Equipment used in Microbiology Laboratory

LIST OF EQUIPMENT IN

MICROBIOLOGY LABORATORY

This Post Include most common equipment that are used in Microbiology Laboratory. However there are many of the equipment that are not listed below. 

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Antibiotics are not a magic bullet

Contamination in Cell Culture

Contamination with microorganisms, such as mycoplasma that cannot be detected by bright field microscopy, as well as cross-contamination with other eukaryotic cells which results in misidentified cell lines, continue to present the most severe challenges afflicting cell culture laboratories worldwide.

• To this day, many researchers consider antibiotics to be a reliable measure to prevent microbial contaminations. Whereas it may not be widely known that most antibiotics used in cell culture do not affect mycoplasma, even the more obvious drawbacks of using antibiotics to control bacterial infections are often ignored. Antibiotics offer a false sense of security: is the prophylactic use of antibiotics in cell culture worth the ensuing disadvantages?

• Development of antibiotic-resistant bacterial strains.
• Low-level contaminations with partially resistant bacteria which are difficult to detect.
• Longer periods of cultivating cells harboring undetected contaminations increase the risk of spreading the contamination throughout the lab.
• Mycoplasma infections may take hold more easily due to the absence of any visible signs of contamination as well as longer cultivation periods.
• A false sense of security may foster poor aseptic technique.
• Antibiotics can have adverse effects on the metabolism of eukaryotic cells.

Quarantine helps avoid the spread of contamination

Tissue materials and cells which have been transferred between laboratories must be considered a potential source of contamination until the absence of microorganisms has been documented by validated methods, or until authentication of the cell line has been performed. Ideally, such materials should be kept in a separate room that is equipped with its own biosafety cabinet and CO₂ incubator. Only after biological materials have tested negative for contamination and cell lines have been authenticated will they be transferred to the general cell culture laboratory.

In general, primary cells or tissue cultures always bear the risk of endogenous contamination. Therefore they should be separated from continuous cell lines. Again, the ideal solution would be a separate cell culture room. If this is not possible, a separate designated quarantine CO₂ incubator and biosafety cabinet will help reduce the risk of spreading contamination.  If the option of a separate room or separate equipment is not given, it is recommended to confine suspect cells to a separate shelf in the incubator and use them last, at the end of the day, after all "clean" cultures have been maintained and all experimental work has been completed.

Get to know your enemy

It is generally not necessary to identify the exact species of a contaminant.  However, if the same type of contamination becomes a frequent occurrence in the lab, it may be worth taking a detailed look at the creatures ruining your cultures. It is hereby important to recognize the type of contamination you are dealing with (e.g. bacterial or yeast contamination). 

Did you ever wonder about mycoplasma, or why the medium turns yellow if your culture is contaminated with bacteria? Find out about the most common types of contaminants identified in cell culture laboratories worldwide. Learn how to recognize and fight contamination.  What does it look like? What impact does it have on your cells?

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Organisation of Human Genome

Organisation of Human Genome

KEY CONCEPTS

• The human genome is subdivided into a large nuclear genome with more than 26,000 genes, and a very small circular mitochondrial genome with only 37 genes. The nuclear genome is distributed between 24 linear DNA molecules, one for each of the 24 different types of human chromosome.
•  Human genes are usually not discrete entities: their transcripts frequently overlap those from other genes, sometimes on both strands.
• Duplication of single genes, subchromosomal regions, or whole genomes has given rise to families of related genes.
• Genes are traditionally viewed as encoding RNA for the eventual synthesis of proteins, but many thousands of RNA genes make functional noncoding RNAs that can be involved in diverse functions.
• Noncoding RNAs often regulate the expression of specifi c target genes by base pairing with their RNA transcripts.
• Some copies of a functional gene come to acquire mutations that prevent their expression. These pseudogenes originate either by copying genomic DNA or by copying a processed RNA transcript into a cDNA sequence that reintegrates into the genome (retrotransposition).
• Occasionally, gene copies that originate by retrotransposition retain their function because of selection pressure. These are known as retrogenes.
• Transposons are sequences that move from one genomic location to another by a cut-andpaste or copy-and-paste mechanism. Retrotransposons make a cDNA copy of an RNA transcript that then integrates into a new genomic location.
• Very large arrays of high-copy-number tandem repeats, known as satellite DNA, are associated with highly condensed, transcriptionally inactive heterochromatin in human chromosomes.

