A suspension method to determine reuse life of chemical disinfectants during clinical use.

In-use testing of disinfectants is necessary to ensure efficacy over time. The current official procedure for testing disinfectants, the Association of Official Analytical Chemists (AOAC) use-dilution method, cannot be adapted to repeated sampling techniques of use-life testing. It is therefore necessary to use an alternative method when evaluating the activity of a disinfectant under actual use. The Clinical Research Associates (CRA) suspension method was developed to fill this need. It consists of adding 0.5 ml of a standard culture to 5.0 ml of test disinfectant and sampling the mixture after 10 min for surviving bacteria. When this test was compared with the AOAC use-dilution method under a simulated use situation, the two methods were generally equivalent in identifying disinfectant inactivation. In addition, the CRA method was less time consuming, easier to perform, and less variable than the AOAC method. Use of the CRA method in a clinical study demonstrated the need for reuse claims to be based on clinical use studies rather than on laboratory testing only.     

Evaluation of Disinfectants for Hospital Housekeeping Use

A use-dilution procedure and screening method are proposed to aid the hospital bacteriologist in selecting the most effective disinfectants. Sixteen disinfectants were tested with and without organic material on six different organisms. Sporadic results usually obtained with quaternaries tested by the procedure of the Association of Official Agricultural Chemists (AOAC) were eliminated by the methods of this study. The use-dilution procedure employs the material upon which the disinfectant is to be used, rather than stainless steel, as in the AOAC use-dilution test. The importance of testing each disinfectant against several organisms and in the presence of organic material is discussed.     

Use-Dilution Test and Newcastle Disease Virus

The use-dilution test for evaluating the effectiveness of disinfectants against bacteria was modified to determine the effectiveness of disinfectants against a group of viruses. Modifications were kept to a minimum to retain the general principles of the test and thereby retain the test's familiarity among testing laboratory personnel. Modifications included the use of a standard allantoic fluid suspension of Newcastle disease virus instead of a standard bacterial culture. The only other modification was the inoculation of six embryonated chicken eggs (10 to 12 days old) with 0.1 ml of nutrient broth into which a carrier ring was transferred after a standard period in diluted disinfectant. The death or survival of 60 embryos, then, is the criterion by which a disinfectant can be judged effective at use-dilution. Experiments are described which establish the validity of the modified test procedure. The effectiveness of nine common disinfectants against Newcastle disease virus as judged by this test procedure is reported.     

An Examination of the Effect of Three Phenolic Disinfectants on Mycobacterium tuberculosis

The effect of a phenolic disinfectant ( o -phenylphenol 45% w/w) with linseed oil soap or with soya oil soap on Mycobacterium tuberculosis was determined by three methods. Neither the geometrical dilution test nor the modified capacity use-dilution test revealed any differences between the two disinfectants. However, paradoxically both methods proved that the highest concentration of the disinfectants tested (3·5% v/v) exhibited a very low germicidal effect on M. tuberculosis , whereas lower concentrations showed a much better effect. When determining the tuberculocidal effects of various concentrations of the two disinfectants at different exposure times, the higher concentrations showed very low effects, even after the longest exposure time. At concentrations of 2·0 and 1·0% (v/v), the disinfectants displayed the most rapid effects. In the present investigation the disinfectant with the linseed oil soap seemed to destroy the cells more quickly than that with the soya oil soap. The third disinfectant containing p -chloro- m -cresol and o -benzyl- p -chlorophenol with a total of 9·2% (w/w) phenols in a detergent system, did not display, when employing the capacity use-dilution test, the same phenomenon in the concentrations used, but the experiment showed that the recommended use-dilution concentration ought to be doubled.     

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.     

Disinfectant Evaluation by a Capacity Use-dilution Test

SUMMARY: Two testing procedures, B.S. 3286: 1960 and Kelsey & Sykes (1969), have been examined in terms of their reproducibility and ease of operation using different types of disinfectants. The capacity use-dilution test realistically simulated practical conditions and gave reasonable results. It is recommended for use-dilution assessments of general purpose disinfectants.     

A Comparative Investigation of the Bactericidal and Fungicidal Effects of Three Phenolic Disinfectants

Two methods, a capacity use-dilution test and another employing geometrical dilution, are used when comparing the bactericidal and fungicidal effects of a phenolic disinfectant containing 45% (w/w) o -phenylphenol in an aqueous soap solution of linseed oil (soap content 6·5% w/v) with the corresponding effects of the same phenolic compound in an aqueous soya oil soap solution (soap content 3·5% w/v). The soya oil soap did not change the disinfectant capacity on the different test organisms used. For the most resistant strain ( Staphylococcus aureus ) the usedilution concentration was evaluated to be slightly above 1%, i.e. 2%, which is much lower than the recommended use-dilution concentration. However, using the same method and test organisms the capacity use-dilution testing of a third phenolic disinfectant, containing p -chloro- m -cresol and o -benzyl- p -chlorophenol with a total of 9·2 (w/w) phenols in a detergent system, indicated that the recommended use-dilution concentration should be doubled.     

