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Introduction Antimicrobial resistance (AMR) is a serious global concern, as infectious diseases become harder to treat [1].The methods covered include disk diffusion assay, well diffusion, spot assay, cross-streaking method, poisoned food method, agar dilution, broth macrodilution and microdilution, resazurin assay, co-culture method, time-kill kinetics, flow cytometry, thin layer chromatography-bioautography (TLC bioautography), bioluminescence assay and impedance measurement.The agar diffusion assay serves as a valuable tool in antimicrobial research, providing qualitative data on the effectiveness of different substances against specific microorganisms.Types of antimicrobial nanoparticles include silver nanoparticles which disrupt bacterial cell membranes and damage intracellular structures [22], zinc oxide nanoparticles which produce reactive oxygen species (ROS) elevating membrane lipid peroxidation that causes membrane leakage of reducing sugars, DNA, proteins, and reduces cell viability [23]; copper nanoparticles generate ROS, disrupt microbial cell walls and membranes, and interact with proteins and DNA [24]; the calcium oxide nanoparticles produce free radicals that damage bacterial cell membrane and the arrangement of polyunsaturated phospholipids [25]; and titanium dioxide nanoparticles which possess photocatalytic properties generating antimicrobial reactive oxygen species [26].Other synthetic antimicrobial agents include daptomycin, a semisynthetic lipopeptide antibiotic that disrupts bacterial cell membranes and is used to treat skin and bloodstream infections caused by Gram-positive bacteria [21]; metronidazole, used to treat infections caused by anaerobic bacteria and parasites by disrupting their DNA synthesis; and others.Among the phytochemicals, polyphenolics such as flavonoids, tannins, quinones, coumarins, and others have shown significant antimicrobial properties by disrupting microbial cell membranes, inhibiting key enzymes, and interfering with key cellular processes [3].Additionally, the arsenal of microbial antimicrobial compounds includes other bioactive substances such as bacteriocin-like inhibitory substance (BLIS), cyclic lipopeptides, and lectins, all of which display potent activity against a wide range of microbial pathogens [14].Synthetic antimicrobials Synthetic antimicrobial compounds are chemically synthesized substances meticulously designed to possess potent antimicrobial properties, providing protection against the escalating challenge of bacterial resistance to conventional antibiotics.This comprehensive overview aims to aid researchers and practitioners in choosing appropriate screening techniques, allowing for the efficient identification of potential antimicrobial agents and contributing to the development of effective therapies against infectious diseases.Terpenoids represent another important group of plant-extract bioactives which exhibit potent antimicrobial activity by disrupting microbial membranes, inhibiting protein synthesis, or interfering with essential metabolic pathways [4].Moreover, alkaloids, another group of plant-derived bioactive compounds, also demonstrated remarkable antimicrobial properties with some alkaloid-based drugs such as quinine, berberine, among others historically utilized for treating infectious diseases [3].Like plants, various animal species including insects, amphibians, reptiles, birds, and mammals, also produce AMPs that can target and disrupt microorganisms' cell membranes, showing broad-spectrum activity against bacteria, fungi, and certain viruses [7].An iconic example is Penicillin, derived from the Penicillium fungus [11], which marked a groundbreaking advancement in modern medicine and paved the way for numerous crucial therapeutic agents to combat bacterial infections.By discussing gaps and limitations in current methodologies, the paper encourages researchers to explore approaches and technologies that enhance the accuracy, sensitivity, and efficiency of antimicrobial detection.Sources of antimicrobials The presence of antimicrobial activity has been documented in a diverse array of sources, encompassing natural origins derived from plants, animals, and microorganisms as well as synthetic compounds, nanoparticles, and so forth.Quinolones, a class of synthetic antimicrobial compounds such as ciprofloxacin and levofloxacin, impede bacterial DNA replication by inhibiting enzymes such as DNA gyrase and topoisomerase IV [19].Methods to evaluate antimicrobial activity The relentless quest for novel and highly effective antimicrobial compounds remains an ongoing pursuit driven by the pressing challenge of antimicrobial resistance, which diminishes the effectiveness of traditional antibiotics.On the other hand, newer methods like flow cytometry, bioluminescence, and impedance measurement offer higher sensitivity and throughput, but they may be costlier and less accessible in resource-limited regions.The phytochemicals employ various mechanisms to inhibit the growth and survival of microorganisms including bacteria, fungi, and viruses that make them promising candidates in developing antimicrobial agents.Microbial sources of antimicrobials Microbial products encompass a remarkable diversity of antimicrobial activity, as microorganisms themselves produce potent compounds to compete for resources and ensure survival in their environments.These small cationic peptides exhibit impressive effectiveness against closely related bacteria, contributing to food preservation by inhibiting spoilage and pathogenic bacteria in fermented foods [12, 13].These methods rely on the diffusion of antimicrobial agents from paper discs, wells, or plugs into the adjacent agar medium, inhibiting the growth of the test microorganism inoculated on the agar surface [27].Defensins penetrate microbial membranes, leading to cell death and preventing infections in various anatomical sites like the respiratory and gastrointestinal tracts [8].Linezolid, another synthetic antibiotic, inhibits protein synthesis in Gram-positive bacteria including Staphylococcus aureus and Streptococcus pneumoniae [18].For each assay, their respective advantages and limitations are presented, addressing issues of cost, accessibility, reproducibility, complexity of the tested samples, and other relevant factors.Nanoparticles exhibit potent antimicrobial activity against bacteria, fungi, and viruses.1.

