concentration and time of exposure was critical for most chemicals. Exposure to 70% ethanol, 1 mg Virkon ml(-1) or 1 mg benzalkonium chloride ml(-1) resulted in death of all zoosporangia after 20 s. The most effective products for field use were Path-X and the quaternary ammonium compound 128, which can be used at dilutions containing low levels (e.g. 0.012 or 0.008%, respectively) of the active compound didecyl dimethyl ammonium chloride. Bleach, containing the active ingredient sodium hypochlorite, was effective at concentrations of 1% sodium hypochlorite and above. Cultures did not survive complete drying, which occurred after <3 h at room temperature. B. dendrobatidis was sensitive to heating, and within 4 h at 37 degrees C, 30 min at 47 degrees C and 5 min at 60 degrees C, 100% mortality occurred. UV light (at 1000 mW m(-2) with a wavelength of 254 nm) was ineffective at killing B. dendrobatidis in culture.     

Inactivation of Bacillus anthracis Spores by Liquid Biocides in the Presence of Food Residue

Biocide inactivation of Bacillus anthracis spores in the presence of food residues after a 10-min treatment time was investigated. Spores of nonvirulent Bacillus anthracis strains 7702, ANR-1, and 9131 were mixed with water, flour paste, whole milk, or egg yolk emulsion and dried onto stainless-steel carriers. The carriers were exposed to various concentrations of peroxyacetic acid, sodium hypochlorite (NaOCl), or hydrogen peroxide (H₂O₂) for 10 min at 10, 20, or 30°C, after which time the survivors were quantified. The relationship between peroxyacetic acid concentration, H₂O₂ concentration, and spore inactivation followed a sigmoid curve that was accurately described using a four-parameter logistic model. At 20°C, the minimum concentrations of peroxyacetic acid, H₂O₂, and NaOCl (as total available chlorine) predicted to inactivate 6 log₁₀ CFU of B. anthracis spores with no food residue present were 1.05, 23.0, and 0.78%, respectively. At 10°C, sodium hypochlorite at 5% total available chlorine did not inactivate more than 4 log₁₀ CFU. The presence of the food residues had only a minimal effect on peroxyacetic acid and H₂O₂ sporicidal efficacy, but the efficacy of sodium hypochlorite was markedly inhibited by whole-milk and egg yolk residues. Sodium hypochlorite at 5% total available chlorine provided no greater than a 2-log₁₀ CFU reduction when spores were in the presence of egg yolk residue. This research provides new information regarding the usefulness of peroxygen biocides for B. anthracis spore inactivation when food residue is present. This work also provides guidance for adjusting decontamination procedures for food-soiled and cold surfaces.     

Inactivation of laboratory animal RNA-viruses by physicochemical treatment

Eight commonly used chemical disinfectants and physical treatments (UV irradiation and heating) were applied to both enveloped RNA viruses (Sendai virus, canine distemper virus) and unenveloped RNA viruses (Theiler's murine encephalomyelitis virus, reo virus type 3) to inactivate infectious virus particles. According to the results, alcohols (70% ethanol, 50% isopropanol), formaldehyde (2% formalin), halogen compounds (52ppm iodophor, 100ppm sodium hypochlorite), quaternary ammonium chloride (0.05% benzalkonium chloride) and 1% saponated cresol showed virucidal effects giving more than 99.95% reduction in the infectivity of virus samples of Sendai virus and canine distemper after 10 minutes exposure. There was no significant difference in the effects on the two enveloped RNA viruses. The susceptibility of unenveloped RNA viruses to chemical disinfectants and physical treatments differed greatly from the enveloped viruses. The two unenveloped viruses showed distinct resistance to 50% isopropanol, 2% formalin, 1% saponated cresol and to physical treatments (heating at 45, 56, 60 degrees C, and UV irradiation). These results indicate that using physicochemical methods to inactivate RNA viruses in laboratory animal facilities should be considered in accordance with the characteristics of the target virus. For practical purposes in disinfecting enveloped RNA viruses, 70% ethanol, 0.05% quaternary ammonium chloride and 1% saponated cresol diluted in hot water (greater than 60 degrees C) are considered as effective as UV irradiation. For unenveloped RNA viruses, halogen compounds, more than 1,000 ppm sodium hypochlorite or 260 ppm iodophor are recommended over a period of 10 minutes for disinfecting particles, although these compounds result in an oxidation problem with many metals.     

Comparative susceptibility of Salmonella Typhimurium biofilms of different ages to disinfectants

There is a general consensus that with increasing age a biofilm shows increased resistance to antimicrobials. In this study the susceptibility of 3-, 5- and 7-day-old Salmonella enterica serovar Typhimurium biofilms to disinfectants was evaluated. It was hypothesized that 7-day-old biofilms would be more resistant to disinfectants compared to 3- and 5-day-old biofilms. Biofilms were formed using the MBEC? system and treated with six chemical disinfectants for 1 and 5 min. Four disinfectants at the highest concentration available showed 100% reduction in viable cells from all ages of biofilms after exposure for 5 min, and ethanol at 70% v/v was the least effective against biofilms, followed by chlorhexidine gluconate (CG). At the recommended user concentrations, only sodium hypochlorite showed 100% reduction in viable cells from all ages of biofilms. Benzalkonium chloride and CG were the least effective against biofilms, followed by quaternary ammonium compound which only showed 100% reduction in viable cells from 5-day-old biofilms. Overall, the results from this study do not display enhanced resistance in 7-day-old biofilms compared to 3- and 5-day-old biofilms. It is concluded that under the conditions of this study, the age of biofilm did not contribute to resistance towards disinfectants. Rather, the concentration of disinfectant and an increased contact time were both shown to play a role in successful sanitization.     

The testing of disinfectants

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.