Bacteria disinfectant tests

Disinfection tests can be classified according to the test organism, ie, whether the test employs certain species of bacteria, fungi, or vimses classified as to whether it is a static test or a cidal test, as in a bactericidal vs bacteriostatic test or sporicidal vs sporistatic test or classified as to whether it is a microbial reduction test or an end-point test where all the organisms in the test are apparently killed. Procedures may be distinguished by in vitro or in vivo testing. Another way to consider tests is whether they are screening tests, practical type laboratory tests, or field tests. 

In the United States, several tests using bacteria as the test organisms have been adopted as standardized methods. The primary test used is the use-dilution method from the AOAC (Association of Official Analytical Chemists). The American Society for Testing and Materials (ASTM) has also developed virucidal test procedures. In western Europe, disinfection tests are published as national standards in France (AFNOR), the United Kingdom (BSI), and Germany (DIN). 

The efficacy tests used are the MIC test and other tests based on the same principle as the disinfectant tests (see Sec. II. B). The bacteria used in the tests are chosen among strains representative of those found in the home for example, bacteria from the Pseudomonas family, the Enterobacteriaceae, yeast such as Candida, molds such as Aspergillus niger, and others. 

Whereas these preparations do not possess the high bacteriostatic activity of quaternary ammonium germicides, they have the alternate advantage of being rapidly functional in acid solution. In comparative experiments of several different disinfectants, the acid—anionic killed bacteria at lower concentration than five other disinfectants. Only sodium hypochlorite and an iodine product were effective at higher dilution than the acid—anionic. By the AO AC use dilution test, the acid—anionic killed Pseudomonas aeruginosa at 225 ppm. Salmonella choleraesuis at 175 ppm, and Staphylococcus aureus at 325 ppm (172). 

Available Chlorine Test. The chlorine germicidal equivalent concentration test is a practical-type test. It is called a capacity test. Under practical conditions of use, a container of disinfectant might receive many soiled, contaminated instniments or other items to be disinfected. Eventually, the capacity of the disinfectant to serve its function would be overloaded due to reaction with the accumulated organic matter and organisms. The chlorine germicidal equivalent concentration test compares the load of a culture of bacteria that a concentration of a disinfectant will absorb and still kill bacteria, as compared to standard concentrations of sodium hypochlorite tested similarly. In the test, 10 successive additions of the test culture are added to each of 3 concentrations of the hypochlorite. One min after each addition a sample is transferred to the subculture medium and the next addition is made 1.5 min after the previous one. The disinfectant is then evaluated in a manner similar to the phenol coefficient test. For equivalence, the disinfectant must yield the same number of negative tubes as one of the chlorine standards. 

Tuberculocidal Test. The tubercle bacillus is resistant to disinfectants because the cells are protected with a waxy coating that is not readily penetrated. The tuberculocidal test is a use dilution practical type test that employs porcelain cylinders. The bacteria are different from those in the use dilution method (Table 10), the incubation time is longer, and the details of the procedure are different. For example, in the tuberculocidal test the test is divided into two parts, a presumptive test and a confirmatory test. The former employs Mycobacterium smegmatis and the latter employs Mycobacterium bovis (BCG). For the presumptive test the incubation time is 12 days, as against 48 hours for other bacteria used in the use-dilution method. For the confirmatory test the incubation time is 60 days, with an additional 30 days in case there is no growth. As shown in Table 10, the concentrations of the phenol standard are higher than used with other bacteria. 

In all antiseptic testing, it is recognized that skin and mucous membranes to which products ate appHed cannot be disinfected or sterilized but it is possible to significantly reduce the population of transient and resident pathogenic bacterial flora. AH in vivo test methods requite a deterrnination of the bacteria on the skin before and after treatment. Because of the normal variation in bacterial population of the skin of different people, a number of people must be tested in order to make a statistical analysis of the results. Different parts of the body are used for different tests. In aH of the tests the details of the protocol ate extremely important and must be strictly adhered to in order to obtain reproducible results. 

Stainless steel disks are contaminated with a bacterial test suspension and dried. The disinfectant is applied on the dried film on the disk and kept at a specified temperature for a defined time. The disk is than transferred to a previously validated neutralization medium to stop the action of the disinfectant. The cfii of surviving bacteria recovered from the surface is determined quantitatively. 

In a parallel test with water instead of disinfectant the colony forming units (cfu) of surviving bacteria are determined and the reduction in viable counts is calculated. 

In the control of chlorine disinfectant systems, the effective use of the chlorine for its intended purpose is assumed if the treated water considerably downstream from the chlorinalor contains a residual of chlorine. Depending upon use. lull-contact tinte may be assumed alter len miuules. or the interval may be extended lo several hnurs. The systems also are usually carefully monitored by bacteriological testing. Normally a dose of I lo 2 milligrams of chlorine per liter is adequate lo destroy all bacteria and leave an effective residual. Residuals of 0.1 to 0.2 milligrams per liter are usually maintained in the diluent streams front water-treatment plants as a factor of safely for consumers. 

PHENOL COEFFICIENT. In determining the effectiveness of a disinfectant using phenol as a standard of comparison, the phenol coefficient is a value obtained by dividing the highest dilution of the test disinfectant by the highest dilution of phenol that sterilizes a given culture of bacteria under standard conditions of time and temperature. 

Chick s early work at the Lister, undertaken with Charles Martin,37 was on the chemical kinetics of the disinfection process. Of particular importance, she found that the temperature dependence of the disinfectant action did not follow the Arrhenius rate expression instead, the rate increased by as much as seven- or eight-fold for a 10°C increase in temperature, thus showing that warm disinfectant solutions were far better for killing bacteria than cold solutions. This led to her being the co-developer of the Chick-Martin Test for the efficacy of a disinfectant. 

The primary purpose of the Sterilamp tube in air-conditioning systems has been to destroy microorganisms (22). Tests on the effect of 1 to 2 p.p.m. by voliune of ozone on E. coli sprayed into an air duct revealed that the organisms were not destroyed. This would confirm the data of Elford and Van den Ende ( ) that ozone is a poor disinfectant of air at low relative humidity. At high relative humidity these authors found that as low as 0.04 p.p.m. by volume destroyed bacteria dispersed in an aerosol. This would also agree with the results reported here, that organisms on surfaces and seeded on Petri plates can be destroyed by minute amounts of ozone. 

Phenol coefficient tests were developed in the early 20th century when typhoid fever was a significant public health problem and phenolics were used to disinfect contaminated utensils and other inanimate objects. Details of such tests can be found in earlier editions of this book. However, as non-phenolic disinfectants became more widely available, tests that more closely paralleled the conditions under which disinfectants were being used (e.g. blood spills) and which included a more diverse range of microbial types (e.g. viruses, bacteria, fungi, protozoa) were developed. Evaluation of a disinfectant s efficacy was based on its ability to kill microbes, i.e. its cidal activity, under environmental conditions mimicking as closely as possible real life situations. As an essential component of each test was a final viability assay, removal or neutralization of any residual disinfectant became a significant consideration. 

Compared with suspended (planktonic) cells, bacteria on surfaces as biofilms are invariably phe-notypically more resistant to antimicrobial agents. With biofilms, suspension tests can be modified to involve biofilms produced on small pieces of an appropriate glass or metal substrate, or on the bottom of microtitre tray wells. After being immersed in, or exposed to the disinfectant solution for the appropriate time interval, the cells from the biofilm are removed, e.g. by sonication, and resuspended in a suitable neutralizing medium. Viable counts are then performed on the resulting planktonic cells. 

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