Introduction
Antimicrobial resistance (AMR) is a serious global concern, as infectious diseases become harder to treat [1]. Multi-resistant bacteria are on the rise, and the development of new antimicrobials is limited, making the situation even more challenging. To address this issue, researchers are exploring both natural and synthetic antimicrobial compounds as potential solutions [2]. Natural products, derived from plants, animals, and microorganisms, offer a rich source of biologically active compounds that show promise in combating infections. Additionally, synthetic antimicrobial compounds designed in laboratories have also demonstrated remarkable potential in fighting microbial infections. To unlock the potential of these compounds, appropriate antimicrobial screening and evaluation methods are essential. The in vitro antimicrobial assays play a crucial role in the discovery and development of new antimicrobial agents, providing crucial insights into their effectiveness and mechanisms of action. Understanding these methods better will help researchers identify potential antimicrobial candidates more efficiently and contribute to addressing the growing threat of AMR. While traditional technologies like disk diffusion and broth dilution methods are widely used, they may have limitations in reproducibility and time. On the other hand, newer methods like flow cytometry, bioluminescence, and impedance measurement offer higher sensitivity and throughput, but they may be costlier and less accessible in resource-limited regions. Thus, a comprehensive review of in vitro assays used to assess the antimicrobial activities of both natural and synthetic compounds is important.

This review provides an overview of the most common in vitro assays used to characterize the antimicrobial activity of promising natural and synthetic compounds. The methods covered include disk diffusion assay, well diffusion, spot assay, cross-streaking method, poisoned food method, agar dilution, broth macrodilution and microdilution, resazurin assay, co-culture method, time-kill kinetics, flow cytometry, thin layer chromatography-bioautography (TLC bioautography), bioluminescence assay and impedance measurement. For each assay, their respective advantages and limitations are presented, addressing issues of cost, accessibility, reproducibility, complexity of the tested samples, and other relevant factors. This comprehensive overview aims to aid researchers and practitioners in choosing appropriate screening techniques, allowing for the efficient identification of potential antimicrobial agents and contributing to the development of effective therapies against infectious diseases. By discussing gaps and limitations in current methodologies, the paper encourages researchers to explore approaches and technologies that enhance the accuracy, sensitivity, and efficiency of antimicrobial detection.

Sources of antimicrobials
The presence of antimicrobial activity has been documented in a diverse array of sources, encompassing natural origins derived from plants, animals, and microorganisms as well as synthetic compounds, nanoparticles, and so forth.

