BACTERIOLOGY LABLECTURE 1:Antimicrobial Sensitivity TestingProf Jason “Ab” Chua, RMTNov 7, 2021For updates and corrections →@mar4riion TwitterPRECISE DEMONSTRATION OF THE KIRBY-BAUERTECHNIQUE●Most clinically important bacteria is capable of acquiringand expressing resistance to antimicrobial agentscommonly used to treat the infections they cause.●Once an organism is isolated in the laboratory,characterization frequently includes tests to detectantimicrobial resistance. In addition to the organisms’identification, the antimicrobial susceptibility profile isanother key component of the laboratory report that isproduced for the position.●The procedures used to detect antimicrobial susceptibilityprofiles and detect resistant agents that may be usedtherapeutically is referred to as“AntimicrobialSusceptibility Testing Methods”.Direct measures of antimicrobial activity are accomplishedusing:●Conventional susceptibility testing methods○Broth dilution○Agar dilution○Disk diffusion●Commercial susceptibility systems●Special screens and indicator testsLearning ResourcesReagents andSolutionsMaterialsEquipment● Mueller-HintonAgar (MHA)●Antibiotic Disk●70% alcohol●0.5 Mc FarlandStandard●Alcohol Lamp●Normal SalineSoluttion (NSS)● Bleach●Gram Stainsolution set●Immerion Oil●Tryptic Soy Broth(TSB)●Antibiotic disk●Sterile swabs● Forcceps●Metric ruler orcaliper●Marker or waxpencil●Beaker inoculatingloop●Test tube racks●Sterile PasteurPipettes● Incubator● Refrigerator● AutoclaveIntroduction●Primary goal of Antimicrobial Susceptibility Testing○Determine whether the bacterial etiology ofconcern is capable of expressing resistance toantimicrobial agents that are potential choicesfor therapy○Because intrinsic resistance are well-known formost organisms○Testing to establish this resistance is notusually necessary and organism identification issufficient●Antimicrobial Susceptibility Testing are assays deignedto determine the extent of an organism’s acquiredresistance for any clinically important organism whoseantimicrobial susceptibility profile is unpredictable●The number of antibiotics available today is larger thanever before. New antibiotics are continuously beingdeveloped and discovered; thus, there is an increasingdemand on the clinical laboratory to determine theantibiotic susceptibility or resistance of variouspathogenic bacteria●One method is theSensitivity Disk Method orBONES●Kirby-Bauer or Disk Diffusion Susceptibility Testing○In this method, antibiotics are impregnated ontopaper disks and the placed on a seededMueller-Hinton Agar (MHA)plate using amechanical dispenser or sterile forceps○MHA contains beef infusions, nucleic acids,vitamins, casein hydrolysate as a peptonesource, cornstarch to neutralize fatty acids, andagar as a solidifying agent○This medium supports the growth of mostbacteria, and can be supplemented with bloodto grow fastidious bacterias such asStreptococcus pneumoniae○Increase concentration of calcium andmagnesium ions in the agar may lead to thedecrease activity of the amino glycosidesagainst pseudomonas aeruginosa, anddecrease activity of tetracyclines against allbacteria○Decrease concentration of calcium andmagnesium salts leads to increase activityagainst these agents, thus the medium musthave acceptable levels of these cations, whichcan be monitored through quality control
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