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Microbiological culture sensitivity test

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Microbiological culture sensitivity test

  1. 1. Microbiological culture sensitivity tests PREPARED BY: DR.C.SUHAS REDDY 1 Microbiology  Microbiology is the study of living organisms that are invisible to the naked eye, such as bacteria, virus and fungi. 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. Sensitivity test  A laboratory test perform to check the effectiveness of a drug against a microorganism, to select the best drug regimen that is effective. Microbiological culture sensitivity tests  It is a method of multiplying microbial organisms in a predetermined culture media with favourable conditions and test perform to check the sensitivity of organism towards the drugs. Purpose of culture sensitivity test  To guide the clinician in selecting the best drug for an individual patient.  To control the use of inappropriate drug in clinical practice.  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. How sensitivity analysis done  Sensitivity analysis starts with a bacterial sample.  The sample is obtained by swabbing the infected area or secretions of the infected area and from any area that has an infection.  Cultures are taken from blood, Urine, Sputum, inside a cervix and a wound. 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 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 (culture media) and the Bacteria in the culture will grow and multiply.
  2. 2. Microbiological culture sensitivity tests PREPARED BY: DR.C.SUHAS REDDY 2  The bacteria will form colonies-large groups of bacteria that will be exposed to different antibiotics.  With a sufficient population of bacteria grown on the plate in the form of a “lawn", the technicians will perform two main operations: i. Identify the species of bacteria  This is done with various techniques, including examination of lawn characteristics-color, texture, growth pattern, etc.  Gram staining, microscopic examination, metabolic requirements and even DNA sequencing.  Bacterial species commonly isolated depends on the location and the cause of the infection. ii. 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 each disk. Methods for Microbiological Culture Sensitivity Tests 1. DILUTION METHODS 2. DISC-DIFFUSION METHOD 3. E-TEST 4. AUTOMATED METHODS 5. MECHANISM-SPECIFIC TESTS 6. GENOTYPIC METHODS 1. DILUTION METHODS (Eg-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 0.1 ml total broth volume and can be conveniently performed in a micro-titer format.  Macro dilution testing uses broth volumes at about 1.0ml in standard test tubes.
  3. 3. Microbiological culture sensitivity tests PREPARED BY: DR.C.SUHAS REDDY 3  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.  Minimum inhibitory concentration (MIC), in microbiology, is the lowest concentration of an antimicrobial that will inhibit the visible growth of a micro- organism after overnight incubation.  Minimum inhibitory concentrations are important in diagnostic laboratories to confirm resistance of microorganisms to an antimicrobial agent and also to monitor the activity of new antimicrobial agents.  The MIC is thus the minimum concentration of the antibiotic that will inhibit the particular isolate.  The test is only valid only if the positive control shows growth and negative control shows no growth.  A procedure similar to broth dilution is agar dilution.  Agar dilution method follow a principle of establishing the lowest concentration of the serially diluted antibiotic concentration at which bacterial growth is still inhibited. Antibiotics are diluted to various dilutions to test the MIC  Test tubes – without red color – no microorganism growth  Test tubes- Red color mark shows – growth of microorganism  1 - Represents agar media & 2,4,8,16,32 - Represents antibiotics.  1/2= one part of agar + 2 parts of antibiotic in one test tube.
  4. 4. Microbiological culture sensitivity tests PREPARED BY: DR.C.SUHAS REDDY 4 2. DISC-DIFFUSION METHOD  Take the sample (microbial) and swab it on to the growth media (Mueller-Hinton agar).  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 2 x 10 colony forming units per ml).  Then, using a dispenser such as the one pictured, antibiotic-impregnated disks 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 disk.  The test antibiotic immediately begins to diffuse outward 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 7 mm.  Zone diameter is reported in millimetres, looked up on the chart, and result reported as S (sensitive), R (resistant), or I (intermediate). Antibiotic Disc Zone of inhibition a. A,B,C-are different antibiotic disc. b. Clear white color zone around the antibiotic disc is zone of inhibition. c. color zone in the remaining areas indicate growth of microorganism Measure the diameter of the zone of inhibition by using the scale.