The human genome comprises two parts: a complex nuclear genome with more than 26,000 genes, and a very simple mitochondrial genome with only 37 genes. The nuclear genome provides the great bulk of essential genetic information and is partitioned between either 23 or 24 different types of chromosomal DNA molecule (22 autosomes plus an X chromosome in females, and an additional Y chromosome in males). Mitochondria possess their own genome—a single type of small circular DNA—encoding some of the components needed for mitochondrial protein synthesis on mitochondrial ribosomes. However, most mitochondrial proteins are encoded by nuclear genes and are synthesized on cytoplasmic ribosomes before being imported into the mitochondria. sequence comparisons with other mammalian genomes and vertebrate genomes indicate that about 5% of the human genome has been strongly conserved during evolution and is presumably functionally important. Protein-coding DNA sequences account for just 1.1% of the genome. The other 4% or so of strongly conserved genome sequences consists of non protein-coding DNA sequences, including genes whose final products are functionally important RNA molecules, and a variety of cis-acting sequences that regulate gene expression at DNA or RNA levels. Although sequences that make non-protein-coding RNA have not generally been so well conserved during evolution, some of the regulatory sequences are much more strongly conserved than protein-coding sequences. Protein-coding sequences frequently belong to families of related sequences that may be organized into clusters on one or more chromosomes or be dispersed throughout the genome. Such families have arisen by gene duplication during evolution. The mechanisms giving rise to duplicated genes also give rise to nonfunctional gene-related sequences (pseudogenes). One of the big surprises in the past few years has been the discovery that the human genome is transcribed to give tens of thousands of different non coding RNA transcripts, including whole new classes of tiny regulatory RNAs not previously identified in the draft human genome sequences published in 2001. Although we are close to obtaining a definitive inventory of human protein-coding genes, our knowledge of RNA genes remains undeveloped. It is abundantly clear, however, that RNA is functionally much more versatile than we previously suspected. In addition to a rapidly increasing list of human RNA genes,we have also become aware of huge numbers of pseudogene copies of RNA genes. A very large fraction of the human genome, and other complex genomes, is made up of highly repetitive non coding DNA sequences. A sizeable component is organized in tandem head-to-tail repeats, but the majority consists of interspersed repeats that have been copied from RNA transcripts in the cell by reverse transcriptase. There is a growing realization of the functional importance of such repeats. In this article we primarily consider the architecture of the human genome. We outline the different classes of DNA sequence, describe briefly what their function is, and consider how they are organized in the human genome. In later article we describe other aspects of the human genome: how it compares with

other genomes, and how evolution has shaped it, DNA sequence variation and polymorphism, and aspects of human gene expression.

Test Methods for Disinfectants

     Test Methods for Disinfectants

Standardized tests for disinfectants have existed worldwide for over 20 years. However, not all tests are the same.

Disinfectants used in hospitals and laboratories must be tested periodically to ascertain its potency and efficacy. As certain disinfectants lose potency on standing and addition of organic matter, their efficacy must be tested. While certain methods help in selecting the right dilution of disinfectant for use others test the efficacy of disinfectant already in use. Some methods compare the performance with that of phenol whereas other methods simply state if the disinfectant is effective or not. There are several methods of testing disinfectants, with their own advantages and disadvantages. All these tests can be allocated to one of the following disinfectant tests: carrier test, suspension test, capacity test, practical test, field test or in-use test. Disinfection process validation is defined as "establishing documented evidence that a disinfection process will consistently remove or inactivate known or possible pathogens from inanimate objects." Robert Koch described a disinfectant test in the article Uber Desinfektion, in 1881. A silk thread was contaminated by submersion in a liquid culture of Bacillus anthracis. After drying, the contaminated thread was immersed in several disinfectant solutions for a given exposure time. The thread was then cultured in a nutrient broth and no growth after incubation indicated activity of the disinfectant. He concluded from the comparisons of disinfectant solutions that mercuric chloride was the most active disinfectant. The results were erroneous as the disinfectants residues were also carried over to the subculture medium. The problem was overcome by Geppert in 1890 by neutralising the disinfectant at the end of exposure period.

