Development of a rhodamine-based fluorescent probe for ATP detection for potential applications in meat freshness assessment.

Adenosine triphosphate (ATP) plays a vital role in physiological processes and is an essential indicator of microbial content in food. Herein, a new sensitive, rapid and water-soluble probe for ATP detection was developed. Rhodamine B and pentaethylenehexamine were employed to design and synthesise the probe rhodamine-pentaethylenehexamine (RP) for selective ATP detection. The synthesised probe RP was characterized using Fourier transform infrared, NMR and dynamic light scattering size distributions. Upon the addition of ATP, the probe exhibited a distinct change in fluorescence intensity, with fluorescence emission at 580 nm. A linear relationship was observed between fluorescence intensity and ATP concentrations at 0-50 µmol/L, with a limit of detection of 10.97 × 10-9 mol/L. The results of the zeta potential and molecular dynamics simulation demonstrated that the detection mechanism of the probe RP is associated with the electrostatic adsorption interaction between the multi-positively charged sites of RP and the negatively charged triphosphate structure of ATP. Our study provides new insights into improving charge site identification in small molecule detection. Furthermore, the successful detection of ATP on meat surfaces indicates that RP has the potential to assess meat freshness.

Size-classified monitoring of ATP bioluminescence for rapid assessment of biological distribution in airborne particulates.

The COVID-19 pandemic ignited massive research into the rapid detection of bioaerosols. In particular, nanotechnology-based detection strategies are proposed as alternatives because of issues in bioaerosol enrichment and lead time for molecular diagnostics; however, the practical implementation of such techniques is still unclear due to obstacles regarding the large research and development effort and investment for the validation. The use of adenosine triphosphate (ATP) bioluminescence (expressed as relative luminescence unit (RLU) per unit volume of air) of airborne particulate matter (PM) to determine the bacterial population as a representative of the total bioaerosols (viruses, bacteria, and fungi) has been raised frequently because of the high reponse speed, resolution, and compatibility with culture-based bioaerosol monitoring. On the other hand, additional engineering attempts are required to confer significance because of the size-classified (bioluminescence for different PM sizes) and specific (bioluminescence per unit PM mass) biological risks of air for providing proper interventions in the case of airborne transmission. In this study, disc-type impactors to cut-off aerosols larger than 1 µm, 2.5 µm, and 10 µm were designed and constructed to collect PM1, PM2.5, and PM10 on sampling swabs. This engineering enabled reliable size-classified bioluminescence signals using a commercial ATP luminometer after just 5 min of air intake. The simultaneous operations of a six-stage Andersen impactor and optical PM spectrometers were conducted to determine the correlations between the resulting RLU and colony forming unit (CFU; from the Andersen impactor) or PM mass concentration (deriving specific bioluminescence).

Assessment of postmanual cleaning adenosine triphosphate tests to prevent the use of contaminated duodenoscopes and linear echoendoscopes: the DETECT study.

BACKGROUND AND AIMS: We investigated whether the use of postmanual cleaning adenosine triphosphate (ATP) tests lowers the number of duodenoscopes and linear echoendoscopes (DLEs) contaminated with gut flora. METHODS: In this single-center before-and-after study, DLEs were ATP tested after cleaning. During the control period, participants were blinded to ATP results: ATP-positive DLEs were not recleaned. During the intervention period, ATP-positive DLEs were recleaned. DLEs underwent microbiologic sampling after high-level disinfection (HLD) with participants blinded to culture results. RESULTS: Using 15 endoscopes of 5 different DLE types, we included 909 procedures (52% duodenoscopes, 48% linear echoendoscopes). During the intervention period, the absolute rate of contamination with gut flora was higher (16% vs 21%). The main analysis showed that contamination was less likely to occur in the intervention period (odds ratio, .32; 95% credible interval [CI], .12-.85). A secondary analysis showed that this effect was based on 1 particular duodenoscope type (estimated probability, 39% [95% CI, 18%-64%] vs 9% [95% CI, 2%-21%]), whereas no effect was seen in the other 4 DLE types. In detail, of the 4 duodenoscopes of this type, 2 had lower contamination rates (69% vs 39% and 36% vs 10%). During the control period, both these duodenoscopes had multiple episodes with ongoing contamination with the same microorganism that ended weeks before the start of the intervention period (ie, they were not terminated by ATP testing). CONCLUSIONS: Postmanual cleaning ATP tests do not reduce post-HLD gut flora contamination rates of DLEs. Hence, postcleaning ATP tests are not suited as a means for quality control of endoscope reprocessing.

