Table 1.

Different screening methods and their respective advantages and disadvantages.

Method
Advantages	Disadvantages
Antibacterial and antifungal screening
Agar disk-diffusion method and variations
Simple, low cost High performance	Only qualitative results Not all fastidious bacteria can be tested
Poisoned food method
Useful for evaluating antifungal effects Quantitative and qualitative results	Poor performance
Thin-layer chromatography (TLC)-bioautography
Simple, low cost Suitable for bioactivity-guided fractionation	Poor efficiency for water-insoluble compounds
Dilution method
Easy interpretation Quantitative results, suitable for MIC calculation Appropriated for fastidious or non-fastidious bacteria, yeast and filamentous fungi	Labor-intensive and time consuming Poor efficiency for water-insoluble compounds
Time-kill test
Suitable for determining synergism or antagonism between bactericidal or fungicidal drugs Useful for determining the time- and concentration-dependent antimicrobial effect	Interlaboratory variability Labor-intensive and time consuming
ATP bioluminescence assay
Fast, especially for antimycobacterial Quantitative results Suitable for testing in vivo	Expensive technique Requires specialized equipment
Flow cytometry
Provide more information: detect antimicrobial resistance and target cell damage Fast	Requires specialized equipment
Antibiofilm and antiquorum-sensing screening
Colorimetric-based assays
Useful to assess the total biomass within a biofilm Highly accurate for large amounts of biofilm	Indirect measurement High detection limit No differentiation between dead and live cells
Laser confocal microscopy
Direct measurement of biofilms 3D representation of biofilms	Fluorophores are required Reporter molecules are limited Fluorophores interference with biofilm Auto-fluorescence might mask fluorophores’ signal
Disc diffusion assay
Simple, low cost Efficient	Qualitative results only Requires specific indicator strains
Flask incubation assay
Simple, low cost Quantitative results	Requires specific indicator strains Indirect measurement
Quorum quenching assay
Simple, low cost Efficient	Qualitative results Requires specific indicator mutated strains
Anti-tropical diseases screening
Kinetoplastid parasites
Target-based screening
High performance assays (HTS)	Very few fully validated drug targets Additional screening is needed for avoiding off-target effects
Phenotypic screening
High performance assays (HTS) and high-content imaging (HCS) in some parasite stages	Complex life cycles challenging to reproduce in laboratory Effectiveness in one parasitic stage does not guarantee the in vivo effect
Helminths
Target-based screening
High performance assays (HTS)	Very few fully validated drug targets Additional screening is needed for avoiding off-target effects
Phenotypic screening
Use of C. elegans (nonparasitic specie) as model High-content imaging (HCS) in larvae	Few screening campaigns to date Complex life cycles challenging to reproduce in laboratory Effectiveness in one parasitic stage does not guarantee the in vivo effect
Malaria
Target-based screening
High performance assays (HTS)	Very few fully validated drug targets Additional screening is needed for avoiding off-target effects
Phenotypic screening
Different methods developed for the different stages of life cycle: asexual erythrocytic-stage, liver stage, gametocyte Improvement in more physiologic in vitro human liver platforms High-content imaging techniques	Complex life cycles challenging to reproduce in laboratory Effectiveness in one parasitic stage does not guarantee the in vivo effect
Anticancer screening
Stained viable cells assay
Less expensive than other anticancer screening methods Quantitative results that are independent of the dye enzymatic conversion	Indirect measurement
Dye exclusion assay
Inexpensive	Indirect measurement Time consuming User biased Low precision
Methods based on metabolic activity
Suitable for HTS (MTT assays) Not cytotoxic for cells (Resazurin assay) High sensitivity (ATP content assay) Quantitative results	Indirect measurement Labor-intensive and time consuming
Protease viability marker assay
Can be used in multiplex High sensitivity Not toxic Quantitative results	Indirect measurement
Clonogenic cell survival assay
Highly precise results Quantitative results	Time consuming Only applicable to tumour cells that grow in culture
DNA synthesis cell proliferation assay
Highly accurate and reliable Suitable for HTS Quantitative results	Use of radioactive labels Time-consuming protocol
Neuroprotectors screening
Stress reduction assays
Allows mimics in vitro some features of neurodegenerative diseases Suitable for the combination of target-based and phenotypic screening High performance assays (HTS) and high-content imaging (HCS)	Too much simplification of the diseases
Neuroprotection assays
Allows mimics in vitro some features of neurodegenerative diseases Suitable for the combination of target-based and phenotypic screening High performance assays (HTS) and high-content imaging (HCS)	Too much simplification of the diseases
Regeneration assays
More physiologic cellular models High-content imaging (HCS)	Challenging techniques and specialized equipment