Carrier tests:

These tests are the oldest tests. The test described by Robert Koch was a carrier test. In these tests, the carrier such as a silk or catgut thread or a penicylinder (a little stick) is contaminated by submersion in a liquid culture of the test organism. The carrier is then dried and is brought in contact with the disinfectant for a given exposure time. After the exposure, it is cultured in a nutrient broth; no growth indicates activity of the disinfectant tested whereas growth indicates a failing. By multiplying the number of test concentrations of the disinfectant and the contact times, a potentially active concentration-time relationships of the disinfectant is obtained. Example of a carrier test is the former use-dilution test of the American Association of Official Analytical Chemists (AOAC, 1990). Limitation of the carrier tests are: a) the number of bacteria dried on a carrier is hard to standardize and b) the survival of the bacteria on the carrier during drying is not constant. The AOAC Use-dilution test is a carrier-based test. The organisms used are Salmonella cholerasuis, S. aureus and P. aeruginosa. Carriers (stainless steel cylinders) are meticulously cleaned, sterilized by autoclaving in a solution of aspargine, cooled and inoculated with a test organism by immersing in one of the culture suspensions. The cylinders are drained on filter paper, dried at 37o C for 40 minutes, exposed to the use-dilution of the disinfectant for 10 minutes, and cultured to assess the survival of the bacteria. A single test involves the evaluation of 60 inoculated carriers (one organism) against one product sample. In addition to the 60 carriers, 6 carriers are required to estimate carrier bacterial load and 6 more are included as extras. Thus, a total of 72 seeded carriers are required to perform a single test. A result showing no growth in all ten tubes confirms the result of phenol coefficient test. If any carrier produces growth, the test must be repeated using a lower dilution of the disinfectant. Use-dilution test is performed to confirm the efficiency of disinfectant dilution derived from phenol coefficient test.

Suspension tests:

In these tests, a sample of the bacterial culture is suspended into the disinfectant solution and after exposure it is verified by subculture whether this inoculum is killed or not. Suspension tests are preferred to carrier tests as the bacteria are uniformly exposed to the disinfectant. There are different kinds of suspension tests: the qualitative suspension tests, the test for the determination of the phenol coefficient (Rideal and Walker, 1903) and the quantitative suspension tests. Initially this was done in a qualitative way. A loopful of bacterial suspension was brought into contact with the disinfectant and again a loopful of this mixture was cultured for surviving organisms. Results were expressed as ‘growth’ or ‘no growth’. In quantitative methods, the number of surviving organisms is counted and compared to the original inoculum size. By subtracting the logarithm of the former from the logarithm of the latter, the decimal log reduction or microbicidal effect (ME) is obtained. An ME of 1 equals to a killing of 90% of the initial number of bacteria, an ME of 2 means 99% killed. A generally accepted requirement is an ME that equals or is greater than 5: at least 99.999% of the germs are killed. Even though these tests are generally well standardized, their approach is less practical.

Determination of phenol coefficient:

Phenol coefficient of a disinfectant is calculated by dividing the dilution of test disinfectant by the dilution of phenol that disinfects under predetermined conditions.

Rideal Walker method:

Phenol is diluted from 1:400 to 1:800 and the test disinfectant is diluted from 1:95 to 1:115. Their bactericidal activity is determined against Salmonella typhi suspension. Subcultures are performed from both the test and phenol at intervals of 2.5, 5, 7.5 and 10 minutes. The plates are incubated for 48-72 hours at 37°C. That dilution of disinfectant which disinfects the suspension in a given time is divided by that dilution of phenol which disinfects the suspension in same time gives its phenol coefficient.

For example, after 7.5 minutes, the test organism was killed by the test disinfectant at a dilution of 1;600. In the same period the test organism was killed by phenol at a dilution of 1:100.