A live-cell assay for the real-time assessment of extracellular ATP levels.

Extracellular ATP (eATP) is a potent damage associated molecular pattern (DAMP) molecule known to exert profound effects on the innate and adaptive immune responses. As such, it has become an important biomarker for studying means to pro-actively modulate inflammatory processes. Unfortunately, traditional methodologies employed for measuring eATP require cumbersome supernatant sampling, onerous time courses, or unnecessary duplication of effort. Here we describe a new reagent that is tolerable to test cells in extended exposures and enables a fully homogeneous assay method for real-time determinations of extracellular ATP levels. The reagent is introduced into assay plates containing cells at the time of stimulus introduction. The real-time feature of the format allows for sensitive, continuous accounting of eATP levels in the test model over at least 24 h. This work details our efforts to create and characterize this new reagent and to validate utility by demonstrating its use with multiple cell lines and chemically diverse eATP induction stimuli.

Implications of Adenylate Metabolism in Hygiene Assessment: A Review.

The assessment of a hygienic state or cleanliness of contact surfaces has significant implications for food and medical industries seeking to monitor sanitation and exert improved control over a host of operations affecting human health. Methods used to make such assessments commonly involve visual inspections, standard microbial plating practices, and the application of ATP-based assays. Visual methods for inspection of hygienic states are inherently subjective and limited in efficacy by the accuracy of human senses, the degree of task-specific work experience, and various sources of human bias. Standard microbial swabbing and plating techniques are limited in that they require hours or even days of incubation to generate results, with such steps as enrichment and colony outgrowth resulting in delays that are often incompatible with manufacturing or usage schedules. Rapid in conduct and considered more objective in operation than visual or tactile inspection techniques, swabbing surfaces using ATP-based assessments are relied on as routine, even standard, methods of hygienic assessment alone or in complement with microbial and visual inspection methods. Still, current ATP methods remain indirect methods of total hygiene assessment and have limitations that must be understood and considered if such methods are to be applied judiciously, especially under increasingly strict demands for the verification of hygiene state. Here, we present current methods of ATP-based bioluminescence assays and describe the limitations of such methods when applied to general food manufacturing or health care facilities.

Evaluation of ATP bioluminescence-based methods for hygienic assessment in fish industry.

AIMS: To evaluate ATP bioluminescence-based hygiene monitoring systems under conditions relevant to fish processing environments. METHODS AND RESULTS: The ATP bioluminescence of fish fractions that are potentially present after insufficient cleaning of fish processing environments was determined. Different fractions and interfering substances representing the stages from slaughtering to smoking were prepared and measured using two different commercial systems (SystemSURE Plus and Clean-Trace). ATP bioluminescence was quenched by acidic liquid smoke and by sodium chloride even at concentrations as low as 0·9% NaCl. Large variations were observed between different types of trout homogenates: the ATP bioluminescence from raw belly fat homogenate was 100-1000 times lower than for trout blood. There were about a 1000-fold lower ATP bioluminescence in raw compared to heat-treated fractions from trout, with the exception of blood. The bioluminescence from Listeria monocytogenes was very low. Results from fish processing plants supported the laboratory findings. CONCLUSIONS: The output from ATP-monitoring instruments depends on the nature of fish soil present, as well as the presence of sodium chloride and low pH. This may lead to considerable under- or overestimation of the level of organic soil. SIGNIFICANCE AND IMPACT OF THE STUDY: ATP bioluminescence instruments are widely used by the fish industry for monitoring hygiene. The monitoring method will only give valuable information about the hygiene if critical limits are set after a validation period, distinguishing between areas with different types of soil and between different hygiene zones.

Building and indoor environmental quality assessment of Nigerian primary schools: A pilot study.

