MICROBIOLOGICAL CULTURE SENSITIVITY TESTS.docx
- 1. MICROBIOLOGICAL CULTURE SENSITIVITY TESTS Microbiology: • Microbiology is the study of living organisms that are invisible to the naked eye , such as bacteria, fungi and virus. Microbiological culture: • It is a method of multiplying microbial organisms by letting them reproduce in predetermined culture media under controlled laboratory conditions. • Microbial cultures are used to determine the type of organism and its abundance in the sample being tested or both. Definition: They are the in-vitro procedures used to detect anti-microbial resistance in individual bacterial isolates. The same can be used for monitoring the emergence and spread of resistant microorganisms in a population. In modern laboratories, bacteria are usually identified by characterization of the genome : identifying the characteristics of the DNA and RNA of the sample species Sensitivity test: • Also called susceptibility testing • A laboratory test perform to check the effectiveness of a drug against a microorganism, to select the best drug regimen that is effective. Purpose of culture sensitivity tests: • To guide the clinician in selecting the best drug for an individual patient. • To control the use of inappropriate drug in clinical practice.
- 2. • To accumulate epidemiological information on the resistance of microorganism of public health importance within the community. • To reveal the changing trends in the local isolates. • To overcome the microbial drug resistance. Why is sensitivity analysis done? Unfortunately, many bacteria are resistant to common antibiotics. Resistant means that the drug cant kill the bacteria. Sensitivity analysis is a useful tool to help quickly determine if bacteria are resistant to certain drugs. Also be used for fungal infections. Examples of antibiotic resistant infections include: A persistent sore throat A recurring urinary tract infection(UTI) An unresponsive case of pneumonia. How sensitivity analysis done? • Sensitivity analysis starts with a bacterial sample. • The sample is obtained by swabbing the infected area and from any area that has infection. • Cultures are taken from blood, Urine, Sputum, inside cervix and a wound. Steps in analysis: • The doctor will send the specimen to a licensed laboratory in a special culture tube for testing. • There the sample of infective material spread onto a plate of nutrient substance and the bacteria in the culture will grow and multiply. • The bacteria will form colonies- large groups of bacteria that will be exposed to different antibiotics.
- 3. • With a sufficient population of bacteria grown on the plate in the form of a lawn, the technicians will perform two main operations: Identify the species of bacteria: This is done with various techniques, including examination of lawn characteristics-color, texture, growth pattern, etc. Bacterial species commonly isolated depends on the location and the cause of the infection. Gram staining, microscopic examination, metabolic requirements and even DNA sequencing. Determine the bacterial populations sensitivity to arrange of antibiotics: This can be done by placing small disks of filter paper or the agar impregnated with various types of antibiotics onto the bacterial lawn. The bacteria are allowed to incubate for a specified days and then the late is examined to see whether the bacterial growth is inhibited or not by the antibiotics on the each disk. Methods for microbiological culture sensitivity test: 1.DILUTION METHODS :(Broth and Agar dilution method) The broth dilution method involves subjecting a series of concentrations of anti-microbial agents in a broth environment. Micro dilution tests uses about 0.05 to 0.1ml total broth volume and can be conveniently performed in a micro-titre format. Macro dilution testing uses broth volumes at about 1.0ml in standard test tubes. For both these dilution methods, the lowest concentration at which the isolate is completely inhibited – as evidenced by the absence of bacterial growth-is recorded as the minimal inhibitory concentration.
- 4. The minimum inhibitory concentration(MIC) is the minimum concentration of the anti-biotic that will inhibit the particular isolate . The test is only valid only if the positive control shows growth and the negative control shows no growth. A procedure similar to broth dilution is agar dilution. Agar dilution method follows the principle of establishing the lowest concentration of the serially diluted antibiotic concentration at which bacterial growth is still inhibited. 2. Disc diffusion method: • Because of convenience, efficiency , the disc diffusion method is most widely used method. • A growth medium , usually Mueller-Hinton agar , is first even seeded throughout the plate with the isolate of interest that has been diluted at a standard concentration (app 1 to 2x10 colony forming units per ml). • Then , using a dispenser such as the one pictured, antibiotic-impregnated discs are placed onto the agar surface . As the bacteria on the lawn grow , they are inhibited to varying degrees by the antibiotic diffusing from the disc.