Phenol coefficient = 600/100 = 6

This result indicates that the test disinfectant can be diluted six times as much as phenol and still possess equivalent killing power for the test organism. Disadvantages of the Rideal-Walker test are: No organic matter is included; the microorganism Salmonella typhi may not be appropriate; the time allowed for disinfection is short; it should be used to evaluate phenolic type disinfectants only.

Chick Martin test:

This test also determines the phenol coefficient of the test disinfectant. Unlike in Rideal Walker method where the test is carried out in water, the disinfectants are made to act in the presence of yeast suspension (or 3% dried human feces) to simulate the presence or organic matter. Time for subculture is fixed at 30 minutes and the organism used to test efficacy is S.typhi as well as S.aureus. The phenol coefficient is lower than that given by Rideal Walker method.

The phenol coefficient test recommended by AOAC included two test organisms (S. aureus and P. aeruginosa) and included the disinfectant inactivators in the recovery medium. The recovery medium Letheen broth contains the inactivator Lecithin and Polysorbate 80. In separate tests, the bacterial suspensions are added to standard dilutions of pure phenol and several dilutions of the test disinfectant. After contact time of 5, 10 and 15 minutes, samples are transferred to the recovery medium by a standard wire loop. When the positive and negative cultures have been recorded, the result of the test is expressed as phenol coefficient. It is calculated by dividing the highest dilution of the disinfectant that kills the test inoculum in ten minutes but not in five minutes by the dilution of phenol that gives the same result.

Disinfectant kill time test

This test was designed to demonstrate log reduction values over time for a disinfectant against selected bacteria, fungi, and/or mold. The most common organisms tested include: Bacillus subtilis, Bacillus atrophaeus, Bacillus thuringiensis, Staphylococcus aureus, Salmonella cholerasuis, Pseudomonas aeruginosa, Aspergillus niger, and Trichophyton mentagrophytes. A tube of disinfectant is placed into a waterbath for temperature control and allowed to equilibrate. Once the tube has reached temperature, it is inoculated to achieve a concentration of approximately 106 CFU/mL. At selected time points (generally five points are used including zero) aliquots are removed and placed into a neutralizer blank. Dilutions of the neutralizer are made and selected dilutions plated onto agar. Colonies are enumerated and log reductions are calculated.

Capacity tests:

Each time a soiled instrument is placed into a container with disinfectant, a certain quantity of dirt and bacteria is added to the solution. The ability to retain activity in the presence of an increasing load is the capacity of the disinfectant. In a capacity test, the disinfectant is challenged repeatedly by successive additions of bacterial suspension until its capacity to kill has been exhausted. Capacity tests simulate the practical situations of housekeeping and instrument disinfection. The best known capacity test is the Kelsey-Sykes test (Kelsey and Sykes, 1969).

Kelsey-Sykes test is a triple challenge test, designed to determine concentrations of disinfectant that will be effective in clean and dirty conditions. The disinfectant is challenged by three successive additions of a bacterial suspension during the course of the test. The duration of test takes over 30 minutes to perform. The concentration of the disinfectant is reduced by half by the addition of organic matter (autoclaved yeast cells), which builds up to a final concentration of 0.5%. Depending on the type of disinfectant, a single test organism is selected from S. aureus, P. aeruginosa, P. vulgaris and E. coli. The method can be carried out under 'clean' or 'dirty' conditions. The dilutions of the disinfectant are made in hard water for clean conditions and in yeast suspension for dirty conditions. Test organism alone or with yeast is added at 0, 10 and 20 minutes interval. The contact time of disinfectant and test organism is 8 min. The three sets of five replicate cultures corresponding to each challenge are incubated at 32o C for 48 hours and growth is assessed by turbidity. The disinfectant is evaluated on its ability to kill microorganisms or lack of it and the result is reported as a pass or a fail and not as a coefficient. Sets that contain two or more negative cultures are recorded as a negative result. The disinfectant passes at the dilution tested if negative results are obtained after the first and second challenges. The third challenge is not included in the pass/fail criterion but positive cultures serve as inbuilt controls. If there are no positive cultures after the third challenge, a lower concentration of the disinfectant may be tested.