A total of 15 classrooms went through on-site assessments/inspections, including measurements of temperature (T), and concentrations of carbon monoxide (CO) and carbon dioxide (CO2 ). In addition, the level of surface biocontamination/cleaning effectiveness was assessed by measuring adenosine triphosphate (ATP) levels on students' desks. Based on the data, the quality of facilities in the buildings was low. Classroom occupancy exceeded ASHRAE 50 person/100 m2 standard in all cases indicating overcrowding. However, concentrations of CO2 remained below 1000 ppm in most classrooms. On the other hand, indoor T was above the recommended levels for thermal comfort in all classrooms. Maximum indoor CO was 6 ppm. Median ATP concentrations on the desk tops were moderately high in all schools. The use of open incinerators and power generator sets near classrooms, which was suspected to be the main source of CO, should be discouraged. Improved hygiene could be achieved by providing the students access to functioning bathroom facilities and cafeteria, and by effective cleaning of high contact surfaces such as desks. Although ventilation seems adequate based on CO2 concentrations, thermal comfort was not attained especially in the afternoon during extreme sunlight. Therefore, installing passive and/or mechanical cooling systems should be considered in this regard.

Assessment of flow cytometry for microbial water quality monitoring in cooling tower water and oxidizing biocide treatment efficiency.

The control of Legionella proliferation in cooling tower water circuits requires regular monitoring of water contamination and effective disinfection procedures. In this study, flow cytometry was assessed to monitor water contamination and disinfection treatment efficiency on bacterial cells regarding nucleic acid injury (SYBR® Green II), cell integrity (SYBR® Green II and propidium iodide) and metabolism activity (ChemChrome V6). A total of 27 cooling tower water samples were analyzed in order to assess water contamination levels regarding viable populations: standard culture, ATP measurement and flow cytometry methods were compared. Flow cytometry and plate counts methods showed a significant correlation for changes in concentrations despite a 1 to 2-log difference regarding absolute quantification. Concerning intracellular activity, the use of two different flow cytometers (FACSCanto™ II and Accuri™ C6) showed no statistical difference while a difference was observed between flow cytometry and usual methods (culture and ATP measurement). The standard culture and flow cytometry methods were also compared for in vitro bacteria inactivation measurements in the presence of 3 different types of oxidizing biocides commonly used for cooling tower disinfection. Reductions observed ranged between 1 and 2 log depending on (1) the detection method, (2) the bacterial population origin and/or (3) the active biocide molecule used. In conclusion, flow cytometry represents an efficient, accurate and fast approach to monitor water contamination and biocide treatment efficiency in cooling towers.

[Importance of cleaning and disinfection of critical surfaces in dental health services. Impact of an intervention program]. / Importancia del proceso de limpieza y desinfección de superficies críticas en un servicio dental. Impacto de un programa de intervención.

Introduction Dental care has become a challenge for healthcare associated infection prevention programs, since the environment, within other factors, plays an important role in the transmission chain. Materials and Methods An intervention program was designed for the Dental Unit of Hospital Militar de Santiago, between years 2014 and 2015. The program contemplated 3 stages: diagnostic, intervention and evaluation stage. Objective To improve the safety of critical surfaces involved in dental healthcare. Results During the diagnostic stage, the cleaning and disinfection process was found to be deficient. The most contaminated critical surface was the instrument holder unit, then the clean area and lamp handle. The surfaces that significantly reduced their contamination, after the intervention, were the clean area and the instrument carrier unit. Conclusion Training in the processes of cleaning and disinfecting surfaces and dental equipment is one of the cost-effective strategies in preventing healthcare-associated infections (HCAI), with simple and easy-to-apply methods.

A novel, efficient, and ecologically relevant bioassay method using aquatic fungi and fungus-like organisms for fungicide ecological effect assessment.

Fungicides are used to control fungal plant pathogens, but they may also be highly toxic to aquatic fungi, which play an important role in natural aquatic ecosystems. However, a bioassay method using aquatic fungi has not been sufficiently developed. In the present study, a novel, efficient, and ecologically relevant bioassay method was developed for the ecological effect assessment of fungicides. Candidate test species were selected by considering the following 4 factors: 1) their ecological relevance (i.e., widely distributed and frequently observed) in freshwater habitats, 2) inclusion of a wide range of taxonomic groups, 3) availability from public culture collections, and 4) suitability for culture experiments using a microplate. The following 5 fungal species were selected: Rhizophydium brooksiaum (Chitridiomycota), Chytriomyces hyalinus (Chitridiomycota), Tetracladium setigerum (Ascomycota), Sporobolomyces roseus (Basidiomycota), and Aphanomyces stellatus (Oomycota, fungus-like organism). An efficient test method using the 5 species was developed based on a microplate assay using a 96-well white microplate and a test duration of 48 h. Fungal biomass was determined as adenosine 5'-triphosphate (ATP) luminescence, which is known to be proportional to live cell density and can be determined with a microplate reader. Test performance was evaluated by conducting bioassays of 3,5-dichlorophenol and malachite green as standard test substances. Fungal species were clearly more sensitive than other species to the fungicide malachite green. Environ Toxicol Chem 2018;37:1980-1989. © 2018 SETAC.

