Microbiology

The discovery of broad-spectrum antiparasitic agents relies on the ability to evaluate drug efficacy under harmonized in vitro conditions across related species. However, current drug screening pipelines for intraerythrocytic parasites are constrained by the use of species-specific media with distinct nutrient compositions and serum sources, which hinder direct comparison of compound potency. To address this gap, we describe a unified erythrocytic culture system based on DMEM/F12 supplemented with 20% fetal bovine serum (DFS20), which supports robust asexual growth of multiple Plasmodium falciparum strains (3D7, Dd2, HB3, V1/S), Babesia duncani, Babesia divergens (Rouen 87), and Babesia MO1. Parasite proliferation and morphology in DFS20 are comparable to those observed in established species-specific media such as RPMI-1640 for P. falciparum and B. divergens and HL-1/Claycomb/DMEM/F12/SFM for B. duncani, while eliminating reliance on undefined or discontinued proprietary components. Importantly, this standardized medium enables cross-species growth inhibition assays for direct comparison of drug efficacy under identical conditions. Using this platform, we recently screened dihydrotriazines and biguanides targeting the conserved DHFR-TS enzymes and identified potent antifolate candidates with broad-spectrum activity against Babesia and Plasmodium species. For B. duncani, which is uniquely supported by both a continuous in vitro human erythrocyte culture system and a lethal in vivo mouse infection model, integration with the in-culture and in-mouse (ICIM) pipeline enables systematic validation of pharmacodynamics, pharmacokinetics, resistance, and toxicity. This unified DFS20-based system establishes a scalable and reproducible protocol for harmonized drug efficacy evaluation across intraerythrocytic parasites and provides a foundation for the development and prioritization of pan-antiparasitic therapies.

Toxoplasma gondii is an apicomplexan parasite that infects a wide variety of eukaryotic hosts and causes toxoplasmosis. The cell cycle of T. gondii exhibits a distinct architecture and regulation that differ significantly from those observed in well-studied eukaryotic models. To better understand the tachyzoite cell cycle, we developed a fluorescent ubiquitination-based cell cycle indicator (FUCCI) system that enables real-time visualization and quantitative assessment of the different cell cycle phases via immunofluorescence microscopy. Quantitative immunofluorescence and live-cell imaging of the ToxoFUCCI^S probe with specific cell cycle markers revealed substantial overlap between cell cycle phases S, G₂, mitosis, and cytokinesis, further confirming the intricacy of the apicomplexan cell cycle.

Most viruses extensively remodel their host cells to establish productive infection. Visualization of virus-induced cellular remodeling by electron microscopy (EM) has been revolutionized in recent years by advances in cryo-focused ion beam (cryo-FIB) milling paired with cryo-electron tomography (cryo-ET). As cryo-FIB/ET becomes more widely available, there is a need for beginner-friendly guides to optimize the preparation of virus-infected mammalian cells on EM grids. Here, we provide an in-house protocol for new users for preparing samples of cells infected with herpes simplex virus 1 (HSV-1) for cryo-FIB/ET. This protocol guides users in how to seed infected cells onto grids, blot, and plunge-freeze grids using basic, manual equipment. It also provides tips on how to screen and prioritize grids for efficient milling and data collection.

Lipid droplets have emerged as dynamic organelles involved in diverse cellular processes beyond simple lipid storage. In plants and cyanobacteria, growing evidence highlights their importance in stress adaptation and signaling, yet methods to study their structure and purity remain limited. Traditionally, in situ transmission electron microscopy (TEM) has been used to visualize lipid droplets within intact cells. While powerful, this approach cannot easily evaluate isolated lipid droplets or confirm their purity. In this protocol, we describe a rapid method for preparing and visualizing cyanoglobule lipid droplets isolated from cyanobacteria. The isolated droplets are directly processed for TEM using negative staining with uranyl acetate, providing a straightforward and efficient workflow. The procedure can be applied broadly to lipid droplets from diverse organisms, independent of species or cellular origin. This protocol offers a simple, fast, and widely applicable approach to assessing lipid droplets, expanding the toolkit for researchers studying their structure and function.

Microbial life cycles are often reconstructed theoretically from fragmentary pieces of evidence. Protocols for the direct and continuous observation of entire microbial life cycles, including sexual reproduction, are scarce, which limits the study of cellular transitions between different life cycle stages and prevents the visualization of cryptic stages. Although sequence-based techniques, such as -omics approaches, can reconstruct cellular transitions at the genetic and biochemical level, these methods are destructive and do not recover information from the same living cell over time. This protocol provides a solution to directly and continuously observe microbial life cycles, including sexual reproduction, by using microfluidics manipulations that expose single cells to nutritional stimuli and selective pressures. As proof of principle, we triggered a life cycle sequence transition in the model yeast Saccharomyces cerevisiae, starting with an arrest of proliferation in an ancestor cell followed by induction of meiosis through starvation, selection of sexually reproducing cells through exposure to a drug cocktail, germination of haploid spores, and mating of haploid individuals, creating a new descendant generation. This protocol offers the possibility to directly compare molecular and cellular behavior across life cycle stages and across sexually reproducing generations.