The capacity test of Kelsey and Sykes gives a good guideline for the dilution of the preparation to be used. Disadvantage of this test is the fact that it is rather complicated.

Test for stability and long-term effectiveness:

Recommended concentrations based on Kelsey Sykes test apply only to freshly prepared solutions but if the solutions are likely to be kept for more than 24 hours, the effectiveness of these concentrations must be confirmed by a supplementary test for stability of unused solution and for the ability of freshly prepared and stale solutions to prevent multiplication of a small number of bacteria that may have survived the short term exposure. P. aeruginosa is used a test organism. Sufficient disinfectant solution is prepared for two tests. One portion is inoculated immediately and tested for growth after holding for seven days at room temperature. The other portion is kept at room temperature for seven days and then inoculated with a freshly prepared suspension of test organism. It is also tested for growth seven days after inoculation. If growth is detected, a higher concentration of disinfectant must be tested in the same way.

Practical tests:

The practical tests under real-life conditions are performed after measuring the time-concentration relationship of the disinfectant in a quantitative suspension test. The objective is to verify whether the proposed use dilution is still adequate in the conditions under which it would be used. The best known practical tests are the surface disinfection tests. Surface tests assess the effectiveness of the selected sanitizer against surface-adhered microorganisms. The test surface (a small tile, a microscopic slide, a piece of PVC, a stainless steel disc, etc.) is contaminated with a standardized inoculum of the test bacteria and dried: then a definite volume of the disinfectant solution is distributed over the carrier; after the given exposure time the number of survivors is determined by impression on a contact plate or by a rinsing technique, in which the carrier is rinsed in a diluent, and the number of bacteria is determined in the rinsing fluid. In order to determine the spontaneous dying rate of the organisms caused by drying on the carrier, a control series is included in which the disinfectant is substituted by distilled water; from the comparison of the survivors in this control series with the test series, the reduction is determined quantitatively.

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 continues to dry during the exposure. Surface tests can reflect in-use conditions like contact times, temperatures, use-dilutions, and surface properties.

Surface Time kill Test

A 24 hour culture in nutrient broth culture is prepared. A volume of microbial culture (usually 0.010 mL to 0.020 mL) is placed onto the center of each of a number of sterile test surfaces. This inoculum can be spread over the sterile test surface in a circular pattern to achieve a thin, uniform coverage with the test microorganism if desired. To measure initial microbial concentrations, one or more untreated, inoculated test surfaces are harvested and microorganisms are enumerated. The remaining inoculated test surfaces are treated with the disinfectant, each for a different length of time. Immediately after the treatment times have elapsed, the test surfaces are placed into a solution that neutralizes the disinfecting action of the product, and microorganisms surviving treatment with the disinfectant or sanitizer are cultured and enumerated. Results of the timekill study are tabulated and reported, usually by charting microbial concentrations on the test surfaces as a function of treatment time with the disinfectant or sanitizer.

In-use test:

A simple to use test was described by Maurer in 1985 that can be used in hospitals and laboratories to detect contamination of disinfectants. A 1 ml sample of the disinfectant is added to 9 ml diluent which also contains an inactivator. Ten drops, each of 0.02 ml volume of the diluted sample are placed on each of two nutrient agar plates. One is incubated at 37o C for three days and the other at room temperature for seven days. Five or more colonies on either plate indicate contamination.

British standard tests for quaternary ammonium compounds:

This test was initially described in 1960 to distinguish bactericidal action from the high level of bacteriostatic activity, which is characteristic of QACs. This test is also applicable to other bactericides such as chlorhexidine and synthetic phenols. The inactivator used contains 2% lecithin and 3% non-ionic detergent (polysorbate 80). The test is performed using suspensions of gram negative and gram positive bacteria with or without the inclusion of organic matter. If a series of samples are taken from a dilution of the disinfectant containing 5 x108 to 5 x 109 bacteria per ml at the start of the test, a death curve may be prepared from the colony counts on the agar containing recovery medium and reduction factors up to 106 (99.99% kill) can be verified. In order to determine the antimicrobial value of QAC, it was revised as the highest dilution of the disinfectant that, under the test conditions, will reduce the microbial population to a colony count not greater than 0.01% of that in the control. In this revision, E. coli was used and the contact time was 10 minutes. The challenge medium is E. coli culture suspension with equal amount of horse serum. One ml of the challenge is added to nine ml of each dilution of the disinfectant along with two control tubes containing diluent alone. At the end of exposure period, one ml each of the mixture is added to 9 ml of inactivator and the surviving bacteria are counted as colony forming units on agar plates. 