Assessment of test methods for evaluating effectiveness of cleaning flexible endoscopes.

BACKGROUND: Strict adherence to each step of reprocessing is imperative to removing potentially infectious agents. Multiple methods for verifying proper reprocessing exist; however, each presents challenges and limitations, and best practice within the industry has not been established. Our goal was to evaluate endoscope cleaning verification tests with particular interest in the evaluation of the manual cleaning step. The results of the cleaning verification tests were compared with microbial culturing to see if a positive cleaning verification test would be predictive of microbial growth. METHODS: This study was conducted at 2 high-volume endoscopy units within a multisite health care system. Each of the 90 endoscopes were tested for adenosine triphosphate, protein, microbial growth via agar plate, and rapid gram-negative culture via assay. The endoscopes were tested in 3 locations: the instrument channel, control knob, and elevator mechanism. RESULTS: This analysis showed substantial level of agreement between protein detection postmanual cleaning and protein detection post-high-level disinfection at the control head for scopes sampled sequentially. CONCLUSIONS: This study suggests that if protein is detected postmanual cleaning, there is a significant likelihood that protein will also be detected post-high-level disinfection. It also infers that a cleaning verification test is not predictive of microbial growth.

Encompassing ATP, DNA, insulin, and protein content for quantification and assessment of human pancreatic islets.

Islet transplantation has made major progress to treat patients with type 1 diabetes. Islet mass and quality are critically important to ensure successful transplantation. Currently, islet status is evaluated using insulin secretion, oxygen consumption rate, or adenosine triphosphate (ATP) measurement. These parameters are evaluated independently and do not effectively predict islet status post-transplant. Therefore, assessing human pancreatic islets by encompassing ATP, DNA, insulin, and protein content from a single tissue sample would serve as a better predictor for islet status. In this study, a single step procedure for extracting ATP, DNA, insulin, and protein content from human pancreatic islets was described and the biomolecule contents were quantified. Additionally, different mathematical calculations integrating total ATP, DNA, insulin, and protein content were randomly tested under various conditions to predict islet status. The results demonstrated that the ATP assay was efficient and the biomolecules were effectively quantified. Furthermore, the mathematical formula we developed could be optimized to predict islet status. In conclusion, our results indicate a proof-of-concept that a simple logarithmic formula can predict overall islet status for various conditions when total islet ATP, DNA, insulin, and protein content are simultaneously assessed from a single tissue sample.

Importancia del proceso de limpieza y desinfección de superficies críticas en un servicio dental: impacto de un programa de intervención / Importance of cleaning and disinfection of critical surfaces in dental health services: impact of an intervention program

Resumen Introducción La atención odontológica se ha transformado en un desafío para los programas de prevención y control de infecciones asociadas a la atención de salud (IAAS), dado que el ambiente, entre otros factores, juega un rol importante en la cadena de transmisión. Método Se realizó un programa de intervención en el Servicio de Dental del Hospital Militar de Santiago, entre los años 2014-2015, que contempló tres etapas: etapa diagnóstica, de intervención y de evaluación. Objetivo Mejorar la seguridad de las superficies críticas involucradas en la atención dental. Resultados Durante la etapa diagnóstica se constató que el proceso de limpieza y desinfección era deficiente. La superficie crítica más contaminada fue la unidad porta-instrumental, luego el área limpia y la manilla de la lámpara. Las superficies que redujeron significativamente su contaminación, posterior a la intervención, fueron el área limpia y la unidad porta-instrumental. Conclusiones La capacitación en relación a los procesos de limpieza y desinfección de superficies y del equipamiento dental es una de las estrategias costo-eficientes en la prevención de las IAAS, sencilla y fácil de aplicar.