Plant sources of antimicrobials
Plants produce a diverse range of bioactive compounds as a part of their natural defense system. Many of these compounds, known as phytochemicals, are highly effective in combatting microbial pathogens. The phytochemicals employ various mechanisms to inhibit the growth and survival of microorganisms including bacteria, fungi, and viruses that make them promising candidates in developing antimicrobial agents. Among the phytochemicals, polyphenolics such as flavonoids, tannins, quinones, coumarins, and others have shown significant antimicrobial properties by disrupting microbial cell membranes, inhibiting key enzymes, and interfering with key cellular processes [3]. Terpenoids represent another important group of plant-extract bioactives which exhibit potent antimicrobial activity by disrupting microbial membranes, inhibiting protein synthesis, or interfering with essential metabolic pathways [4]. Some terpenoids such as essential oils have been found highly effective against a range of microorganisms rendering them valuable contenders for antimicrobial applications. Saponins, glycosides found in numerous plant species, also exhibit antimicrobial properties by disrupting microbial cell membranes [5]. Moreover, alkaloids, another group of plant-derived bioactive compounds, also demonstrated remarkable antimicrobial properties with some alkaloid-based drugs such as quinine, berberine, among others historically utilized for treating infectious diseases [3]. Additionally, plants also produce certain antimicrobial peptides (AMPs) such as defensins which also contribute to their antimicrobial activity by targeting microbial cell membranes [6]. Some plants produce lectins, which are carbohydrate-binding proteins with antimicrobial properties, offering another line of defense against pathogens. These diverse bioactive compounds derived from plant extract highlight their remarkable potential as sources of natural antimicrobial agents.

Animal sources of antimicrobials
The animal kingdom serve as a valuable resource for antimicrobial peptides and proteins which participate in their natural defense against harmful microorganisms. Like plants, various animal species including insects, amphibians, reptiles, birds, and mammals, also produce AMPs that can target and disrupt microorganisms' cell membranes, showing broad-spectrum activity against bacteria, fungi, and certain viruses [7]. For instance, amphibians like frogs and toads produce AMPs in their skin secretions, acting as a powerful defense against microbes. Defensins, the largest AMP family, are also produced by many animals, including mammals, birds, and insects. Defensins penetrate microbial membranes, leading to cell death and preventing infections in various anatomical sites like the respiratory and gastrointestinal tracts [8]. Additionally, animal sources provide other antimicrobial molecules like lysozymes, lactoferrin and related proteins [9]. Lysozymes, found in secretions like tears, saliva, and milk, break down bacterial cell walls causing bacterial cell lysis. Lactoferrin, an iron-binding protein in milk and bodily fluids, restricts microbial growth by sequestering essential nutrients like iron. Crustaceans and fish produce AMPs like piscidins and crustins which have demonstrated activity against bacteria, fungi, and viruses [10].

Microbial sources of antimicrobials
Microbial products encompass a remarkable diversity of antimicrobial activity, as microorganisms themselves produce potent compounds to compete for resources and ensure survival in their environments. Among them, antibiotics stand out as the most renowned and extensively studied natural antimicrobial compounds. Produced by certain bacteria and fungi, antibiotics possess the remarkable ability to inhibit the growth of other microorganisms. An iconic example is Penicillin, derived from the Penicillium fungus [11], which marked a groundbreaking advancement in modern medicine and paved the way for numerous crucial therapeutic agents to combat bacterial infections. Microorganisms also produce a large group of antimicrobial peptides known as bacteriocins. These small cationic peptides exhibit impressive effectiveness against closely related bacteria, contributing to food preservation by inhibiting spoilage and pathogenic bacteria in fermented foods [12, 13]. Additionally, the arsenal of microbial antimicrobial compounds includes other bioactive substances such as bacteriocin-like inhibitory substance (BLIS), cyclic lipopeptides, and lectins, all of which display potent activity against a wide range of microbial pathogens [14].