- 5. • The test antibiotic immediately begins to diffuse outwards from the discs, creating a gradient of antibiotic concentration. • Place the metric ruler across the zone of inhibition, at the widest diameter, and measure from one edge of the zone to the other edge. Holding the plate up to the light might help. • The disc diameter will actually be part of that number . If there is NO zone at all , report it as 0…..even though the disc itself is around 7mm. • Zone diameter is reported in millimeters , looked up on the chart , and result reported as S(sensitive), R(resistant), or I(intermediate). 3.E-Test • E-Test (AB Bio disk, solna, Sweden) is a commercially available test that utilizes a plastic test strip impregnated with a gradually decreasing concentration of particular antibiotic • The strip also displays a numerical scale that corresponds to the antibiotic concentration contained therein • This method provides for a convenient quantitative test of antibiotic resistance of clinical isolate • However , separate strip is needed for each antibiotic, and therefore the cost be high .
- 6. 4.Genotypic methods • Although nucleic acid –based detection systems are generally rapid and sensitive , it is important to remember that the presence of a resistance gene does not necessarily equate to treatment failure , because resistance is also dependent on the mode and level of expression of these genes • Some of the most common molecular techniques utilized for Anti microbial resistance detection are as follows: • Polymerization chain reaction: • It is one of the most commonly used molecular techniques for detecting certain DNA sequence of interest. This involves several cycles of denaturation of sample DNA, annealing of specific primers to the target sequence and the extension of this sequence, in an exponential manner, to a point ehinch will be visible detectable by gel electrophoresis with the aid of a DNA-intercalating Chemicals fluorescence under UV light. DNA hybridization • This is based on the fact the DNA pyrimidines (cytosine and thymidine) specifically pair up with purines (guanine and adenine; or uracil for RNA) • Therefore, a labelled probe with a know specific sequence can pair up opened or dentures DNA from the Test sample, as long as their sequence complement each other.
- 7. • if this hybridization occurs, the probe labels this with a detectable radioactive isotope, antigenic substrate, enzyme or chemilumnincenst compound. • Whereas if no target sequence is present or the isolate does not have the specific gene of interest, no attachment of probes will occur, and therefore no signals will be detected. • Modifications of the PCR and DNA hybridization with these basic principles, several modifications have been introduced which further improve the sensitivity and specificity of the procedure. • Examples of such development the use of flouresces –labeled oligonucleotides, the development of molecular bacons, development of DNA arrays and DNA chips, Among many others How the method is selected • Selection of the appropriate method will depend on the intended. Degree of accuracy, convenience, urgency, bioavailability of sources, availability of expertise and cost What are the results for a sensitivity analysis • The colonies show up as: • Susceptible, resistance or intermediate Susceptible :in this case, a clear, circular “ halo” –technically known as a” plaque “ or zone of inhibition –will. Appear around the antibiotic disc , indicating an absence of bacteria. The antibiotic has inhibited their growth and /or killed them, means that they can’t grow if the drug is present. This. Indicates an effective antibiotic • The doctor can use the results to determine the best antibiotic to treat your infection Resistant: in the case, the filter paper will have no discernable plaque around it , meaning that the bacteria can grow even in the presence of antibiotic. This indicates an ineffective antibiotic.
- 8. Intermediate :a somewhere cloudy plaque indicates that not all the bacteria in the area around the disc have been killed. This means that a higher dose of the antibiotic is needed to prevent the growth What are the risk of sensitivity analysis • Few risk are associated with this test blood collection comes with small risk . • Rare risk of taking a blood sample include: • Sightedness or fainting. • Infection usually prevented by the skin being cleaned before the needle is inserted. • Excessive bleeding –bleeding for a long period afterwards may indicate a more serious bleeding condition and should be reported to the doctor. • Hematoma –a bruise where blood accumulates under the skin. • The doctor will advise the patient about the potential risk associated with the sample. Conclusion : • An antibiotic that bacteria, fungus, or another microorganisms shows resistance to shouldn’t be used to treat infection. Your doctor will decide which drug is best if several antibiotics are shown to be effective in killing the microorganisms causing the infection. Applications : • To provide a reliable prediction of whether an infection caused by a bacterial isolated will respond therapeutically to a particular antibiotic treatment. • This may be utilized as guidelines for chemotherapy, or at the population level as indicator of emergence and spread of resistance based on passive or active surveillance. Drawbacks :
- 9. • Because of the required culture time, sensitivity testing may Tai several days , which is not ideal in critical cases demanding urgency • Practitioners switch over to anti-bio grams. Common medias used : • The culture media are prepared according to the specifications of the USP , European, pharmacopoeia, British pharmacopoeia and you India pharmacopoeia. • The antibiotic media are identified numerically with names assigned by Grove and Randall seed agar . • Base agar • Assay broth • Yeast beef agar Quality control : • Aspects of a quality control include: • Standardized bacteriaI inoculum size • Culture conditions growth medium , pH , cation concentration • Blood and serum supplements and thymidine contents • Incubation conditions-atmosphere, temperature, duration • Concentration of anti microbials for testing.