  5. 5. Microbiological culture sensitivity tests PREPARED BY: DR.C.SUHAS REDDY 5 3. E-TEST  E-test is an antimicrobial gradient technique in which 15 reference MIC dilutions of an antibiotic have been repackaged with innovative dry chemistry technology onto a plastic strip.  E-Test(AB Biodisk, Solna, Sweden) is a commercially available test that utilizes a plastic test strip impregnated with a gradually decreasing concentration of a 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 a clinical isolate.  However, a separate strip is needed for each antibiotic, and therefore the cost of this can be high. 4. AUTOMATED METHODS  Most automated antimicrobial susceptibility testing systems provide automated inoculation, reading and interpretation.  These systems have the advantage of being rapid and convenient, but one major limitation for most laboratories is the cost entailed in initial purchase, operation and maintenance of the machinery. The strips are impregnated with various concentration of Antibiotics Zone of inhibition a. Two white color strips are impregnated with different antibiotic concentration. b. Clear colorless zone around the antibiotic disc is zone of inhibition. c. MIC- Minimum dose of a drug required to stop the microorganism growth. d. Color zone in the remaining areas MIC
  6. 6. Microbiological culture sensitivity tests PREPARED BY: DR.C.SUHAS REDDY 6 5. MECHANISM-SPECIFIC TESTS (such as Beta-lactamase detection test and Chromogenic Cephalosporin test)  Resistance may also be established through tests that directly detect the presence of a particular resistance mechanism.  For example, beta lactamase detection can be accomplished using an assay such as the chromogenic cephalosporinase test. 6. GENOTYPIC METHODS (such as PCR and DNA hybridization methods)  Although nucleic acid-based detections 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 expressions of these genes.  Some of the most common molecular techniques utilized for anti-microbial resistance detection are PCR and DNA hybridization methods. i. Polymerization chain reaction:  It is one of the most commonly used molecular techniques for detecting certain DNA sequences of interest.  This involves several cycles of denaturation of sample DNA, annealing of a specific primers to the target sequence and the extension of this sequence as facilated by a thermostable polymerase leading to replication of a duplicate DNA sequence, in an exponential manner, to a point which will be visibly detectable by gel electrophoresis with the aid of a DNA- intercalating chemical fluoresces under UV light. ii. DNA hybridization method:  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 known specific sequence can pair up with opened or denatured DNA from the test sample, as long as their sequences complement each other.  If this hybridization occurs, the probe labels this with a detectable radioactive isotope, antigenic substrate, enzyme or chemiluminescent 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.
  7. 7. Microbiological culture sensitivity tests PREPARED BY: DR.C.SUHAS REDDY 7  Modifications of the PCR and DNA hybridization. With these basic principles, several modifications have been introduced which further improve the sensitivity and Specificity of these procedures.  Examples of such development the use of fluorescence-labelled oligonucleotides, the development of molecular bacons, development of DNA arrays and DNA chips, Among many others. What are the results for a sensitivity analysis  The colonies show up as: Susceptible and/or Resistant and/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 not grow if the drug is present. This indicates an effective antibiotic.  Resistant: in this case, the filter Paper will have no discernable plaque around it, meaning that the bacteria can grow even in the presence of a antibiotic. This indicates an ineffective anti-biotic  Intermediate: a somewhat 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.  The doctor can use the results to determine the best antibiotic to treat your infection. What are the risks of a sensitivity analysis  Few risks are associated with this test. Blood collection comes with small risks.  Rare risks of taking a blood sample include:  Fainting.  Hematoma- a bruise where blood accumulates under the skin.  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.  The doctor will advise the patient about the potential risks associated with the sample. Applications  To provide a reliable prediction of whether an infection caused by a bacterial isolate 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.

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