Introduction Dental care has become a challenge for healthcare associated infection prevention programs, since the environment, within other factors, plays an important role in the transmission chain. Materials and Methods An intervention program was designed for the Dental Unit of Hospital Militar de Santiago, between years 2014 and 2015. The program contemplated 3 stages: diagnostic, intervention and evaluation stage. Objective To improve the safety of critical surfaces involved in dental healthcare. Results During the diagnostic stage, the cleaning and disinfection process was found to be deficient. The most contaminated critical surface was the instrument holder unit, then the clean area and lamp handle. The surfaces that significantly reduced their contamination, after the intervention, were the clean area and the instrument carrier unit. Conclusion Training in the processes of cleaning and disinfecting surfaces and dental equipment is one of the cost-effective strategies in preventing healthcare-associated infections (HCAI), with simple and easy-to-apply methods.

Longitudinal assessment of reprocessing effectiveness for colonoscopes and gastroscopes: Results of visual inspections, biochemical markers, and microbial cultures.

BACKGROUND: Flexible endoscopes are currently reused following cleaning and high-level disinfection. Contamination has been found on endoscopes, and infections have been linked to gastrointestinal, respiratory, and urologic endoscopes. METHODS: This longitudinal study involved visual inspections with a borescope, microbial cultures, and biochemical tests for protein and adenosine triphosphate to identify endoscopes in need of further cleaning or maintenance. Three assessments were conducted over a 7-month period. Control group endoscopes reprocessed using customary practices were compared with intervention group endoscopes subjected to more rigorous reprocessing. RESULTS: At final assessment, all endoscopes (N = 20) had visible irregularities. Researchers observed fluid (95%), discoloration, and debris in channels. Of 12 (60%) endoscopes with microbial growth, 4 had no growth until after 48 hours. There were no significant differences in culture results by study group, assessment period, or endoscope type. Similar proportions of control and intervention endoscopes (~20%) exceeded postcleaning biochemical test benchmarks. Adenosine triphosphate levels were higher for gastroscopes than colonoscopes (P = .014). Eighty-five percent of endoscopes required repair due to findings. CONCLUSIONS: More rigorous reprocessing was not consistently effective. Seven-day incubation allowed identification of slow-growing microbes. These findings bolster the need for routine visual inspection and cleaning verification tests recommended in new reprocessing guidelines.

A simplified ATP method for the rapid control of cell viability in a freeze-dried BCG vaccine.

We propose a simple and cost-effective ATP method for controlling the specific activity of a freeze-dried BCG vaccine. A freeze-dried BCG vaccine is reconstituted with 1ml saline and incubated for 15min at room temperature and then for 1h at 37°C. The vaccine is then treated with apyrase to remove extracellular ATP. After that, the cells are lysed with DMSO and the ATP content in the lysate is measured by the bioluminescence method. To implement the method, we developed a kit that requires no time-consuming preparation before the analysis. We demonstrated the linear relationship between the experimental values of the specific activity (106CFU/mg) and intracellular ATP content (ATP, pmol/mg) for different batches of the studied BCG vaccines; the proportionality coefficient was К=0.36±0.02. We proposed a formula for calculating the specific activity from the measured content of intracellular ATP (ATP, pmol/mg). The comparison of the measured and calculated values of the specific activity (106CFU/mg) shows that these values are similar; their differences fall within the allowable range of deviations for the specific activity values of the BCG vaccine.

Assessment of disinfection of hospital surfaces using different monitoring methods.

OBJECTIVE: to assess the efficiency of cleaning/disinfection of surfaces of an Intensive Care Unit. METHOD: descriptive-exploratory study with quantitative approach conducted over the course of four weeks. Visual inspection, bioluminescence adenosine triphosphate and microbiological indicators were used to indicate cleanliness/disinfection. Five surfaces (bed rails, bedside tables, infusion pumps, nurses' counter, and medical prescription table) were assessed before and after the use of rubbing alcohol at 70% (w/v), totaling 160 samples for each method. Non-parametric tests were used considering statistically significant differences at p<0.05. RESULTS: after the cleaning/disinfection process, 87.5, 79.4 and 87.5% of the surfaces were considered clean using the visual inspection, bioluminescence adenosine triphosphate and microbiological analyses, respectively. A statistically significant decrease was observed in the disapproval rates after the cleaning process considering the three assessment methods; the visual inspection was the least reliable. CONCLUSION: the cleaning/disinfection method was efficient in reducing microbial load and organic matter of surfaces, however, these findings require further study to clarify aspects related to the efficiency of friction, its frequency, and whether or not there is association with other inputs to achieve improved results of the cleaning/disinfection process.