Testing schemes:

The antimicrobial efficiency of a disinfectant is examined at three stages of testing. The first phase concerns laboratory tests in which it is verified whether a chemical compound or a preparation possesses antimicrobial activity: for these preliminary screening tests essentially quantitative suspension tests are considered. The second stage is still carried out in the laboratory but in conditions simulating real-life conditions. Not disinfectants, but disinfection procedures are examined. It is determined in the practical tests in which conditions and at which use-dilution after a given contact time the preparation is active. The third phase comprises the field tests or pilot studies, and the variant of in-use tests. In these tests it is verified whether, after a normal period of use, germs in the disinfectant solution are still killed. Most studied are the bactericidal tests in which the activity towards vegetative bacteria is examined. AOAC has schedules that are applicable for fungi and yeasts too (fungicidal tests), for mycobacteria (tuberculocidal tests), for viruses (virucidal tests) and for spores of bacteria (sporicidal tests). 

Bactericidal tests:

A bactericidal test must include the following sequence of steps: 1. The test organism is exposed to a suitable concentration of the disinfectant 2. Samples are taken at specified times and added immediately to a diluent or culture medium containing the appropriate disinfectant inactivator 3. The treated samples are cultured for surviving microorganisms.

Test organisms:

Specified strains (usually ATCC) of S. aureus, P. aeruginosa, P. vulgaris and E. coli are usually recommended. A synthetic broth is recommended for preparing a series of subcultures to be used in the tests. The 24-hour broth culture may be used without further treatment; however, it is usually filtered (to remove slime) and centrifuged. The washed bacteria are resuspended in hard water to which autoclaved yeast or serum may be added to simulate dirty conditions. Finally, the suspension is shaken with glass beads on a vortex mixer and a viable count is set up immediately before performing the test.

The disinfectant:

The concentration or dilution of the disinfectant to be tested may be based on manufacturer’s recommendations. The solutions should be prepared on the day of test. Distilled water or standard hard water is used to make dilutions. Tap water is unsuitable because it may contain chemicals that may precipitate with some disinfectants.

References:

The testing of disinfectants: Gerald Reybrouck, International Biodeterioration & Biodegradation 41 (1998) 269-272

Introduction to sterilization and disinfection control, 2nd edition, Churchill Livingstone Joan F.Gardner, Margaret M Peel. 1991

Testing of Disinfectant

Testing of Disinfectant

Introduction

Disinfection describes a process that eliminates many or all pathogenic microorganisms, except bacterial spores, on inanimate objects. In health-care settings, objects usually are disinfected by liquid chemicals or wet pasteurization. Each of the various factors that affect the efficacy of disinfection can nullify or limit the efficacy of the process. Factors that affect the efficacy of both disinfection and sterilization include prior cleaning of the object; organic and inorganic load present; type and level of microbial contamination; concentration of and exposure time to the germicide; physical nature of the object (e.g., crevices, hinges, and lumens); presence of biofilms; temperature and pH of the disinfection process; and in some cases, relative humidity of the sterilization process (e.g., ethylene oxide). Unlike sterilization, disinfection is not sporicidal. A few disinfectants will kill spores with prolonged exposure times (3–12 hours); these are called chemical sterilants. At similar concentrations but with shorter exposure periods (e.g., 20 minutes for 2% glutaraldehyde), these same disinfectants will kill all microorganisms except large numbers of bacterial spores; they are called high-level disinfectants. Low level disinfectants can kill most vegetative bacteria, some fungi, and some viruses in a practical period of time. Intermediate-level disinfectants might be cidal for mycobacteria, vegetative bacteria, most viruses, and most fungi but do not necessarily kill bacterial spores. Germicides differ markedly, primarily in their antimicrobial spectrum and rapidity of action.