Test Method for the Evaluation of Virucidal Efficacy of Three Common Liquid Surface Disinfectants on a Simulated Environmental Surface

The Association of Official Analytical Chemists bacterial use-dilution test for evaluating liquid surface disinfectants was modified to determine the efficacy of three common environmental germicide compounds against representatives of four virus groups. Modifications were made to conform to the Environmental Protection Agency guidelines on virucidal testing procedures. Alterations included: the use of a concentrated tissue culture preparation of virus instead of a standard bacterial culture; HEp-2 tissue culture cells as a visible test system in place of standard nutrient broth; and controls to measure the virus titer end point and the toxicity of the disinfectant to the cell cultures. Comparison of control end points with results from the test proper were the measure of the effectiveness of the germicides against the viruses. Results are described which agree with those based on other methods previously reported.     

Pseudomonas pellicle in disinfectant testing: electron microscopy, pellicle removal, and effect on test results.

Pseudomonas aeruginosa ATCC 15442 is a required organism in the Association of Official Analytical Chemists use-dilution method for disinfectant efficacy testing. When grown in a liquid medium, P. aeruginosa produces a dense mat or pellicle at the broth/air interface. The purpose of this investigation was to examine the pellicle by scanning electron microscopy, to evaluate three pellicle removal methods, and to determine the effect of pellicle fragments on disinfectant efficacy test results. The efficacies of three methods of pellicle removal (decanting, vacuum suction, and filtration) were assessed by quantifying cell numbers on penicylinders. The Association of Official Analytical Chemists use-dilution method was used to determine whether pellicle fragments in the tubes used to inoculate penicylinders affected test results. Scanning electron micrographs showed the pellicle to be a dense mass of intact, interlacing cells at least 10 microns thick. No significant differences in pellicle removal methods were observed, and the presence of pellicle fragments usually increased the number of positive tubes in the use-dilution method significantly.     

Pseudomonas pellicle in disinfectant testing: electron microscopy, pellicle removal, and effect on test results

Pseudomonas aeruginosa ATCC 15442 is a required organism in the Association of Official Analytical Chemists use-dilution method for disinfectant efficacy testing. When grown in a liquid medium, P. aeruginosa produces a dense mat or pellicle at the broth/air interface. The purpose of this investigation was to examine the pellicle by scanning electron microscopy, to evaluate three pellicle removal methods, and to determine the effect of pellicle fragments on disinfectant efficacy test results. The efficacies of three methods of pellicle removal (decanting, vacuum suction, and filtration) were assessed by quantifying cell numbers on penicylinders. The Association of Official Analytical Chemists use-dilution method was used to determine whether pellicle fragments in the tubes used to inoculate penicylinders affected test results. Scanning electron micrographs showed the pellicle to be a dense mass of intact, interlacing cells at least 10 microns thick. No significant differences in pellicle removal methods were observed, and the presence of pellicle fragments usually increased the number of positive tubes in the use-dilution method significantly.     

Comparative testing and evaluation of hard-surface disinfectants

Summary The activity of eleven disinfectants againstStaphylococcus aureus, Pseudomonas aeruginosa, andSaccharomyces cerevisiae was determined using a method based on the A.O.A.C. germicidal and detergent sanitizer assay. Based on the activity against the test organisms after 30-and 60-s exposures to each disinfectant, the disinfectant containing chlorine dioxide had the highest biocidal activity in this assay, on a mg/l basis. In addition, a disinfectant containing sodium hypochlorite and a disinfectant containing sodium chlorite performed well, at concentrations below label specifications. The results illustrate the importance of testing disinfectants in the context of their intended use.     

A Practical Approach to Evaluation of the Germicidal Efficiency of a General Purpose Military Disinfectant

The bactericidal activity of a general purpose disinfectant consisting of 25% sodium-o-phenylphenolate and 75% sodium-4- and 6-chloro-2-phenylphenolate was evaluated by a simulated in-use, surface-square dilution method. Common floor (asphalt, rubber, and unglazed tiles) and wall (stainless steel tile, ceramic tile, and painted wood) surfaces of various porosities and compositions were selected to simulate actual-use conditions.