Candida albicans is the pathogenic fungus that most frequently causes infections in humans. It is part of the microbiota commonly found in the skin, gastrointestinal tract, and vaginal mucosa. However, certain conditions, including immunosuppression, excessive use of antibiotics, hormonal changes, the use of medical devices in patients, and individual nutritional status, promote the development of opportunistic infections caused by this fungus. One of the main fungal structures interacting with the host is the cell wall, which is principally composed of chitin, glucan, and proteins. The cell wall plays key functions for the cell, such as osmotic protection; it is also responsible for cellular shape and acts as a signaling hub in response to environmental changes. Cell wall proteins participate in diverse cellular functions, such as attachment to surfaces and cell wall structure; some possess catalytic or transport activities. In this protocol, we show the methodology for isolating cell wall proteins covalently linked or not to cell wall components that can be previously labeled with [¹⁴C]-L-lysine by the action of the fungal transglutaminase localized in the cell wall. We use an extraction method by mechanical cell disruption and washing with 2 M NaCl, whose ionic strength eliminates contaminating proteins from other organelles, through subsequent serial treatments with SDS, chitinase, and zymolyase.

The ribosome, a complex macromolecular machine, plays a vital role in cellular translation. To investigate its structure and conduct in vitro experiments, isolating the ribosomes from cells is the first step. While isolating ribosomes from bacterial cells is routine, obtaining them from mycobacteria proves challenging due to the protective mycolic acid layer, which hinders cell lysis. In this study, we present a straightforward and efficient protocol for isolating ribosomes from Mycobacterium smegmatis. Additionally, we introduce a co-sedimentation assay using density gradient ultracentrifugation, providing a simple yet powerful method for studying ribosome–protein interactions. The re-association assay also offers a practical approach for obtaining tRNA-free 70S ribosomes and evaluating the anti-association properties of potential ligands. While these assays are commonly used, our protocol stands out for its simplicity, requiring limited specialized instruments. These methods can also be scaled up or down per requirement. By employing sonication for cell rupture and utilizing basic lab equipment for ultracentrifugation-based assays, our method greatly simplifies ribosome isolation and related research.

Orthoflavivirus is an enveloped, positive-stranded RNA virus that buds into the endoplasmic reticulum (ER) lumen. The budded virus particles are subsequently transported to the Golgi apparatus and secreted into the extracellular environment via the conventional secretion pathway. In this protocol, we describe a method for monitoring the secretion of Orthoflavivirus particles from the ER. To visualize intracellular membrane trafficking, we combine two distinct imaging techniques: the retention using selective hooks (RUSH) system and the split green fluorescent protein (GFP) system. In this approach, GFP11, a peptide tag fused to prME, the outer coat structural protein of Japanese encephalitis virus particles, was co-expressed in HeLa cells along with two additional components: GFP1-10 fused to a streptavidin-binding peptide and a hook construct consisting of streptavidin fused to the ER retention sequence KDEL. Time-lapse imaging was performed after the addition of biotin, which releases the captured GFP-labeled subviral particles from the ER. This method enables synchronized visualization of intracellular subviral particle trafficking and serves as a valuable tool for analyzing the maturation process of Orthoflavivirus particles within cells.

Microbial biofilms are structured communities of microorganisms embedded in a self-produced extracellular matrix, adhering to surfaces. These biofilms enhance bacterial resistance to antibiotics, immune responses, and environmental stress. Current microscopy techniques, such as scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and fluorescence microscopy, are commonly used to visualize and differentiate biofilms. However, their high cost and complexity often render them impractical. In contrast, simpler methods like crystal violet and Congo red staining are limited in distinguishing bacterial cells from the biofilm matrix. This study introduces a cost-effective, dual-staining method using Maneval’s stain to visualize and differentiate microbial biofilms. It requires only basic equipment and minimal reagents, making it ideal for routine use in clinical diagnosis and microbial research.

Cricket paralysis virus (CrPV), a member of the family Dicistroviridae, is a single-stranded positive-sense RNA virus that primarily infects arthropods. Some members of the dicistrovirus family, including the honey bee viruses Israeli acute paralysis virus and Acute bee paralysis virus and the shrimp-infecting Taura syndrome virus, pose significant threats to agricultural ecosystems and economies worldwide. Dicistrovirus infection in Drosophila is used as a model system to study fundamental insect–virus–host interactions. The availability of a CrPV infectious clone allows controlled manipulation of the viral genome at a molecular level. Effective viral propagation and titration techniques are crucial for understanding the pathogenesis and epidemiology of dicistrovirus infections. Traditional methods for assessing viral titers, such as plaque assays, are unsuitable for CrPV, since Drosophila tissue culture cells like Schneider 2 cells cannot readily form adherent plaques. Here, we present a streamlined protocol for generating a recombinant virus from a CrPV infectious clone, propagating the virus in S2 cells and titering the virus by an immunofluorescence-based focus-forming assay (FFA). This protocol offers a rapid and reliable approach for generating recombinant viruses, viral amplification, and determining CrPV titers, enabling efficient investigation into viral biology and facilitating the development of antiviral strategies.