Title 19. MICROBIAL CULTURE SENSITIVITY TESTING.pdf ✅

PHARMD GURU Page 1 INTRODUCTION TO MICROBIAL CULTURE SENSITIVITY TESTING (INTERPRETATION OF RESULTS):  Microbiology is the study of living organisms that are invisible to the naked eye - such as bacteria, virus, and fungi.  In microbiological test - microbial organisms are let to reproduce in predetermined culture media under controlled laboratory conditions.  Microbial culture is used to determine the type of organism and its abundance in the sample being tested or both.  A laboratory test is performed to check the effectiveness of a drug against microorganisms. This is used to select the best effective drug regimen.  The purpose of culture sensitivity test is to guide the clinician in selecting the best drug for an individual patient, to control use of inappropriate drug in clinical practice, to reveal changing trends in the local isolate, to overcome the microbial drug resistance.  Sensitivity analysis starts with a bacterial sample. Culture is taken from blood, urine sputum and a wound.  Selection of the appropriate method will depend on the intended degree of bioavailability of sources, availability of expertise and cost steps in analysis.  Minimum inhibitory concentration in microbiology is the lowest concentration of an antimicrobial that will inhibit the visible growth of microorganisms after overnight incubation.  Minimum inhibitory concentration is important in diagnostic laboratories to confirm resistance of microorganisms to an antimicrobial agent.  Agar dilution method follows 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.  The sample is swabbed onto the growth media.  A growth medium usually Mueller- Hinton agar is first even seeded throughout the plate with isolate of interest that has been diluted at standard concentration. Then using a dispenser such as the one pictured, antibiotic impregnated disc is placed on the agar surface. MICROBIAL CULTURE SENSITIVITY TESTING
PHARMD GURU Page 2  The test antibiotic immediately begins to diffuse outward from the discs, creating a gradient of antibiotic concentration.  Most automated antimicrobial susceptibility testing systems provide automated inoculation, reading and interpretation. PRINCIPLES AND METHODS OF DIFFERENT MICROBIOLOGICAL ASSAYS: (or) Microbiological assay is the technique in which the potency or concentration of a compound is assessed by determines its effect on microorganism. It is a legal quality control requirement for the assay of a number of antibiotics in both the British Pharmacopoeia and United States Pharmacopoeia. Many therapeutic agents that are either inhibit the growth of microorganisms (antibiotics) or essential for their growth (vitamins and amino acids) is standardized by microbiological assays. The microbiological assay of an antibiotic is based upon a comparison of the inhibition of growth of microorganisms by measured concentrations of the antibiotics under examination with that produced by known concentration of a standard preparation of the antibiotic having a known activity. Microbiological assay of vitamins is a type of biological assay performed with the help of microorganisms. APPLICATIONS: 1. This method determines the potency. 2. The method controls antimicrobial chemotherapy. 3. Good in-vitro and in-vivo correlations are provided by this method. 4. This method is accurate, in expensive and convenient. 5. This method is easy to interpret results.
PHARMD GURU Page 3 METHODS OF DIFFERENT MICROBIOLOGICAL ASSAYS: Two methods are used for the microbiological assay namely cylinder plate or cup plate method and tube assay method or turbidity method. METHOD A: CYLINDER PLATE METHOD (FIG. 4.9) Principle: This method depends on the diffusion of an antibiotic from a vertical cavity through the solidified agar layer in the petri plate. The growth of test microorganism is inhibited entirely in a zone around the cylinder containing antibiotic solution. Procedure:  The nutrient agar is melted, cooled and poured into petri dish.  0.2 ml of known concentration of inoculum is spread on the surface of solidified agar by spread plate technique.  Holes or cavities are made by sterile borer.  0.2, 0.4, 0.6, 0.8 and 1 ml of antibiotic is poured into the cup of agar plate and then incubated in 37°C for 24 hours.  Zone of inhibition is observed for antibiotic that has antimicrobial activity.  Zone of inhibition is measured by scale from the back side of the plate from the center of the cavity or hole. One Level Assay:
PHARMD GURU Page 4 L = The calculated zone diameter for the lowest concentration of the standard curve response line. H = The calculated zone diameter for the highest concentration of the standard curve response line. c = Average zone diameter of 36 readings of the reference point standard solution. a, b, d and e = Corrected average values for the other standard solutions, lowest to highest concentrations, respectively. Two Level Factorial Assay: Parallel dilutions containing two levels of both the standard (S1, and S2) and unknown (U1 and U2) are prepared. Cavities of each of plate are poured with diluted test samples along with standard. Finally, they are kept in the incubator and zone of inhibition is measured. The potency is calculated by the following formula If the potency of the sample is less than 60 per cent or more than 150 per cent of the standard, the assay is invalid and should be repeated using higher or lower dilutions of the same solution. The potency of the sample may be calculated from the expression.