The method used consisted of inoculating the surfaces of 1-in. square sections of floor and wall covering with a test organism, air-drying the inoculated surface, applying the disinfectant, allowing it to act for 10 min, and recovering the survivors by plating. Confirmatory results of the standard phenol coefficient and use-dilution tests indicated 700 ppm of the disinfectant to be a safe use concentration. The in-use surface-square dilution studies have shown that this is a more than adequate safe concentration for stainless steel, both glazed and unglazed ceramic tile, and nonwaxed surface of asphalt tile. However, concentrations ranging between 2,500 and 6,000 ppm for plastic-fortified rubber tile, 1,500 and 2,000 ppm for waxed asphalt tile, and 2,000 ppm for painted wood were required to achieve 99.9% reduction of either Salmonella choleraesuis or Salmonella schottmuelleri. These results indicate that a disinfectant concentration derived from the Association of Official Agricultural Chemists use-dilution test cannot always be relied upon to provide a dependable index to actual safe use-dilution when a disinfectant is supplied to certain wall or floor surfaces.

     

A rapid method for assessing the suitability of quenching agents for individual biocides as well as combinations

AIMS: To develop a novel, rapid method for testing the ability of quenching agents to neutralize disinfectants. METHODS AND RESULTS: Tests were performed to determine the suitability of different neutralizers for a range of disinfectants, using a new method based on the Bioscreen optical density analyser. Results showed that during disinfection tests, efficacy could be over-estimated due to poor, or no, neutralization of the disinfectant after a specified time of exposure to the bacteria. The failure to distinguish adequately between bacteriostatic and bactericidal effects can lead to false results during disinfectant testing. Experiments also showed that dilution of the disinfectant, following exposure to the bacteria, was not always sufficient to stop the activity of the disinfectant for chemicals with low dilution coefficients. CONCLUSIONS: The quench test proved to be very quick and easy to perform, with results being available within 18 h. Using the Bioscreen, the test is automated and determines whether dilution into a particular neutralizer is able to inactivate a disinfectant within 30 s. SIGNIFICANCE AND IMPACT OF THE STUDY: This new approach allows the efficacy of quenching agents to be determined, prior to undertaking each disinfection study, and can help in the development of more suitable quenching solutions. The test has also been used to find suitable neutralizers for mixtures of disinfectants which might be used during studies on synergistic biocide combinations.     

Modified quantitative association of official analytical chemists sporicidal test for liquid chemical germicides

The Association of Official Analytical Chemists (AOAC) test for sporicidal activity of disinfectants (966.04) is used in the United States as the legal criteria for classifying a liquid as a chemical sterilant and also as the indicator of the highest level of disinfectant. This qualitative test contains procedures that may cause inaccurate results. A modified, quantitative version of the AOAC sporicidal test has been developed that uses a specified minimum number of spores (2.0 x 10(sup5)) of Bacillus subtilis or Clostridium sporogenes dried onto porcelain penicylinders. This modified test was validated with three commercial sterilant chemicals tested on three separate groups of spore-labeled cylinders.     

Effect of methodology, dilution, and exposure time on the tuberculocidal activity of glutaraldehyde-based disinfectants

The Association of Official Analytical Chemists (AOAC) test for assessing the tuberculocidal activity of disinfectants has been shown to be variable. A modified AOAC test, which substituted Middlebrook 7H9 broth as the primary subculture medium and used neutralization by dilution, was compared with the standard AOAC method to assess the mycobactericidal activity of three glutaraldehyde-based disinfectants at 20 degrees C and various exposure times. These changes had a marked effect on results, with the modified AOAC test providing more positive penicylinders per 10 replicates in 12 of the 13 comparisons that provided positive results. These differences were observed with both Mycobacterium bovis (ATCC 35743) and a clinical isolate of Mycobacterium tuberculosis. The effects of various exposure times to and dilutions of the glutaraldehyde-based disinfectants were also examined. The minimum exposure time needed to inactivate reliably M. bovis or M. tuberculosis with 2% glutaraldehyde was 20 min at 20 degrees C. Diluting 2% glutaraldehyde caused a significant decline in mycobactericidal activity. Modification of the standard AOAC test to improve its sensitivity in detecting the failure of disinfectants to inactivate mycobacteria is indicated.     

Effect of methodology, dilution, and exposure time on the tuberculocidal activity of glutaraldehyde-based disinfectants.

The Association of Official Analytical Chemists (AOAC) test for assessing the tuberculocidal activity of disinfectants has been shown to be variable. A modified AOAC test, which substituted Middlebrook 7H9 broth as the primary subculture medium and used neutralization by dilution, was compared with the standard AOAC method to assess the mycobactericidal activity of three glutaraldehyde-based disinfectants at 20 degrees C and various exposure times. These changes had a marked effect on results, with the modified AOAC test providing more positive penicylinders per 10 replicates in 12 of the 13 comparisons that provided positive results. These differences were observed with both Mycobacterium bovis (ATCC 35743) and a clinical isolate of Mycobacterium tuberculosis. The effects of various exposure times to and dilutions of the glutaraldehyde-based disinfectants were also examined. The minimum exposure time needed to inactivate reliably M. bovis or M. tuberculosis with 2% glutaraldehyde was 20 min at 20 degrees C. Diluting 2% glutaraldehyde caused a significant decline in mycobactericidal activity. Modification of the standard AOAC test to improve its sensitivity in detecting the failure of disinfectants to inactivate mycobacteria is indicated.     

