Indians Make up Over 90% HIV Infected Kids and Teens in South Asia

This World AIDS Day, a report by UNICEF gives a reality check on how far the world is from fulfilling its pledge of ending AIDS by 2030. While commending the remarkable progress made in the past decade among children aged 0-9 years, it sheds light on the lack of efforts to prevent HIV among adolescents.

"A staggering 360,000 adolescents are projected to die of AIDS-related diseases between 2018 and 2030 without additional investment in HIV prevention, testing and treatment programs."The World Today

• 3.0 million children and adolescents are living with HIV. Nearly nine in ten live in sub-Saharan Africa.
• 430,000 children and adolescents became newly infected with the virus in 2017.
• 130,000 children and adolescents died from AIDS-related causes in 2017.

The World in 2030

• 1.9 million children and adolescents are projected to be living with HIV
• 270,000 children and adolescents are projected to become newly infected with the virus annually
• 56,000 children and adolescents are projected to die from AIDS-related causes annually
• 2.0 million new HIV infections could be averted between 2018 and 2030 if global goals are met – 1.5 million of these would be averted among adolescents

HIV in South Asia & India

An estimated 1,20,000 children and adolescents aged 0-19 were living with HIV in India in 2017, the highest number in South Asia.

An estimated 1,20,000 children and adolescents aged 0-19 were living with HIV in India in 2017, the highest number in South Asia, followed by Pakistan’s 5,800, Nepal’s 1600 and Bangladesh’s less than 1000.

However, the report also mentions that South Asia has made substantial progress in reducing HIV risks and vulnerability among children, adolescents, pregnant women, and mothers.

In 2017, the estimated number of children under 5 years old newly diagnosed with HIV was 43 per cent lower than the comparable estimate in 2010 — a decline greater than the 35 per cent recorded globally.

Also, the estimated share of those aged 0–14 living with HIV who had been initiated on lifesaving antiretroviral therapy (ART) was 73 percent in 2017, an increase of nearly 50 percentage points from 2010.

After an overall analysis of the world by major geographical areas, the report makes the need for more efforts and programmes towards HIV prevention and treatment evident.

In the words of UNICEF chief Henrietta Fore, "The report makes it clear, without the shadow of a doubt, that the world is off track when it comes to ending AIDS among children and adolescents by 2030.”

BACTERIA TOO CAN BECOME EXTINCT

Bacteria do become extinct at substantial rates, finds a new study, contradicting widely held scientific thinking that the microbes rarely die because of their very large population.

For the study, published in Nature Ecology and Evolution, the team used massive DNA sequencing and big data analysis to create the first evolutionary tree encompassing a large fraction of Earth's bacteria over the past billion years.

"Bacteria rarely fossilise, so we know very little about how the microbial landscape has evolved over time," said lead author Stilianos Louca, from University of British Columbia (UBC) in Canada.

"Sequencing and math helped us fill in the bacterial family tree, map how they have diversified over time, and uncover their extinctions," she added.

Despite the frequent, steady extinction of individual species, the study shows that overall bacteria have been diversifying exponentially without interruption.

The team estimated 1.4-1.9 million bacterial lineages exist on Earth today. However, 45,000 to 95,000 extinctions occurred in the last million years.

"While modern bacterial diversity is undoubtedly high, it's only a tiny snapshot of the diversity that evolution has generated over Earth's history," Louca said.

The researchers suspected that competition between bacterial species drive the high rate of microbial extinctions, leaving them less prone to sudden mass, multi-species extinctions.

Infection

Infection

in·fec·tion

•Invasion and multiplication of pathogenic microorganisms in a bodily part or tissue, which may produce subsequent tissue injury and progress to overt disease through a variety of cellular or toxic mechanisms.

•The pathological state resulting from having been infected.

What is an Infection?

A harmful invasion and spread of foreign species, or pathogen, in a host. Such as

–VIRUS

• Small pox, measles, influenza, Ebola

–PRIONS

•Cow’s disease

–BACTERIA

•Tuberculosis, pneumonia, salmonella, anthrax

–FUNGUS

•Athlete’s foot, ring worm

–PROTISTS

•Malaria, toxoplasmosis, Algae

•  Disease and Infectious Disease

–Disease

•Any deviation from a condition of good health and

well-being

–Infectious Disease

A disease condition caused by the presence or growth

of infectious microorganisms or parasites

•Pathogenicity and Virulence

–Pathogenicity

•The ability of a microbe to cause disease

•This term is often used to describe or compare

species

–Virulence 

•The degree of pathogenicity in a microorganism

•This term is often used to describe or compare strains

 within a species

Principles of Infection

•Understanding the basic principles of infection is 

essential for any health care worker in any field of 

health care.

Disease transmission

Prevention of disease transmission

Nature of Microorganisms

•Microorganisms (microbes) are small, living 

organisms that are not visible to the naked eye.

•Pathogens (germs) are microorganisms that cause 

disease.

Non-pathogens are microorganisms that do not 

cause disease; can be beneficial

Nature of  Microorganisms 

•Saprophytes

•Parasites

•Pathogens 

Signs & Symptoms of Infection

 Redness

 Swelling
• Tenderness
• Warmth
• Drainage

• Red streaks leading away from the wound

Development of Infection: Clinical Signs and Symptoms

Local signs

Inflammation

Purulent exudate if bacterial infection; serous exudate if viral

Tissue necrosis

Lymphadenopathy

Respiratory effects

Systemic signs

Fever, fatigue, headache, nausea

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. 

For any other information or queries comment below or inbox us on Microbiology

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?

Comment below or inbox us to more information and queries.

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