Disinfectants are antimicrobial agents that are applied to non-living objects to destroy microorganisms that are living on the objects. Disinfection does not necessarily kill all microorganisms, especially resistant bacterial spores; it is less effective than sterilization, which is an extreme physical and/or chemical process that kills all types of life. Disinfectants are different from other antimicrobial agents such as antibiotics, which destroy microorganisms within the body, and antiseptics, which destroy microorganisms on living tissue. Disinfectants are also different from biocides — the latter are intended to destroy all forms of life, not just microorganisms. Disinfectants work by destroying the cell wall of microbes or interfering with the metabolism.

Sanitizers are substances that simultaneously clean and disinfect. Disinfectants are frequently used in hospitals, dental surgeries, kitchens, and bathrooms to kill infectious organisms.

Bacterial endospores are most resistant to disinfectants, but some viruses and bacteria also possess some tolerance. In wastewater treatment, a disinfection step with chlorine, ultra-violet (UV) radiation or ozonation can be included as tertiary treatment to remove pathogens from wastewater, for example if it is to be reused to irrigate golf courses. An alternative term used in the sanitation sector for disinfection of waste streams, sewage sludge or fecal sludge is sanitisation or sanitization.

Disinfectants used in hospitals and laboratories must be tested periodically to ascertain its potency and efficacy.

Properties

A perfect disinfectant would also offer complete and full microbiological sterilisation, without harming humans and useful form of life, be inexpensive, and noncorrosive. However, most disinfectants are also, by nature, potentially harmful (even toxic) to humans or animals. Most modern household disinfectants contain Bitrex, an exceptionally bitter substance added to discourage ingestion, as a safety measure. Those that are used indoors should never be mixed with other cleaning products as chemical reactions can occur. The choice of disinfectant to be used depends on the particular situation. Some disinfectants have a wide spectrum (kill many different types of microorganisms), while others kill a smaller range of disease-causing organisms but are preferred for other properties (they may be non-corrosive, non-toxic, or inexpensive). There are arguments for creating or maintaining conditions that are not conducive to bacterial survival and multiplication, rather than attempting to kill them with chemicals. Bacteria can increase in number very quickly, which enables them to evolve rapidly. Should some bacteria survive a chemical attack, they give rise to new generations composed completely of bacteria that have resistance to the particular chemical used. Under a sustained chemical attack, the surviving bacteria in successive generations are increasingly resistant to the chemical used, and ultimately the chemical is rendered ineffective. For this reason, some question the wisdom of impregnating cloths, cutting boards and worktops in the home with bactericidal chemicals.

Measurements of effectiveness

One way to compare disinfectants is to compare how well they do against a known disinfectant and rate them accordingly. Phenol is the standard, and the corresponding rating system is called the "Phenol coefficient". The disinfectant to be tested is compared with phenol on a standard microbe (usually Salmonella typhi or Staphylococcus aureus). Disinfectants that are more effective than phenol have a coefficient > 1. Those that are less effective have a coefficient < 1.

The standard European approach for disinfectant validation consists of a basic suspension test, a quantitative suspension test (with low and high levels of organic material added to act as ‘interfering substances’) and a two part simulated-use surface test. A less specific measurement of effectiveness is the United States Environmental Protection Agency (EPA) classification into either high, intermediate or low levels of disinfection. "High-level disinfection kills all organisms, except high levels of bacterial spores" and is done with a chemical germicide marketed as a sterilant by the U.S. Food and Drug Administration (FDA). "Intermediate-level disinfection kills mycobacteria, most viruses, and bacteria with a chemical germicide registered as a 'tuberculocide' by the Environmental Protection Agency. Low-level disinfection kills some viruses and bacteria with a chemical germicide registered as a hospital disinfectant by the EPA."

An alternative assessment is to measure the Minimum inhibitory concentrations (MICs) of disinfectants against selected (and representative) microbial species, such as through the use of microbroth dilution testing.