Disinfection in Food Processing – Efficacy Testing of Disinfectants

The key to effective cleaning and disinfection of food plants is the understanding of the type of the soil to be removed from the surfaces. An efficient cleaning and disinfection procedure consists of a sequence of rinses using good quality water with application of detergents and disinfectants. Disinfection is required in food plant operations, where wet surfaces provide favourable conditions for the growth of microbes. The efficacy of disinfectants is usually determined in suspensions, which do not mimic the growth conditions on surfaces where the agents are required to inactivate the microbes. Therefore, the suspension tests do not give adequate information and reliable carrier tests, which mimic surface growth, are needed. In developing a proposal for the testing of disinfectants on surfaces to an analytical standard, it is important to identify the major sources of variation in the procedure. In response to the need for a relatively realistic, simple and reliable test for disinfectant efficacy a method for culturing laboratory model biofilms has developed. The use of artificial biofilms i.e. biofilm-constructs inoculated with process contaminants in disinfectant testing can also be used for screening the activity of various disinfectants on biofilm cells. Both biofilm carrier tests showed clearly that the biofilm protects the microbes against the disinfectants. The chemical cleanliness is also essential in food plants. The total cleanliness of the process lines is mainly based on measuring the microbial load using culturing techniques. These results can give an incorrect picture of the total cleanliness, because the viable microbes do not grow when disinfectants are left on the surface. The luminescent bacteria light inhibition method offers a useful alternative for testing chemical residue left on surfaces after cleaning and disinfection operations.     

Comparative testing of disinfectants using proposed European surface test methods

A surface test method for disinfectants currently under development as a standard European test method is described. The test involves determining the number of viable organisms recoverable from a contaminated stainless steel surface before and after application of disinfectant. Evaluation of a range of disinfectant agents and products currently used in the UK indicated that products at recommended use concentrations produced log reductions in viable count ranging from < 1.0 to > 6.4 (i.e. no detectable survivors) after a 5 min contact period against Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecium and Candida albicans. Increased activity was observed by increasing the disinfectant contact time to 30 min. Addition of 3% w/v albumin to the test suspension used to inoculate surfaces caused a substantial reduction in activity.     

Efficacy of some commerical disinfectants against the bacterial isolates from a poultry slaughterhouse in Turkey

In this study, efficacy of seven different commercial disinfectant preparations was investigated against characteristic bacteria of a poultry slaughterhouse in Ankara (Turkey) by using paper disc-agar diffusion method and surface effectiveness test. According to paper disc-agar diffusion method, some disinfectants had wide efficacy against the test bacteria. The disinfectant effectiveness generally increased by the increasing of disinfectant concentration. However, some disinfectants were ineffective even though at their highest concentrations. The most effective disinfectant was B which contains QAC as active agent.Staphylococcus spp.,Staphyloccus aureus andEscherichia coli were the most sensitive bacteria to the disinfectants. Some pathogenic isolates, especiallySalmonella spp. andCampylobacter spp., were the most resistant ones to many of the disinfectants tested. The results of paper disc-agar diffusion method indicated the importance of characteristic bacterial strains of food plants as the test bacteria for disinfectant efficacy tests. Conversely of paper disc-agar diffusion method, all disinfectants were effective against the isolates by surface effectiveness test depending on exposure time. The disinfectants, except A and F, produced at least 3 log unit reduction during 5 min of exposure. However, all disinfectants at their lowest concentrations were effective against all tested bacteria during 15 and 30 min by this test.     

Neutralization of the activity of eight disinfectants by organic matter

The effect of organic matter on the activity of eight disinfectants was evaluated. Three types of interfering substrates (whole milk powder, dried beef blood and fish meal) were tested according to the method of Whitmore and Miner adapted to the AOAC use-dilution method. Glutaraldehyde and to a certain extent, chlorhexidine acetate and the amphoteric surfactant kept their disinfecting activity after contact with high concentrations of organic matter. The quaternary ammonium compound as well as the quaternary ammonium-glutaraldehyde complex were more readily neutralized whereas anionic acid, iodophor and sodium hypochlorite did not tolerate the presence of organic matter. The neutralizing activity of powders was correlated to their solubility and composition.