Assessment of disinfection of hospital surfaces using different monitoring methods / Avaliação da desinfecção de superfícies hospitalares por diferentes métodos de monitoramento / Evaluación de la desinfección de superficies hospitalarias por diferentes métodos de monitorización

to assess the efficiency of cleaning/disinfection of surfaces of an Intensive Care Unit.

avaliar a eficiência da limpeza/desinfecção de superfícies de uma Unidade de Terapia Intensiva.

evaluar la eficiencia de la limpieza/desinfección de superficies de una Unidad de Terapia Intensiva.

Assessment of an innovative antimicrobial surface disinfectant in the operating room environment using adenosine triphosphate bioluminescence assay.

Terminal cleaning in the operating room is a critical step in preventing the transmission of health care-associated pathogens. The persistent disinfectant activity of a novel isopropyl alcohol/organofunctional silane solution (ISO) was evaluated in 4 operating rooms after terminal cleaning. Adenosine triphosphate bioluminescence documented a significant difference (P < .048) in surface bioburden on IOS-treated surfaces versus controls. RODAC plate cultures revealed a significant (P < .001) reduction in microbial contamination on IOS-treated surfaces compared with controls. Further studies are warranted to validate the persistent disinfectant activity of ISO within selective health care settings.

Adenosine tri-phosphate (ATP)-based cleaning monitoring in health care: how rapidly does environmental ATP deteriorate?

BACKGROUND: Ensuring cleaning compliance of housekeeping staff is critical to ensure adequate application of surface disinfectants. Adenosine triphosphate (ATP) testing has been recommended as a way to monitor cleaning compliance; however, little is known about the stability of ATP on environmental surfaces. AIM: To assess the stability of ATP from various sources to determine if it is stable for sufficient time to be an effective means of assessing environmental cleaning and disinfection in health care. METHODS: Purified ATP, ATP derived from ATS-T (blood-based test soil) and ATP derived from 10(7) colony-forming units/site of micro-organisms (Pseudomonas aeruginosa, Enterococcus faecalis, Candida albicans) were evaluated in liquid suspension and dried on to surfaces to assess stability over 29 days. Cleaners and disinfectants were sprayed on to surface-dried material with no wiping to determine their effect on microbial viability and ATP stability. FINDINGS: Surface-dried P. aeruginosa, E. faecalis and C. albicans retained 65-96% of their original ATP level on Day 29, despite reduced or no viability. Surface-dried ATS-T had 100% and 3% of its original ATP on Days 4 and 29, respectively. Deterioration of the ATP signal was most pronounced for suspensions. Purified ATP was stable over 29 days in suspension or dried on to a surface. CONCLUSIONS: ATP residuals from organic material and micro-organisms (dead or alive) are stable when dried on to surfaces. In the absence of cleaning and disinfection, the relative light unit signal will not deteriorate rapidly, making ATP a good marker to monitor cleaning.

Assessment of hospital daily cleaning practices using ATP bioluminescence in a developing country.

UNLABELLED: Visual assessment of surfaces may not be enough to document the level of cleanliness in the hospital setting. It is necessary to introduce quantitative methods to document the results of this practice. OBJECTIVE: To evaluate the efficacy of hospital terminal cleaning procedures, using an adenosine triphosphate (ATP) bioluminescence method in a teaching hospital. METHOD: During 2008 we conducted an evaluation using ATP bioluminescence LIGHTNING MVP™ (Arquimed) of external and internal housekeeping service. After conducting an initial evaluation we implemented education of cleaning practices and finally we did a post intervention evaluation. Using chi-square method we compared prior versus after cleaning, quality of cleaning performed by external versus internal personnel, single versus double terminal cleaning procedures and prior versus after intervention. A finding of three RLU or less was considered a clean surface. RESULTS: We performed 198 evaluations in 33 patient units and nine OR. Internal personnel accomplished 25.37% of clean surfaces before and 80% after the education intervention (p=0.01). In contrast, external personnel obtained 68.8% before and 73.33% after intervention (p=0.3). CONCLUSIONS: This study suggests that visual assessment is not enough to ensure quality of the process and it is necessary to document the level of cleanliness by quantitative methods.