Some archaea and protists also produce antimicrobial compounds. For example, certain species of archaea produce antimicrobial peptides known as archaeocins which are active against other archaea and some bacteria, and are thought to play a role in inter-microbial competition [15]. Some species of protists, such as amoebae, also produce antimicrobial peptides and proteins that are active against bacteria [16].

Synthetic antimicrobials
Synthetic antimicrobial compounds are chemically synthesized substances meticulously designed to possess potent antimicrobial properties, providing protection against the escalating challenge of bacterial resistance to conventional antibiotics. Triclosan is an example of synthetic antimicrobial compounds that exhibit broad-spectrum antibacterial and antifungal activity [17]. Frequently used in personal care products like soaps and toothpaste, it disrupts bacterial cell membranes. Linezolid, another synthetic antibiotic, inhibits protein synthesis in Gram-positive bacteria including Staphylococcus aureus and Streptococcus pneumoniae [18]. Quinolones, a class of synthetic antimicrobial compounds such as ciprofloxacin and levofloxacin, impede bacterial DNA replication by inhibiting enzymes such as DNA gyrase and topoisomerase IV [19]. Additionally, synthetic peptides have been developed to mimic the natural antimicrobial peptides found in animals and plants. Pexiganan, one such synthetic peptide, exhibits antimicrobial activity against both Gram-positive and Gram-negative bacteria by disrupting bacterial cell membranes [20]. Other synthetic antimicrobial agents include daptomycin, a semisynthetic lipopeptide antibiotic that disrupts bacterial cell membranes and is used to treat skin and bloodstream infections caused by Gram-positive bacteria [21]; metronidazole, used to treat infections caused by anaerobic bacteria and parasites by disrupting their DNA synthesis; and others.

Antimicrobial nanoparticles
Nanoparticles, ranging from 1 to 100 nm in size, possess unique physicochemical properties due to their small size and large surface area. They can be produced synthetically, or naturally by microorganisms, plants, and animals. Nanoparticles exhibit potent antimicrobial activity against bacteria, fungi, and viruses. Their small size enhances the interaction with pathogens making them highly effective antimicrobial agents. Types of antimicrobial nanoparticles include silver nanoparticles which disrupt bacterial cell membranes and damage intracellular structures [22], zinc oxide nanoparticles which produce reactive oxygen species (ROS) elevating membrane lipid peroxidation that causes membrane leakage of reducing sugars, DNA, proteins, and reduces cell viability [23]; copper nanoparticles generate ROS, disrupt microbial cell walls and membranes, and interact with proteins and DNA [24]; the calcium oxide nanoparticles produce free radicals that damage bacterial cell membrane and the arrangement of polyunsaturated phospholipids [25]; and titanium dioxide nanoparticles which possess photocatalytic properties generating antimicrobial reactive oxygen species [26].

Methods to evaluate antimicrobial activity
The relentless quest for novel and highly effective antimicrobial compounds remains an ongoing pursuit driven by the pressing challenge of antimicrobial resistance, which diminishes the effectiveness of traditional antibiotics. To assess potential antimicrobial compounds, whether recently discovered or well-known, researchers employ a wide array of antimicrobial assay techniques. Among these, the following antimicrobial assay methods are extensively used for the identification and evaluation of antimicrobial activity.

1. Agar diffusion based screening of antimicrobial activity
   Agar diffusion based assays, including the disk diffusion, well diffusion, agar plug, and agar spot assays, are widely utilized and cost-effective techniques in antimicrobial research for determining the antimicrobial activity of test compounds. These methods rely on the diffusion of antimicrobial agents from paper discs, wells, or plugs into the adjacent agar medium, inhibiting the growth of the test microorganism inoculated on the agar surface [27]. By measuring the resulting zone of inhibition (Fig. 1), which represents the area where microbial growth is prevented or inhibited by the agent, researchers can assess the relative potency of the test compound against the specific microorganism under investigation [28]. The agar diffusion assay serves as a valuable tool in antimicrobial research, providing qualitative data on the effectiveness of different substances against specific microorganisms.