The observer (via AST): observes no colonies or absence of turbidity. More often the planktonic cells at MBC cannot grow if plated without antimicrobial agent or upon their removal. guarantee therapeutic success or significantly improve the therapeutic outcome . The gold standard method is a universally accepted, definitive, and dependable technique for measuring a specific phenomenon or outcome. It is generally regarded as the most accurate and trustworthy method of measuring a particular phenomenon, and it is often used as a benchmark against which other methods or measurements are evaluated. The use of a gold standard allows researchers to assess the accuracy and efficacy of new methods by comparing them to a known, established standard. In the context of antimicrobial susceptibility testing (AST), there are several gold standard methods that are considered the most accurate and dependable ways to determine the susceptibility of bacterial strains to antibiotics. The initial methods developed for AST are phenotypic in nature and involve techniques that rely on the growth of the microorganism being tested. For those microbes that do not grow readily, special, or enriched media containing growth factors must be used. Among the commonly used phenotypic methods are broth dilution, agar dilution, disk diffusion, and gradient diffusion (Etest®). The first AST method was the ditch plate technique, which was introduced by Alexander Fleming in 1924 . This was modified by cutting wells into agar, and the principle of this technique was developed further by the cylinder plate (also known as the Oxford cup or Heatley cup assay ). Following this, Fleming's second contribution to modern AST was the broth dilution technique using turbidity as an end-point , and later using pH as an indicator instead of turbidity . In the 1940s, Schmith & Reymann described an agar dilution AST method for gonococci . However, the MIC estimates from agar dilution were too time-consuming and cumbersome. Subsequently, many variants of diffusion and dilution techniques were reported by various researchers . Thus, the AST gradually evolved from the ditch plate method, and it became evident that there was a need to standardize AST as many variables affected the test results. In 1968, the National Committee for Clinical Laboratory Standards (NCCLS), now known as the Clinical and Laboratory Standards Institute (CLSI), was established to create a consensus standard, guidelines, and best practices for AST in clinical laboratories. Although there are several gold standard techniques used for the determination of antimicrobial susceptibility testing, these methods are generally comprised of either manual or automated approaches. Phenotypic gold antimicrobial susceptibility testing involves techniques that rely on the growth of the microorganism, and those microbes that do not readily grow require the use of special or enriched media containing growth factors. In summary, the gold standard methods for AST are recognized as the most accurate and reliable ways to determine the susceptibility of bacterial strains to antibiotics, and they provide a benchmark against which other methods are compared to determine their validity and reliability .